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https://openalex.org/W4288074933	https://discovery.ucl.ac.uk/id/eprint/10154870/1/bjo-2021-320887.full.pdf	English	null	Cost-effectiveness of primary surgical versus primary medical management in the treatment of patients presenting with advanced glaucoma	British journal of ophthalmology	2,022	cc-by	10,331	Clinical science Clinical science Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 202 WHAT THIS STUDY ADDS ⇒Trabeculectomy was associated with higher costs and slightly greater quality of life outcomes, medication is more likely to be considered cost effective at a 2-year time horizon. ⇒Trabeculectomy was associated with higher costs and slightly greater quality of life outcomes, medication is more likely to be considered cost effective at a 2-year time horizon. Received 6 December 2021 Accepted 29 June 2022 Received 6 December 2021 Accepted 29 June 2022 ⇒These results were consistent across different health-related quality of life measurement tools. Cost-effectiveness of primary surgical versus primary medical management in the treatment of patients presenting with advanced glaucoma Ashleigh Kernohan ‍ ‍ ,1 Tara Homer,1 Hosein Shabaninejad,1 Anthony J King ‍ ‍ ,2 Jemma Hudson,3 Gordon Fernie,4 Augusto Azuara-Blanco ‍ ‍ ,5 Jennifer Burr ‍ ‍ ,6 John M Sparrow ‍ ‍ ,7 David Garway-Heath,8 Keith Barton,8 John Norrie,9 Graeme Maclennan,4 Luke Vale1,10 ►Additional supplemental material is published online only. To view, please visit the journal online (http://dx. doi.org/10.1136/bjo-2021- 320887). ABSTRACT Synopsis Advanced glaucoma is associated with sight loss. This within-trial economic evaluation compares medical and surgical management strategies. At 2 years, medication appears more cost-effective though longitudinal outcomes are an important subject in future research. WHAT IS ALREADY KNOWN ON THIS TOPIC ⇒Those who present with advanced glaucoma have a high risk for developing sight loss.i ⇒Those who present with advanced glaucoma have a high risk for developing sight loss.i ⇒There are significant resource implications associated with glaucoma management and currently a lack of evidence as to the most cost-effective management strategy for those presenting with advanced glaucoma. For numbered affiliations see end of article. Background/aims Open angle glaucoma (OAG) is a progressive optic neuropathy. Approximately 25% of newly diagnosed patients with OAG present with advanced disease in at least one eye. The vision loss associated with OAG can lead to significant impacts on vision, quality of life and health care resources. The Treatment of Advanced Glaucoma Study is a randomised controlled trial comparing the effectiveness of primary surgical and medical management for newly diagnosed advanced patients with OAG. An economic evaluation was carried out to understand the costs and benefits of each strategy. Correspondence to Ashleigh Kernohan, Population Health Sciences Institute, Newcastle University, Newcastle upon Tyne, UK; ashleigh.kernohan@newcastle. ac.uk Correspondence to Ashleigh Kernohan, Population Health Sciences Institute, Newcastle University, Newcastle upon Tyne, UK; ashleigh.kernohan@newcastle. ac.uk ►Additional supplemental material is published online only. To view, please visit the journal online (http://dx. doi.org/10.1136/bjo-2021- 320887). HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY Protected by copyright. . Protected by copyright. . Protected by copyright. . Protected by copyright. . Protected by copyright. . Protected by copyright. . Protected by copyright. . Protected by copyright. . Protected by copyright. . Protected by copyright. . Protected by copyright. . Protected by copyright. To cite: Kernohan A, Homer T, Shabaninejad H, et al. Br J Ophthalmol Epub ahead of print: [please include Day Month Year]. doi:10.1136/ bjophthalmol-2021-320887 HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY Methods A cost utility analysis was carried out from a National Health Service perspective over a 2-year time horizon inclusive of patient costs. The primary outcome was patient health-related quality of life measured by the EQ-5D-5L, Health Utilities Index 3 (HUI3) and Glaucoma Utility Index (GUI). Results were expressed as incremental cost per QALY gained. ⇒This study provides evidence as to the costs and benefits of surgical and medical management of advanced glaucoma. Future research can focus on the cost and benefits of different strategies over a longer time horizon. p g Results Trabeculectomy was associated with higher costs and greater effect, the EQ-5D-5L results have an incremental cost per QALY of £45,456. The likelihood of surgery being cost-effective at a £20, 000, £30,000 and £50,000 QALY threshold is 0%, 12% and 56%, respectively. The results for the HUI3, GUI and inclusion of patient costs do not change the conclusions of the study. optic nerve results in visual field loss which is progressive if untreated. However, in the early stages, the disease glaucoma is usually asymptom- atic until the vision field loss is severe. Glaucoma can affect many aspects of daily living and can have a profound effect on health-related quality of life, more markedly in those with advanced disease.3 Those who present with a high IOP or severe visual field defect are most likely to lose sight despite treatment4 and advanced presentation is the greatest risk factor for lifetime blindness.5 In the UK, it is estimated that about 25% glaucoma suffers have advanced glaucoma in at least one eye at presentation.6 7 In addition, there are significant resource implications associated with manage- ment of the disease. It has been estimated that the cost of the management of a glaucoma patient is $2746±1560 (US$2015) over three years.8 With pressure on eye care services increasing9 it becomes essential to understand the management strategies Conclusion This is the first study to evaluate management strategies for those presenting with advanced glaucoma. At a 2-year time horizon, medication is the more cost-effective approach for managing glaucoma. Future research can focus on the costs and benefits of the treatments over a longer time horizon. © Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ. . Protected by copyright. . Protected by copyright. . Protected by copyright. . Protected by copyright. . Protected by copyright. . Protected by copyright. . Patient costs In addition to healthcare costs, participants’ out-of-pocket expenses were calculated so that the perspective could be expanded beyond that of the UK NHS. To inform this analysis trial participants completed a time and travel questionnaire. The responses to this questionnaire were used to estimate unit costs to patients and their families of the time and travel cost of accessing each type of healthcare provider. These unit costs were combined with information on the frequency of use of services collected using the CRFs and PQ described above. Current guidelines in the UK recommend those presenting with advanced OAG are offered augmented trabeculectomy,10 however this is not widely offered by ophthalmologists because of a lack of evidence supporting this recommendation11 The Treatment of Advanced Glaucoma Study (TAGS) is a pragmatic UK-based multicentre randomised controlled trial comparing the effectiveness of primary surgery (trabeculectomy) versus medical management (eye drops) in patients presenting with advanced glaucoma in at least one eye.12 Patients who presented with advanced glaucoma (as classified by the Hodapp-Parrish- Anderson (HPA) criteria13) were randomised to either a primary surgical or medical arm. The TAGS trial concluded that at 2 years trabeculectomy and medical management had similar quality of life, safety and vision outcomes, but trabeculectomy achieved a significantly lower IOP. As part of TAGS, a within- trial cost-utility analysis (CUA) was carried out to assess the differential effects on quantity and quality of life as well as the resource implications associated with the management of glau- coma.14 The aim of this study is to compare the costs and the benefits associated with primary surgical management compared with primary medical management of patients presenting with advanced glaucoma. on August 31, 2022 at UCL Library Services. Protected by copyright. http://bjo.bmj.com/ irst published as 10.1136/bjo-2021-320887 on 26 July 2022. Downloaded from With respect to travel unit costs, if the journey was under- taken using public transport the fare was used to represent travel costs. If a journey was undertaken by private car, then a fuel rate of £0.45 per mile was applied based on the business and self-employed expenses rate per mile.20 The cost of participant time was valued using average costs from the Office of National Statistics.21 Paid work, childcare, caring for relative or friend and voluntary work was valued as £13.88 an hour, housework and leisure activities were valued at £10.10 an hour. Data analysis Th i The regression analysis chosen for the TAGS within-trial cost- effectiveness analysis was a seemingly unrelated regression (SUREG). A SUREG is a regression model which permits the simultaneous estimation of costs and effects, calculated at an individual level, that could affect both costs and effects and lead to potential correlation between these two dependent variables. There is evidence that a SUREG improves precision surrounding cost-effectiveness estimation in trial-based economic evalua- tions.27 Trial data were used to derive a total NHS cost per partic- ipant. A SUREG was used to identify any difference between the surgical and medical arm of the trial while controlling for any modifying factors such the participant’s age and their baseline utility score. This method was also used to determine incre- mental costs and effects for all sensitivity analyses. es. Protected by copyright. Patient costs Time spent in unemployment, retirement or in full time education was valued as £6.04 an hour. Finally, private out of pockets expenses (eg, private appointments, spectacles) as captured on the PQ were included in patient time and travel costs. A table describing each of the unit costs is included in online supplemental tables A1–A5. Health care costs P i i Participant resource use, in terms of the type and frequency of resources used, was measured using bespoke case report forms (CRFs) which captured the use of resources such as intervention costs, surgery procedures, medications, post-surgery interven- tions and management of any resulting complications. The CRF was completed at baseline, 4 months, 12 months and 24 months, An additional specific surgery CRF was completed to capture actual surgery costs. In addition, the CRF asked about secondary healthcare resources utilised including; visits to the ophthal- mology outpatient clinic and outpatient procedures which the participants utilised during the trial. Costs in this study were given in GBP (2018). Unit costs for procedures and outpatient visits were derived from National Reference Costs 2017/201815 and the British National Formulary.16 In addition to the CRF’s an additional patient questionnaire (PQ) was administered to participants at 4, 12 and 24 months follow-up. The PQ asked participants about primary care services they have accessed, including general practitioner visits, community optometrist visits and community nurse appointments. Unit cost for these were derived from the Unit Costs of Health and Social Care 2018.17–19 Estimation of effects Th i f The impact of treatments on health-related quality of life (HRQoL) were captured using three tools: the EQ-5D-5L, the Health Utilities Index Mark 3 (HUI3) and the Glaucoma Utility Index (GUI).22–24 In this base case analysis, the primary outcome was based on the results of the EQ-5D-5L, as this is a measure recommended by National Institute for Health and Care Excellence (NICE) for technology appraisal.25 The HUI3 and GUI were investigated as part of additional sensitivity anal- ysis. Responses to the EQ-5D-5L were converted into utilities using the cross-walked values from the EQ-5D-3L dataset22 and were used to estimate quality-adjusted life years (QALYs) using the under the curve approach.26 The quality of life instruments was administered at seven time points during the course of the trial; baseline, 1, 3, 6, 12, 18 and 24 months post-randomisation. In a sensitivity analysis, QALYs were also estimated using the responses to the HUI3 and GUI instruments. Both costs and outcomes were discounted at a rate of 3.5%. The TAGS trial was carried out in 27 National Health Service (NHS) secondary care glaucoma departments. Each partici- pating department had at least one fellowship-trained glaucoma specialist. The outcomes were collected over a 24-month period of follow-up.12 Study participants had severe glaucomatous field loss (HPA classification) in one or both eyes, were over 18 years old and able to consent to participate. Participants were excluded if they were unable to undergo a trabeculectomy, had a high risk of surgical failure, had secondary glaucoma, were pregnant or trying to conceive. Where patients had both eyes eligible the eye with the least amount of visual field loss was deemed the index eye for analysis of clinical outcomes. The within-trial CUA was carried out from both a health care system alone and including a patient perspective with a time horizon of 2 years. on August 31, 2022 at UCL Library Services. Protected by copyright. ://bjo.bmj.com/ INTRODUCTION Open angle glaucoma (OAG) is a progressive pressure-related optic neuropathy and a major cause of blindness in the UK and worldwide.1 2 Intraocular pressure (IOP)related damage to the Kernohan A, et al. Br J Ophthalmol 2022;0:1–6. doi:10.1136/bjophthalmol-2021-320887 1 Clinical science which are both effective for the patient and provide optimal use of healthcare resources. Sensitivity analysis T dd h b To address the robustness of the economic conclusions of the study both stochastic and deterministic sensitivity analyses were Kernohan A, et al. Br J Ophthalmol 2022;0:1–6. doi:10.1136/bjophthalmol-2021-320887 Kernohan A, et al. Br J Ophthalmol 2022;0:1–6. doi:10.1136/bjophthalmol-2021-320887 2 Clinical science undertaken. First, to assess the robustness of the study sampling, non-parametric bootstrapping was carried out. Bootstrapping is a statistical procedure that resamples a single dataset to create many simulated samples to assess statistical precision.28 In this study, 1000 iterations of the bootstrapping procedure of were performed. This simulation process created a sample of boot- strapped means for costs and QALYs with distributions for each. The means and other parametric statistics were then calculated for the bootstrap distribution. Bootstrap estimates of the differ- ence in costs and QALYs to pay between the experimental and control arms were used to populate the cost-effectiveness plane and cost-effectiveness acceptability curve (CEAC).29 A cost- effectiveness plane and CEAC are ways of demonstrating the probability of an intervention’s cost-effectiveness across a range of different values. The former in the form of a scatter plot and the latter with these points expressed on an X and Y axis.30 The results of the bootstrapping were used to estimate the probability of each management strategy being considered cost-effective at different societal willingness to pay for a QALY. For example, the NICE cost-effectiveness threshold of £30 000 per QALY.25 In terms of deterministic sensitivity analysis, QALYs were recal- culated using both the utility values generated from the HUI3 and GUI quality of life tools to see if this changed the economic conclusions. The inclusion of patient time and travel costs were included to assess the impact potential conclusions. Handling missing data Wi h With respect to costs, cost CRF data were reported as missing under two circumstances, first where either the section reporting medications taken or the procedures undertaken were completely unreported (no values were given, positive or negative in either section). Second, costs were also reported as missing if the total costs for healthcare resources reported on the CRF was 0 and the response to the question ‘Has participant completed the TAGS Participant Questionnaire?’ was no. With respect to the estimation of QALYs for this base case analysis, those who had four of the seven data points were included for analysis. First, to account for the missing data points, the assumption that the previous utility remained stable was assumed. This meant the weighted average of the two utility scores around the missing values was used to calculate the missing data. After this, multiple imputation was used to estimate missing utility values for QALY scores.31 on August 31, 2022 at UCL Library Services. Protected by copyright. //bjo.bmj.com/ Sensitivity analysis T dd h b Table 2 Utility values, QALYs for each utility measure by study arm along with differences in QALYs at 24 months Treatment Trabeculectomy Medical management Mean (SD) (n) Mean (SD) (n) Effectiveness EQ-5D-5L baseline (n=444) 0.84 (0.18) (222) 0.84 (0.18) (222) EQ-5D-5L 1 month (n=397) 0.84 (0.18) (194) 0.81 (0.20) (203) EQ-5D-5L 3 months (n=365) 0.84 (0.17) (186) 0.81 (0.20) (179) EQ-5D-5L 6 months (n=381) 0.85 (0.18) (186) 0.82 (0.20) (195) EQ-5D-5L 12 months (n=420) 0.84 (0.18) (211) 0.82 (0.16) (209) EQ-5D-5L 18 months (n=365) 0.83 (0.19) (181) 0.79 (0.22) (184) EQ-5D-5L 24 months (n=409) 0.81 (0.18) (203) 0.80 (0.19) (206) Complete QALYs over 24 months using EQ-5D-5L (n=290) 1.65 (0.24) (144) 1.59 (0.28) (146) HUI3 baseline (n=428) 0.81 (0.20) (214) 0.80 (0.20) (214) HUI3 1 month (n=377) 0.79 (0.23) (184) 0.79 (0.23) (193) HUI3 3 months (n=359) 0.80 (0.22) (180) 0.78 (0.22) (179) HUI3 6 months (n=362) 0.81 (0.22) (180) 0.78 (0.22) (182) HUI3 12 months (n=400) 0.83 (0.19) (204) 0.80 (0.20) (196) HUI3 18 months (n=343) 0.80 (0.21) (169) 0.75 (0.26) (174) HUI3 24 months (n=391) 0.79 (0.23) (198) 0.75 (0.25) (193) Complete QALYs over 24 months using HUI3 (240) 1.61 (0.30) (123) 1.54 (0.36) (117) GUI baseline (n=441) 0.88 (0.13) (219) 0.86 (0.13) (222) GUI 1 month (n=399) 0.86 (0.14) (194) 0.85 (0.16) (205) GUI 3 months (n=377) 0.85 (0.13) (187) 0.84 (0.16) (190) GUI 6 months (n=377) 0.84 (0.16) (186) 0.85 (0.14) (191) GUI 12 months (n=413) 0.86 (0.14) (209) 0.86 (0.14) (204) GUI 18 months (n=365) 0.85 (0.14) (181) 0.83 (0.16) (184) GUI 24 months (n=407) 0.85 (0.15) (205) 0.83 (0.18) (202) Complete QALYs over 24 months using GUI (n=293) 1.67 (0.20) (144) 1.64 (0.24) (149) GUI, Glaucoma Utility Index; HUI3, Health Utilities Index Mark 3; QALY, Quality- Adjusted Life-Year. Table 2 Utility values, QALYs for each utility measure by study arm along with differences in QALYs at 24 months on August 31, 2022 at UCL Library Servic http://bjo.bmj.com/ n 26 July 2022. Downloaded from Completeness of data The results for the QALY scores produced by the three quality of life instruments are summarised in table 2. For the EQ-5D-5L, there was evidence of slightly higher QALYs at 24 months for the trabeculectomy compared with the medical arm in the unad- justed mean difference. Data completion in this trial was generally very high as can be seen in online supplemental table A6. No patterns were observed in the missing QALY data, so the data were imputed randomly. Kernohan A, et al. Br J Ophthalmol 2022;0:1–6. doi:10.1136/bjophthalmol-2021-32088 Table 1 Total unadjusted costs in each arm in first and second 12 months of trial follow-up Total cost Trabeculectomy Medical management Mean SD Mean SD Total cost to the NHS over 24 months (£) 3826 1648 1685 1401 Total cost to the NHS between baseline and 12 months (£) 3157 1299 1067 1299 Total cost to the NHS between 12 and 24 months (£) 669 977 618 632 NHS, National Health Service. Table 1 Total unadjusted costs in each arm in first and second 12 months of trial follow-up Cost estimates The unadjusted costs are summarised in table 1. The mean cost per patient in the trabeculectomy arm was £3826 (95% CI 3600 to 4050) and in the medical arm was £1685 (95% CI 1490 to 1880), there was a significant difference in costs between the two arms £2141 (p=<0.01). Comparison with other glaucoma studies Comparison with other glaucoma studies QALY threshold is 0%, 12% and 56% respectively. The CEAC in figure 2 shows that at 2 years follow-up, surgery is unlikely to be considered cost-effective over the range of values that society might be willing to pay for a QALY. There have been no previous studies comparing cost-effectiveness between medications and surgery as a primary treatment for advanced glaucoma. There have been prior studies which have estimated the cost-effectiveness of glaucoma treatments in different populations. For example, in the LIGHT study selective laser trabeculectomy (SLT) was compared with medical manage- ment of ocular hypertension and early glaucoma. The study found SLT produced similar clinical results at a lower cost than medication.32Stein et al33 also compared medications with laser trabeculoplasty (LTP) for the treatment of patients with newly diagnosed mild OAG using a Markov model with a 25-year time horizon. The results of which suggested medication was superior to LTP.33 These two studies differ from the TAGS as they focused on treatments for patients in the earlier stages of glaucoma. Only one study was identified that considered more severe glaucoma, Guedes et al34 used a Markov model to identify the most cost- effective treatment strategy for each severity of glaucoma.34 The results of this study found that the surgery was cost-effective in participants who are less than 70 years old. Meaning this model found that those who will have a longer life expectancy appear to accrue benefit from the additional costs of trabeculectomy surgery over a longer period. The results of the cost-effectiveness analysis based on QALYs derived from responses to the HUI3 and GUI do not change the conclusion of the analysis (see online supplemental table A7, figures A1, A2 and table A8, figures A3, A4). In both instances, medication was more likely to be cost-effective at the 24-month follow-up, with the surgical arm displaying a small amount of extra effect for each instrument The inclusion of participant costs (which is shown in online supplemental table A9 and figures A5, A6) also did not change the overall conclusions of the study. DISCUSSION Over the 24-month follow-up of the trial, surgical treatment was more costly and more effective than medical management. The results of the stochastic analysis suggest that any increase in QALYs over 24 months follow-up is unlikely to be sufficient to compensate for the increased costs of the surgery. The principal driver of this result was the higher surgical and outpatient costs in the surgical arm. Economic evaluation The results of the CUA are presented in table 3, figures 1 and 2. The results of the SUREG display that the trabeculectomy arm is on average more costly and more effective than medical manage- ment. Using the larger multiple imputed data set, the incremental cost per QALY at 2 years is £45 456. The effectiveness plane (figure 1) demonstrates the difference in costs and QALYs for surgery compared with medical management are almost entirely in the quadrant which represents greater effect at greater cost for surgery compared with medical management. The probability of surgery being cost-effective at a £20 000, £30 000 and £50 000 es. Protected by copyright. 3 Clinical science Clinical science Table 3 Complete and multiple imputation EQ-5D-5L results EQ-5D-5L data Intervention Unadjusted cost (£) (95% CI) Adjusted* incremental cost (£) (95% CI) Unadjusted† QALY (95% CI) Adjusted incremental QALY (95% CI) ICER (ΔCost/ ΔQALY) (£) Probability of being cost-effective at different thresholds for a QALY £0 £20 000 £30 000 £50 000 Complete case data (n=290) Trabeculectomy (n=144) 3686 (3435 to 3937) 2089 (1762 to 2416) 1.65 (1.61 to 1.69) 0.03 (0.01 to 0.08) 64 303 0 0% 6% 35% Medical management (n=146) 1605 (1390 to 1820) 1.59 (1.55 to 1.64) 100% 100% 94% 65% Imputation data (n=403) Trabeculectomy (n=199) 3622 (3372 to 3872) 2013 (1699 to 2327) 1.61 (1.57 to 1.65) 0.04 (−0.01 to 0.08) 45 456 0 0% 12% 56% Medical management (n=204) 1605 (1409 to 1801) 1.56 (1.52 to 1.60) 100% 100% 88% 44% Adjusted results are based on the results of the SUREG. †Unadjusted results are based on the trial data. ICER, Incremental Cost-Effectiveness Ratio ; QALY, Quality-Adjusted Life-Year. Table 3 Complete and multiple imputation EQ-5D-5L results on August 31, 2022 at UCL Library Services. Protected by copyright. http://bjo.bmj.com/ l: first published as 10.1136/bjo-2021-320887 on 26 July 2022. Downloaded from Adjusted results are based on the results of the SUREG. †Unadjusted results are based on the trial data. ICER, Incremental Cost-Effectiveness Ratio ; QALY, Quality-Adjusted Life-Year. Provenance and peer review Not commissioned; externally peer reviewed. Another consideration for the interpretation of the economic evaluation is the inclusion of the costs of managing disease in the non-index eye. When estimating total costs, both the costs for the index eye and non-index eye were included. This was done because the outcome measure of principle interest (QALYs) were not specific to the index eye but to the vision across both eyes. Also, the prognosis of one eye may affect decisions about the management of the other eye. The challenge that this represented for the analysis was that cost and benefits could be undervalued or overvalued if there was any imbalance in severity of disease in the non-index eye between the two arms. However, this was not the case in TAGS and that means that the impact of management of disease in the non-index eye was equally spread between the two arms and should not materially affect the marginal differ- ences between the two randomised groups. Data availability statement Data are available upon reasonable request. Data will be available beginning 10 months and ending four years after publication of this paper. Data will be available for researchers who provide a methodologically sound scientific proposal, which has been approved by an ethics committee. Proof of the latter should be provided. Analyses should achieve the aims reported in the approved proposal. Requests for data sharing should be made to the corresponding author at ashleigh.kernohan@newcastle.ac.uk. on August 31, 2022 at UCL Library Services. Protected by copyright. ://bjo.bmj.com/ Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. REFERENCES 1 Quartilho A, Simkiss P, Zekite A, et al. Leading causes of certifiable visual loss in England and Wales during the year ending 31 March 2013. Eye 2016;30:602–7. 1 Quartilho A, Simkiss P, Zekite A, et al. Leading causes of certifiable visual loss in England and Wales during the year ending 31 March 2013. Eye 2016;30:602–7. 2 Flaxman SR, Bourne RRA, Resnikoff S, et al. Global causes of blindness and distance vision impairment 1990-2020: a systematic review and meta-analysis. Lancet Glob Health 2017;5:e1221–34. Strengths and limitations h l 4Centre for Healthcare Randomised Trials (CHaRT), Health Services Research Unit, University of Aberdeen, Aberdeen, UK 4Centre for Healthcare Randomised Trials (CHaRT), Health Services Research Unit, University of Aberdeen, Aberdeen, UK y 5Centre for Public Health, Queen’s University Belfast, Belfast, UK 6 y p , y p , , 8National Institute for Health Research (NIHR) Biomedical Research Centre, Moorfields Eye Hospital NHS Foundation Trust, London, UK 9Edinburgh Clinical Trials Unit, Usher Institute, University of Edinburgh, Edinburgh, UK 10 The primary strength of this study was that it contains a large sample of homogeneous patients all of whom had advanced glau- coma. Another strength is the use of multiple measures HRQoL: the EQ-5D-5L, the HUI3 and a glaucoma specific measure, the GUI, allowing benefits to be captured by three different metrics. As the conclusion did not change dependent on metric then confidence in them is increased. The study also compared three different methods of assessing HRQoL in relation to eye disease. Interestingly, the EQ-5D-5L proved to be the most likely to detect a difference in HRQoL in glaucoma patients, though the difference between the all the metrics was small. This is despite the fact that the HUI3 had a specific question relating to partic- ipants’ vision and that the GUI was developed specifically for use in glaucoma patients and had a value set purposely devel- oped for this trial population. The LIGHT trial also used the EQ-5D to measure quality of life. This trial reported a similar small difference in EQ-5D scores. This difference was smaller than that identified in TAGS.32 However, this aligns with the findings of Bozzani et al,35 which found the sensitivity of the EQ-5D to detect differences varied according to the stages of disease. Bozzani et al35 went on to conclude that there is a need for future research to assess the measurement in terms of sensi- tivity and generalisabilty for measurement of eliciting HRQoL in patients with eye disease.35 10NIHR Applied Research Collaboration North East and North Cumbria, NIHR, Newcastle, UK ORCID iDs Ashleigh Kernohan http://orcid.org/0000-0002-5514-3186 Anthony J King http://orcid.org/0000-0002-3091-911X Augusto Azuara-Blanco http://orcid.org/0000-0002-4805-9322 Jennifer Burr http://orcid.org/0000-0002-9478-738X John M Sparrow http://orcid.org/0000-0003-3704-0105 Provenance and peer review Not commissioned; externally peer reviewed. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. CONCLUSION This is the first study to evaluate accurately and prospectively the cost of treating newly diagnosed advanced glaucoma with the two currently standard treatment approaches. For treating those with advanced OAG both medical and surgical are viable treatment options in terms of HRQoL outcomes. At a 2-year time horizon, medication is the more cost-effective approach at managing glaucoma. Further research will follow the longitu- dinal benefits of surgical and medical intervention beyond the 2-year time horizon, as there is evidence from previous economic modelling studies that surgery could be considered cost-effective over a longer time horizon. Twitter Ashleigh Kernohan @ashkerno Contributors The economic evaluation data analysis was carried out by AK with support from TH, LV and HS. All the authors were involved of the interpretation of the economic evaluation. The TAGS clinical trial from which the data was derived was designed by AJK, JB, AA-B, JMS, KB, TG-H, JN, LV and GM and managed by AJK and GF with input and oversight from all the authors. AK wrote the first draft of the manuscript, which was reviewed, modified and approved by all of the authors. All the authors vouch for the accuracy and completeness of the data reported. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. AK is the guarantor. on August 31, 2022 at UCL Library http://bjo.bmj.com/ 0887 on 26 July 2022. Downloaded from Funding This work was supported by the National Institute for Health Research Health Technology Assessment Programme, grant number [12/35/38]. Competing interests All authors have completed the ICMJE uniform disclosure form at http://www.icmje.org/coi_disclosure.pdf and declare: support from the NIHR-HTA programme for the submitted work; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years, no other relationships or activities that could appear to have influenced the submitted work. Patient consent for publication Not applicable. Ethics approval This study involves human participants and was approved by East Midlands – Derby Research Ethics Committee (reference number 13/EM/0395). Participants gave informed consent to participate in the study before taking part. Provenance and peer review Not commissioned; externally peer reviewed. Kernohan A, et al. Br J Ophthalmol 2022;0:1–6. doi:10.1136/bjophthalmol-2021-320887 Strengths and limitations h l For the economic evaluation, one of the key limitations of the within-trial analysis was the limited follow-up in the trial 4 Kernohan A, et al. Br J Ophthalmol 2022;0:1–6. doi:10.1136/bjophthalmol-2021-320887 y the within-trial analysis was the limited follow-up in the trial Figure 1 Cost-effectiveness plane for trabeculectomy versus medical management—adjusted bootstrapped replications for CUA for EQ-5D- 5L results. CUA, cost-utility analysis. QALY, quality-adjusted life year. Figure 2 Cost-effectiveness curves for the trabeculectomy and medical management using the results from the EQ-5D-5L using the multiple imputation data. QALY, quality-adjusted life year. 2022 at UCL Library Services. Protected by copyright. ernohan A, et al. Br J Ophthalmol 2022;0:1–6. doi:10.1136/bjophthalmol-2021-320887 Figure 2 Cost-effectiveness curves for the trabeculectomy and medical management using the results from the EQ-5D-5L using the multiple imputation data. QALY, quality-adjusted life year. rnohan A, et al. Br J Ophthalmol 2022;0:1–6. doi:10.1136/bjophthalmol-2021-320887 Figure 2 Cost-effectiveness curves for the trabeculectomy and medical management using the results from the EQ-5D-5L using the multiple imputation data. QALY, quality-adjusted life year. Figure 2 Cost-effectiveness curves for the trabeculectomy and medical management using the results from the EQ-5D-5L using the multiple imputation data. QALY, quality-adjusted life year. Figure 1 Cost-effectiveness plane for trabeculectomy versus medical management—adjusted bootstrapped replications for CUA for EQ-5D- 5L results. CUA, cost-utility analysis. QALY, quality-adjusted life year. Figure 1 Cost-effectiveness plane for trabeculectomy versus medical management—adjusted bootstrapped replications for CUA for EQ-5D- 5L results. CUA, cost-utility analysis. QALY, quality-adjusted life year. Kernohan A, et al. Br J Ophthalmol 2022;0:1–6. doi:10.1136/bjophthalmol-2021-320887 Kernohan A, et al. Br J Ophthalmol 2022;0:1–6. doi:10.1136/bjophthalmol-2021-320887 4 on August 31, 2022 at UCL Library Services. Protected by http://bjo.bmj.com/ Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 2022. Downloaded from Clinical science Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 2022 (24 months). As OAG is a chronic, lifelong condition the full benefit (and costs) of each randomised intervention is unlikely to be captured within this time frame, as demonstrated by Guedes et al.34 Future research is underway to focus on the longitu- dinal costs and outcomes of the interventions over a longer time horizon. Clinical science 21 Office for National StatisticsSmith R, ed. Employee earnings in the UK: 2018, in statistical bulletin, 2018: 1–16. 4 Peters D, Bengtsson B, Heijl A. Factors associated with lifetime risk of open-angle glaucoma blindness. Acta Ophthalmol 2014;92:421–5. 22 van Hout B, Janssen MF, Feng Y-S, et al. Interim scoring for the EQ-5D-5L: mapping the EQ-5D-5L to EQ-5D-3L value sets. Value Health 2012;15:708–15. g 5 Mokhles P, Schouten JSAG, Beckers HJM, et al. A systematic review of end-of-life visual impairment in open-angle glaucoma: an epidemiological autopsy. J Glaucoma 2016;25:623–8. 23 Horsman J, Furlong W, Feeny D, et al. 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Oxford: Oxford University Press, 2015. 6/bjo p 11 Stead R, Azuara-Blanco A, King AJ. on August 31, 2022 at UCL L http://bjo.bmj.com/ Br J Ophthalmol: first published as 10.1136/bjo-2021-320887 on 26 July 2022. Downloaded from Clinical science Clinical science Attitudes of consultant ophthalmologists in the UK to initial management of glaucoma patients presenting with severe visual field loss: a national survey. Clin Exp Ophthalmol 2011;39:858–64. 30 Fenwick E, Marshall DA, Levy AR, et al. Using and interpreting cost-effectiveness acceptability curves: an example using data from a trial of management strategies for atrial fibrillation. BMC Health Serv Res 2006;6:52. 30 Fenwick E, Marshall DA, Levy AR, et al. Using and interpreting cost-effectiveness acceptability curves: an example using data from a trial of management strategies for atrial fibrillation. BMC Health Serv Res 2006;6:52. y p p 12 King AJ, Hudson J, Fernie G, et al. Primary trabeculectomy for advanced glaucoma: pragmatic multicentre randomised controlled trial (tags). BMJ 2021;373:n1014. 31 Faria R, Gomes M, Epstein D, et al. A guide to handling missing data in cost-effectiveness analysis conducted within randomised controlled trials. Pharmacoeconomics 2014;32:1157–70. 31 Faria R, Gomes M, Epstein D, et al. A guide to handling missing data in cost-effectiveness analysis conducted within randomised controlled trials. Pharmacoeconomics 2014;32:1157–70. 13 Hodapp E, Parrish RK, Anderson DR. Clinical decisions in glaucoma. 1993: Mosby Incorporated. on August 31, 2022 at UCL Library http://bjo.bmj.com/ 0887 on 26 July 2022. Downloaded from 14 Fiscella RGet al. Cost of illness of glaucoma. Pharmacoeconomics 2009;27:189–98. 32 Gazzard G, Konstantakopoulou E, Garway-Heath D, et al. Selective laser trabeculoplasty versus eye drops for first-line treatment of ocular hypertension and glaucoma (light): a multicentre randomised controlled trial. Lancet 2019;393:1505–16. 14 Fiscella RGet al. Cost of illness of glaucoma. Pharmacoeconomics 2009;27:189–98. 15 NHS trust and NHS foundation trusts. Reference cost collection: national schedule of reference costs 2018. National Health Service. 16 Joint Formulary Committee, British National Formulary (online Association & The Royal Pharmaceutical Society: UK, 2018. 16 Joint Formulary Committee, British National Formulary (online Association & The Royal Pharmaceutical Society: UK, 2018. 16 Joint Formulary Committee, British National Formulary (onlin 33 Stein JD, Kim DD, Peck WW, et al. Cost-Effectiveness of medications compared with laser trabeculoplasty in patients with newly diagnosed open-angle glaucoma. Arch Ophthalmol 2012;130:497–505. Association & The Royal Pharmaceutical Society: UK, 2018 y y 17 Curtis L, Burns A. Community based health care stuff, in unit costs of health and social care. P.S.S.R.U, University of Kent, 2018: 153–67. 17 Curtis L, Burns A. Community based health care stuff, in unit costs of health and social care. P.S.S.R.U, University of Kent, 2018: 153–67. Author affiliations 1 l l h i 1Population Health Sciences Institute, Newcastle University, Newcastle upon Tyne, UK 2Department of Ophthalmology, Nottingham University Hospital, Nottingham, UK 3Health Services Research Unit, University of Aberdeen, Aberdeen, UK 3 Sotimehin AE, Ramulu PY. Measuring disability in glaucoma. J Glaucoma 2018;27:939-949. Kernohan A, et al. Br J Ophthalmol 2022;0:1–6. doi:10.1136/bjophthalmol-2021-320887 5 Clinical science p 34 Guedes RAP, Guedes VMP, Gomes CEdeM, et al. Maximizing cost-effectiveness by adjusting treatment strategy according to glaucoma severity. Medicine 2016;95:e5745. y 18 Curtis LA, Burns A. Unit Costs of Health and Social Care 2015. PSSRU, University of Kent: Canterbury, 2015. 19 Curtis LA, Burns A. Unit Costs of Health and Social Care 2017. PSSRU, University of Kent: Canterbury, 2017. 35 Bozzani FM, Alavi Y, Jofre-Bonet M, et al. A comparison of the sensitivity of EQ-5D, SF-6D and TTO utility values to changes in vision and perceived visual function in patients with primary open-angle glaucoma. BMC Ophthalmol 2012;12:43. 35 Bozzani FM, Alavi Y, Jofre-Bonet M, et al. A comparison of the sensitivity of EQ-5D, SF-6D and TTO utility values to changes in vision and perceived visual function in patients with primary open-angle glaucoma. BMC Ophthalmol 2012;12:43. 20 Department of Transport,. TAG Data Book Transport Appraisal and Strategic Modelling (TASM) Division, Editor. London: Department of Transport, 2018. Clinical science 6 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance terial placed on this supplemental material which has been supplied by the author(s) Br J Ophthalmol BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Br J Ophtha BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Br J Ophthalmol Additional Tables for Within Trial Economic Evaluation Paper Table A1: Unit costs for hospital appointments Hospital Appointments Item £ Unit Reference Notes Inpatient 448 Per night National Reference Costs 2017/2018 Ward costs per night Outpatient appointment 105.09 Per appointment National Reference Costs 2017/2018 Consultant led Ophthalmology outpatient costs Additional Tables for Within Trial Economic Evaluation Paper Table A2: Unit costs for glaucoma procedures Ophthalmic Procedure Source HRG Code Unit Cost Comments Trabeculectomy (Day Case) National Reference Costs 2017/2018 BZ92B 1639.44 Very Major, Glaucoma or Iris Procedures, with CC Score 0-1 Trabeculectomy (Inpatient) National Reference Costs 2017/2018 BZ92B 2184.07 Very Major, Glaucoma or Iris Procedures, with CC Score 0-1 Massage National Reference Costs 2017/2018 BZ24G 143.29 Minor, Glaucoma or Iris Procedures Adjustment / suturelysis / releasable release National Reference Costs 2017/2018 BZ95 143.29 Minor, Glaucoma or Iris Procedures 5-FU injection National Reference Costs 2017/2018 BZ95Z 149.69 Minor, Glaucoma or Iris Procedures + Unit Costs of Fluorouracil Steroid Injections National Reference Costs 2017/2018 BZ95Z 150.16 Minor, Glaucoma or Iris Procedure + Unit Prednisolone Needling + 5-FU injection National Reference Costs 2017/2018 BZ94B 149.69 Minor, Glaucoma or Iris Procedures + Unit Costs of Fluorouracil Revision of bleb NEC National Reference Costs 2017/2018 BZ91B 1347.56 Complex, Glaucoma or Iris Procedures, with CC Score 0-1 Reformation of anterior chamber of eye National Reference Costs 2017/2018 BZ94B 127.90 Intermediate, Glaucoma or Iris Procedures, with CC Score 0 Phaco and IOL National Reference Costs 2017/2018 BZ32B 878.61 Intermediate, Cataract or Lens Procedures, with CC Score 0-1 Table A2: Unit costs for glaucoma procedures doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Clinical science Kernohan A BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Br J Ophthalmol Supplemental material Medication Dose Administration Unit cost (£) Source Comments Saflutan 15 micrograms/m l Single dose unit eye drop 12.20 BNF 2018 (Online) Overall costs per box Latanoprost 50 micrograms/m l Eye drop 1.53 BNF 2018 (Online) Overall costs per 2.5ml bottle Bimatoprost 300 micrograms/m l Eye drop 10.30 BNF 2018 (Online) Overall costs per 3ml bottle, Travoprost 40 micrograms/m l Eye drop 7.27 BNF 2018 (Online) Overall costs per 2.5ml Average Cost PA 7.83 Carbonic Anhydrase Inhibitors (CAI) Brinzolamide 10mg/ml Eye drop 1.89 BNF 2018 (Online) Overall costs per 5ml bottle Dorzolamide 20mg/ml Eye drop 1.55 BNF 2018 (Online) Overall costs per 5ml bottle Average Cost CAI 1.72 Alpha-2 Adrenergic Agonists (AA) Brimonidine 0.2% Eye drop 1.13 BNF 2018 (Online) Overall costs per 5ml bottle Iopidine 5mg/ml Eye drop 10.88 BNF 2018 (Online) Overall costs per 5ml bottle Average Cost of AA 6.01 Beta Blockers (BB) Timolol 0.25% Eye drop 0.78 BNF 2018 (Online) Overall costs per 5ml bottle Betoptic 0.5% Eye drop 1.90 BNF 2018 (Online) Overall costs per 5ml bottle Average Cost of BB 1.34 Parasympathetic Drops (Para) Pilocarpine hydrochloride 1% Eye drop 20.78 BNF 2018 (Online) Overall costs per 10ml bottle Average Cost of Para 20.78 Oral Glaucoma Medications Carbonic Anhydrase Inhibitors (CAI) doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A Clinical science Kernohan A BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Br J Ophthalmol Medication Dose Administration Unit cost (£) Source Comments Acetazolamide 250mg Tablet 16.66 BNF 2018 (Online) Overall cost per box (30) Combination Glaucoma Medications (comb) Bimatoprost with timolol (AZARGA) 10mg/ml Eye drops 11.05 BNF 2018 (Online) Overall cost per 5ml bottle Bimatoprost with timolol (Ganfort) 5mg/m Eye drops 14.16 BNF 2018 (Online) Overall cost per 3ml bottle Brinzolamide with brimonidine (Simbrinza) 2mg/ml Eye drops 9.23 BNF 2018 (Online) Overall cost per 5ml bottle Dorzolamide with timolol (Cosopt) 5mg/ml Eye drops 1.50 BNF 2018 (Online) Overall cost per 5ml bottle Dorzolamide with timolol unit dose (Cosopt) 5mg/ml Single dose unit eye drop 28.59 BNF 2018 (Online) Unit Dose,60 doses Dorzolamide with timolol (Eylamdo) 5mg/ml Eye drop 14.29 BNF 2018 (Online) Overall cost per 5ml bottle Travoprost with timolol (DuoTrav) 5mg/ml Eye drop 13.95 BNF 2018 (Online) Overall cost per 5ml bottle Average Cost of Comb 13.25 Non-Glaucoma Medications Steroid Eye Drops Dexamethasone with hypromellose, neomycin and polymyxin B (Maxitrol) Dexamethason e 1 mg per 1 gram Neomycin (as Neomycin sulfate) 3500 unit per 1 gram Polymyxin B sulfate 6000 unit per 1 gram Eye drop 1.68 BNF 2018 (Online) Overall cost per 5ml bottle Betamethasone sodium phosphate 1 mg per 1 ml Eye drop 2.32 BNF 2018 (Online) Overall cost per 5ml bottle Dexamethasone (Maxidex) 1 mg per 1 ml Eye drop 1.42 BNF 2018 (Online) Overall cost per 5ml bottle Prednisolone (Pred Forte) 10 mg per 1 ml Eye drop 1.82 BNF 2018 (Online) Overall cost per 5ml bottle Medication Dose Administration Unit cost (£) Source Comments Acetazolamide 250mg Tablet 16.66 BNF 2018 (Online) Overall cost per box (30) Combination Glaucoma Medications (comb) Bimatoprost with timolol (AZARGA) 10mg/ml Eye drops 11.05 BNF 2018 (Online) Overall cost per 5ml bottle Bimatoprost with timolol (Ganfort) 5mg/m Eye drops 14.16 BNF 2018 (Online) Overall cost per 3ml bottle Brinzolamide with brimonidine (Simbrinza) 2mg/ml Eye drops 9.23 BNF 2018 (Online) Overall cost per 5ml bottle Dorzolamide with timolol (Cosopt) 5mg/ml Eye drops 1.50 BNF 2018 (Online) Overall cost per 5ml bottle Dorzolamide with timolol unit dose (Cosopt) 5mg/ml Single dose unit eye drop 28.59 BNF 2018 (Online) Unit Dose,60 doses Dorzolamide with timolol (Eylamdo) 5mg/ml Eye drop 14.29 BNF 2018 (Online) Overall cost per 5ml bottle Travoprost with timolol (DuoTrav) 5mg/ml Eye drop 13.95 BNF 2018 (Online) Overall cost per 5ml bottle Average Cost of Comb 13.25 Non-Glaucoma Medications Non-Glaucoma Medications doi: 10.1136/bjo-2021-320887 –6. doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A Clinical science :1 0 2022; Br J Ophthalmol , et al. Kernohan A BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance ental material placed on this supplemental material which has been supplied by the author(s) Br J Ophthalmol BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Br J Ophthalmol Supplemental material Medication Dose Administration Unit cost (£) Source Comments Antibiotic Eye Drops Azithromycin (azyter) 15 mg per 1 gram Eye drop 1.17 BNF 2018 (Online) Unit Dose, 6 doses Choloramphenicol 5 mg per 1 ml Eye drop 1.14 BNF 2018 (Online) Overall cost per 10ml bottle Celluvisc Unit Dose 1% Single dose unit eye drop 4.80 BNF 2018 (Online) Unit dose, 30 doses Hylo-Forte unit dose 0.2% Single dose unit eye drop 5.60 BNF 2018 (Online) Unit dose, 30 doses Sodium hyaluronate (Vismed Multi) 0.18% Eye drop 6.87 BNF 2018 (Online) Overall cost per 10ml bottle Mydriatics Cyclopentolate (Mydrilate) 5 mg per 1 ml Eye drops 8.08 BNF 2018 (Online) Overall Cost per 5ml bottle Atropine 10 mg per 1 ml Eye drops 15.10 BNF 2018 (Online) Unit Dose, 20 doses NSAID Bromfenac (Yellox) 900 microgra m per 1 ml Eye drops 8.50 BNF 2018 (Online) Overall cost per 5ml bottle Sympathomimetic Phenylephrine 50 microgram per 1 ml Eye drops 11.87 BNF 2018 (Online) Unit Dose, 20 doses Average Cost of Non Glaucoma Drops 7.27 doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A Clinical science Kernohan A BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance upplemental material placed on this supplemental material which has been supplied by the author(s) Br J Ophthalmol BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Br J Ophthalmol Supplemental material Table A4: Unit costs for community appointments Table A4: Unit costs for community appointments Item Unit £ Comments GP GP visit at their practice Per 9.22 minute appointment 37 PSSRU 2018 GP home visit 11.4 minute Per appointment 45.98 PSSRU 2015 (most recent info) 11.4 minutes (2015 Health and Social Care) x 2017 hourly rate (£242) (no travel costs) Telephone triage with GP Cost per call 8.10 PSSRU 2017 (most recent info) 15.5 minutes x2015 hourly rate (£67) (not including travel) Nurse Practice Nurse consultation 15.5 minutes per consultation 10.84 PSSRU 2018 15.5 minutes (length of appointment Unit Costs 2015) x 2017 hourly rate (£42) District Nurse 25 minutes per consultation 17.29 PSSRU 2017 15.5 minutes x2015 hourly rate (£67) (not including travel) Optician Optometrist in practice Per examination 21.31 Department of Health and Social Care Eye exam fee (As participants have glaucoma all will be entitled to NHS eye examinations) Optometrist at home Per examination 58.87 Department of Health and Social Care Eye exam fee + domiciliary fee (As participants have glaucoma all will be entitled to NHS eye examinations) doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance rial placed on this supplemental material which has been supplied by the author(s) Br J Ophthalmol BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Br J Ophthalmol Table A5. Costs for different resources in each arm. Table A5. Costs for different resources in each arm. Table A5. Costs for different resources in each arm. Resource Total cost (£) in the Trabeculectomy Total cost (£) in the Medical management Mean Median Standard Deviation Mean Median Standard Deviation GP Surgery consultations 26 0 103 13 0 34 GP Home consultations 3 0 20 2 0 18 GP telephone consultations 22 0 63 32 0 97 Practice Nurse consultations 26 0 49 45 0 128 District Nurse consultations 10 0 81 8 0 33 Optometrist consultations 29 21 38 21 21 25 Ophthalmology consultations 1313 1129 615 593 407 524 Other Consultations 41 0 246 69 0 311 Nights in Hospital 28 0 166 10 0 78 Releasable release 101 0 160 17 0 59 Ocular Massage 97 0 192 5 0 30 Trabeculectomy 1821 1639 850 394 0 800 5-Fluorouracil injection 30 0 95 8 0 37 Steroid injection 18 0 59 6 0 44 Needling + 5-Fluorouracil injection 37 0 99 10 0 54 Bleb resuturing 20 0 160 7 0 94 Anterior Chamber reformation 3 0 20 1 0 9 Bleb revision 45 0 242 7 0 94 Phacoemulsification 63 0 309 81 0 374 Prostaglandin Analogues 181 178 141 286 320 120 Carbonic Anhydrase Inhibitor 7 0 15 18 0 25 doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Br J BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance upplemental material placed on this supplemental material which has been supplied by the author(s) Br J Ophthalmol BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Br J Ophthalmol Supplemental material Table A6. doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A Table A5. Costs for different resources in each arm. Data completion in each arm Beta Blockers 9 0 16 19 10 22 Alpha Agonists 5 0 19 15 0 50 Pilocarpine 0 0 5 1 0 21 Combinations 4 0 22 1 0 10 Diamox 1 0 10 1 0 18 Others 0 0 4 0 0 0 Data response rates Trial arm Trabeculectomy %(n) Medical management %(n) Case Report Form 4-months 99% (226) 99% (223) 12-months 99% (226) 100% (226) 24-months 96% (217) 97% (219) EQ-5D-5L Baseline 98% (222) 98% (222) 1-month 85% (194) 90% (203) 3-months 82% (186) 79% (179) 6-months 82% (186) 86% (195) 12-months 93% (211) 92% (209) 18-months 80% (181) 81% (184) 24-months 91% (206) 90% (203) Resource Use Questionnaire 4-months 93% (210) 96% (216) 12-months 94% (213) 92% (208) 24-months 92% (208) 90% (204) Time and Travel Questionnaire 68% (154) 65% (148) Beta Blockers 9 0 16 19 10 22 Alpha Agonists 5 0 19 15 0 50 Pilocarpine 0 0 5 1 0 21 Combinations 4 0 22 1 0 10 Diamox 1 0 10 1 0 18 Others 0 0 4 0 0 0 Table A6. Data completion in each arm Data response rates Trial arm Trabeculectomy %(n) Medical management %(n) Case Report Form 4-months 99% (226) 99% (223) 12-months 99% (226) 100% (226) 24-months 96% (217) 97% (219) EQ-5D-5L Baseline 98% (222) 98% (222) 1-month 85% (194) 90% (203) 3-months 82% (186) 79% (179) 6-months 82% (186) 86% (195) 12-months 93% (211) 92% (209) 18-months 80% (181) 81% (184) 24-months 91% (206) 90% (203) Resource Use Questionnaire 4-months 93% (210) 96% (216) 12-months 94% (213) 92% (208) 24-months 92% (208) 90% (204) Time and Travel Questionnaire 68% (154) 65% (148) Table A6. Data completion in each arm doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Table A5. Costs for different resources in each arm. Kernohan A BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Br J Ophthalmol Supplemental material Table A7: Complete and MI HUI 3 Cost Utility Analysis Results HUI 3 Data Intervention Unadjusted Cost (£) Adjusted Incremental Cost (£) Unadjusted QALY Adjusted Incremental QALY ICER (ΔCost/ ΔQALY) (£) Probability cost-effective at threshold £0 £20,000 £30,000 £50,000 Complete case data (n=240) Surgery 3819 2129 1.61 0.06 33,758 0% 9% 38% 76% Medication 1691 1.54 100% 91% 62% 24% Imputation data (n=382) Surgery 3688 2040 1.54 0.06 36,130 0% 4% 32% 72% Medication 1640 1.48 100% 96% 68% 28% Table A7: Complete and MI HUI 3 Cost Utility Analysis Results doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A Br J Ophthalmol Supplemental material Figure A1: Cost effectiveness curves for the surgical and medical arms using the results from the imputed HUI3 sample 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 10000 20000 30000 40000 50000 Probability cost-effective Willingness to pay for a QALY (£) Surgery Medication Surgery Medication Figure A1: Cost effectiveness curves for the surgical and medical arms using the results from the imputed HUI3 sample doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Br J Ophthalmol Supplemental material Figure A2: Cost effectiveness curves for the surgical and medical arms using the results from the imputed HUI3 sample 0 500 1000 1500 2000 2500 3000 -0.050 0.000 0.050 0.100 0.150 Incremental cost Incremental QALYs Surgery vs Medication Average Figure A2: Cost effectiveness curves for the surgical and medical arms using the results from the imputed HUI3 sample 0 500 1000 1500 2000 2500 3000 -0.050 0.000 0.050 0.100 0.150 Incremental cost Incremental QALYs Surgery vs Medication Average Average Figure A2: Cost effectiveness curves for the surgical and medical arms using the results from the imputed HUI3 sample doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A doi: 10.1136/bjo-2021-320887 –6. Table A5. Costs for different resources in each arm. :1 0 2022; Br J Ophthalmol , et al. Kernohan A BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Br J Ophthalmol Supplemental material Table A8: Complete and MI GUI Cost Utility Results Adjusted difference is in favour of medication in this instance not surgery. GUI Data Intervention Unadjusted Cost (£) Adjusted Incremental Cost (£) Unadjusted QALY Adjusted Incremental QALY ICER (ΔCost/ΔQALY) (£) Probability cost-effective at threshold £0 £20,000 £30,000 £50,000 Complete case data (n=293) Surgery 3683 2138 1.67 0.01 111,117 0% 9% 38% 76% Medication 1541 1.64 100% 91% 62% 24% Imputation data (n=398) Surgery 3617 1995 1.64 0.00 350,149 0% 0% 0% 0% Medication 1615 1.62 100% 100% 100% 100% Table A8: Complete and MI GUI Cost Utility Results Adjusted difference is in favour of medication in this instance not surgery. Adjusted difference is in favour of medication in this instance not surgery. *Adjusted difference is in favour of medication in this instance not surgery. doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A Br J Ophthalmol Supplemental material 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 10000 20000 30000 40000 50000 Probability cost-effective Willingness to pay for a QALY (£) Surgery Medication Figure A3: Cost effectiveness curves for the surgical and medical arms using the results from the imputed GUI sample Surgery Medication Figure A3: Cost effectiveness curves for the surgical and medical arms using the results from the imputed GUI sample doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Table A5. Costs for different resources in each arm. Kernohan A Br J Ophthalmol Supplemental material 0 500 1000 1500 2000 2500 3000 -0.080 -0.060 -0.040 -0.020 0.000 0.020 0.040 0.060 Incremental cost Incremental QALYs Surgery Vs Medication Average Figure A4: Cost effectiveness curves for the surgical and medical arms using the results from the imputed GUI sample 0 500 1000 1500 2000 2500 3000 -0.080 -0.060 -0.040 -0.020 0.000 0.020 0.040 0.060 Incremental cost Incremental QALYs Surgery Vs Medication Average Figure A4: Cost effectiveness curves for the surgical and medical arms using the results from the imputed GU Figure A4: Cost effectiveness curves for the surgical and medical arms using the results from the imputed GUI sample doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Br J Ophthalmol Supplemental material Table A9: EQ-5D-5L Data with patient time and travel costs included Cost Utility Results EQ-5D-5L Data with patient time and travel costs included Intervention Unadjusted Cost with TT (£) Adjusted Incremental Cost (£) Unadjusted QALY Adjusted Incremental QALY ICER (ΔCost/ ΔQALY) (£) Probability cost-effective at threshold £0 £20,000 £30,000 £50,000 Complete case data (n=290) Surgery 4453 2412 1.65 0.03 75,347 0% 1% 2% 25% Medication 2046 1.59 100% 99% 98% 75% Imputation data (n=403) Surgery 4419 2359 1.61 0.04 54,197 0% 1% 5% 42% Medication 2052 1.56 100% 99% 95% 58% Table A9: EQ-5D-5L Data with patient time and travel costs included Cost Utility Results doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Table A5. Costs for different resources in each arm. Kernohan A Br J Ophthalmol Supplemental material 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 10000 20000 30000 40000 50000 Probability cost-effective Willingness to pay for a QALY (£) Surgery Medication Figure A5: Cost effectiveness curves for the surgical and medical arms using the results from the imputed ED-5D-5L using the time and travel data Surgery Figure A5: Cost effectiveness curves for the surgical and medical arms using the results from the imputed ED for the surgical and medical arms using the results from the imputed ED-5D-5L using the time and travel data Figure A5: Cost effectiveness curves for the surgical and medical arms using the results from the imputed ED-5D-5L using the time and travel data doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A Br J Ophthalmol Supplemental material Figure A6: Cost-effectiveness plane for adjusted bootstrapped replications for Cost-utility analysis from the imputed ED-5D-5L results with time and travel costs 0 500 1000 1500 2000 2500 3000 3500 -0.020 0.000 0.020 0.040 0.060 0.080 0.100 0.120 Incremental cost Incremental QALYs Survery Vs… Average 0 500 1000 1500 2000 2500 3000 3500 -0.020 0.000 0.020 0.040 0.060 0.080 0.100 0.120 Incremental cost Incremental QALYs Survery Vs… Average Figure A6: Cost-effectiveness plane for adjusted bootstrapped replications for Cost-utility analysis from the im Figure A6: Cost-effectiveness plane for adjusted bootstrapped replications for Cost-utility analysis from the imputed ED-5D-5L results with time and travel costs doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A doi: 10.1136/bjo-2021-320887 –6. :1 0 2022; Br J Ophthalmol , et al. Kernohan A
https://openalex.org/W2099165403	https://www.scielo.br/j/bar/a/j5CfGtd4YyG7kmqQCDnznVs/?lang=en&format=pdf	English	null	Relationships between learning, context and competency: a multilevel study	BAR - Brazilian Administration Review	2,012	cc-by	12,634	* Corresponding author: Hugo Pena Brandão SCES Trecho 2, Lote 22, Ed. Presidente Tancredo Neves, 1º andar, Brasília, DF, 70200-002, Brazil. Available online at http://www.anpad.org.br/bar BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 Available online at http://www.anpad.org.br/bar Relationships between Learning, Context and Competency: a Multilevel Study(1) Hugo Pena Brandão * E-mail address: hugopb@brturbo.com.br Banco do Brasil, Fundação Getúlio Vargas – FGV Management Brasília, DF, Brazil. Hugo Pena Brandão * E-mail address: hugopb@brturbo.com.br Banco do Brasil, Fundação Getúlio Vargas – FGV Management Brasília, DF, Brazil. Jairo Eduardo Borges-Andrade E-mail address: jairo@unb.br Universidade de Brasília – IP/UnB Brasília, DF, Brazil. Katia Puente-Palacios E-mail address: kep.palacios@gmail.com Universidade de Brasília – IP/UnB Brasília, DF, Brazil. Katia Puente-Palacios E-mail address: kep.palacios@gmail.com Universidade de Brasília – IP/UnB Brasília, DF, Brazil. Jacob Arie Laros E-mail address: jalaros@gmail.com Universidade de Brasília – IP/UnB Brasília, DF, Brazil. Copyright © 2012 Brazilian Administration Review. All rights reserved, including rights for translation. Parts of this work may be quoted without prior knowledge on the condition that the source is identified. H. P. Brandão, J. E. Borges-Andrade, K. Puente-Palacios, J. A. Laros 2 BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 Abstract This study aims to identify the predictor variables for three types of management competencies: financial business management, process management and socio-environmental management. It was hypothesized that attributes of bank managers (such as the learning strategies they use) and those of the branches where they work (such as organizational support) are associated with the expression of management competencies at work. Structured questionnaires were used for data collection. Multilevel regression analyses (HLM) were performed to test the theoretical model of investigation. The sample was made up of 775 managers from the Bank of Brazil, spread among 239 bank branches. The greatest relative contribution to explain the management competencies came from intrinsic and extrinsic reflection, a learning strategy employed by managers at work. The managers’ perceptions regarding the company’s performance management practices, one dimension of organizational support, appeared as another important predictor. Other learning strategies and dimensions of organizational support, as well as the number of hours spent on training activities, revealed less expressive effects regarding management competencies. Practical recommendations are presented and the study’s limitations and contributions are discussed. Key words: management competencies; learning strategies at work; perceived organizational support; multilevel analysis; Bank of Brazil. BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar Relationships between Learning, Context and Competency 3 Relationships between Learning, Context and Competency Introduction Researchers and organizations have shown a growing interest in the topics of learning and competency at work (Calhoun, Vincent, Calhoun, & Brandsen, 2008; Gonczi, 1999). Despite efforts, especially in the theoretical field, to explain the relationship between these constructs and the influence that context has on them, many questions remain unanswered. There are theoretical assumptions and constructions that still lack empirical sustentation (Brandão, 2007). Furthermore, despite the fact that organizations constitute integrated systems, characterized by having a multiple level structure (Kozlowski & Klein, 2000), the great majority of research done in this field, including that done on learning and competency, does not consider this assumption and simply studies each organizational level in isolation (individual, team, unit or organization as a whole, for example). Due to this, there is a shortage of investigations which not only strive to empirically examine the relationships between these constructs, but which also adopt a multilevel perspective in their analyses (Kreft & Leeuw, 1998; Van Yperen, Van den Berg, & Willering, 1999). The study presented here had as its objective the development and testing of a theoretical model of investigation, with the expression of management competencies at work as its criterion variable. Based on a multilevel theoretical model and making use of a compatible analysis tool (Hox, 2010; Snijders, 2008), the intention was to empirically verify the existence of predictive relationships between: (a) on one hand, bank manager attributes (such as the informal learning strategies they employ and the number of hours they spend on formal learning activities) and the attributes of the bank branches where they work (such as organizational support, for example); and (b) on the other hand, the expression of competencies at work. o de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar Background Literature and Theoretical Model Although competency(2) constitutes a complex and multifaceted concept (Gonczi, 1999; McLagan, 1997), it is traditionally understood as the individual’s capability to perform a role in a particular professional context (Brandão & Borges-Andrade, 2008). It is derived from personal attributes, such as knowledge, skills and attitudes (Durand, 2000; Gonczi, 1999), which become evident through the individual’s behavior at work (Whiddett & Hollyforde, 1999). Competencies are revealed as people behave in the professional situations they come up against (Zarifian, 1999). They are generally described in terms of behavioral patterns, which represent the organization’s expectations regarding the performance of its employees (Gonczi, 1999). The behaviors expected from those people while exercising managerial functions are usually called management competencies (Boak & Coolican, 2001; Cockerill, 1994). Competencies are developed through the learning process (Sonnentag, Niessen, & Ohly, 2004), which involves acquiring knowledge, skills and attitudes (Durand, 2000; Illeris, 2004). Learning may be defined as a relatively lasting change in a person’s capacity or behavior that is transferable to new situations (Pozo, 2000). It constitutes the process through which the competency is developed (Gonczi, 1999; Le Boterf, 1999), while the expression of competencies represents a manifestation of what the person learned (Freitas & Brandão, 2006). Learning processes may be formal or informal, in such a way that the individual is able to learn just as much from training and development activities (T&D) that are formally undertaken by the organization as from learning practices informally used in the workplace (Sonnentag et al., 2004). These informal activities that people adopt in their professional context – in order to gain knowledge and skills – are referred to as learning strategies at work (Sonnentag et al., 2004). According to Holman, Epitropaki and Fernie (2001), it is possible to empirically distinguish six types of informal learning strategies: AR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar www.anpad.org.br/bar BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 H. P. Brandão, J. E. Borges-Andrade, K. Puente-Palacios, J. A. Laros 4 . reproduction: mental repetition of information, without reflecting on its meaning; . intrinsic reflection: formulating mental structures which interrelate the component elements of work; . extrinsic reflection: formulating mental structures that connect work to other aspects of the company; . interpersonal help seeking: obtaining assistance from other people; . interpersonal help seeking: obtaining assistance from other people; . Background Literature and Theoretical Model seeking help from written material: searching documents, manuals, databases, books and other non- social sources; and . practical application: experimentation, trying things out in practice. The first three strategies are classified as cognitive, while the last three are considered behavioral. Theorists from this area defend that the use of these strategies is positively associated with the effectiveness of the learning process (Warr & Downing, 2000), or in other words, with the acquisition of knowledge, skills and attitudes. Some studies (Felix, 2005; Sonnentag & Kleine, 2000) show that the amount of time dedicated by the individual to training activities or to practices of informal learning affects the domain and the expression of competencies at work. Learning and expression of competencies can be influenced by employee attributes as much as it can be by contextual variables (Le Boterf, 1999; Sonnentag et al., 2004). Characteristics of the work team (such as managerial support and organizational climate) and of the organization (such as culture, rules and other aspects) can facilitate or restrict the development of competencies as well as the possibility to apply them at work (Abbad, Freitas, & Pilati, 2006; DeNisi, 2000). Studies carried out on this subject show that the employee’s perception on the organizational support can predict the expression of competencies at work (Abbad, Pilati, & Borges-Andrade, 1999; Rhoades & Eisenberger, 2002). Perceived organizational support refers to the worker’s perception of the quality of the treatment he or she receives as compensation for the efforts made at work (Eisenberger, Huntington, Hutchison, & Sowa, 1986). It is related to the employee’s opinions about how much the company values the employee’s contributions and how much it takes care of the employee’s well-being (Abbad et al., 1999). Such opinions about organizational support can be classified into four dimensions of the work context (Abbad et al., 1999): . performance management practices: describes the actions taken by the organization related to improving work processes, to professional training, to stimulating employee participation, and to the availability of orientation, among others; . performance management practices: describes the actions taken by the organization related to improving work processes, to professional training, to stimulating employee participation, and to the availability of orientation, among others; . workload: involves goals and time limits, workday, and work volume; . workload: involves goals and time limits, workday, and work volume; . Background Literature and Theoretical Model This approach defends that the lowest level of an organization (the individual, for example) is contained in the higher levels (team, unit and organization) and that people can modify organizational processes just as much as groups and the organization itself can influence the performance of individuals (DeNisi, 2000). The multilevel approach permits the construction of explanatory models containing data that represents different levels, thus making it possible to assess the relative importance of individual and organizational attributes in the relationships between the studied variables (Elovainio, Kivimaki, Steen, & Kalliomaki-Levanto, 2000; Hox, 2010). According to Kozlowski and Klein (2000), by admitting the investigation of a phenomenon, analyzing it at multiple levels, such an approach favors a more integrated and vast understanding of different constructs, making it possible to build an organizational science that is theoretically rich and may have practical relevance. From a mathematical point of view, multilevel analysis permits the examination of particular contributions of predictor variables from different levels towards explaining the phenomenon of interest, as well as investigating the presence of random coefficients (Hox, 2010; Kreft & Leeuw, 1998), which indicates that the magnitude of the effect produced by these predictors varies according to the different scenarios in which they occur (for example, in different teams or work units). Its utilization is recommended not only for theoretical reasons, but also because this approach uses procedures which improve the reliability of statistical tests. By taking the variance within each group and between the groups into consideration, multilevel models produce more precise predictive estimates, in addition to providing a more adequate description of the relationships between the variables studied (Van der Vegt, Emans, & Van de Vliert, 2001). For this reason, a multilevel approach was chosen to serve as the foundation for constructing and testing this theoretical model. The main purpose of this study was to test a theoretical model of the expression of management competencies at bank branches, which was formulated on the basis of the theoretical and empirical foundations reviewed in this section. The variables included in this study and the hypothesized predictive relationships (direct and moderated) are described in Figure 1. This model is structured into two levels of analysis (Elovainio et al., 2000), in which individual employees holding management positions (level 1) at the Bank of Brazil are grouped in organizational units called bank branches (level 2). Background Literature and Theoretical Model material support: meaning the availability, quantity and quality of material, equipment, tools, furniture, and other elements needed to work; and . promotion and reward practices: includes organizational policies regarding incentives, professional ascent, and compensation and employee appreciation. According to Abbad, Freitas and Pilati (2006), even though the learning activities undertaken by the individual have promoted the acquisition of knowledge, skills and attitudes, it is possible that this learning process does not generate favorable effects on the expression of competencies at work, if the individual doesn’t have the adequate organizational support in the context of work. So, organizational support variables can moderate the relationship between the acquisition and the expression of competencies at work (Brandão & Borges-Andrade, 2008). The influence that these variables have on the work context characterizes one of the main presuppositions in the organizational behavior field of study: that an organization composes an Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.an www.anpad.org.br/bar Relationships between Learning, Context and Competency 5 integrated and multilevel system, since its results are influenced by processes occurring on various levels, which interact with each other in an interdependent manner (DeNisi, 2000). integrated and multilevel system, since its results are influenced by processes occurring on various levels, which interact with each other in an interdependent manner (DeNisi, 2000). According to Kozlowski and Klein (2000), if one level of the organization influences and is influenced by other levels, this should be taken into consideration in theoretical models and their empirical tests; given that no single level can solely explain, in a complete and appropriate manner, the nature of organizational behavior phenomena. Despite the fact that it is assumed that the development and expression of an individual’s competencies at work can be influenced just as much by the person’s own attributes as by the characteristics of the team and organization to which the person belongs (Abbad et al., 2006), many times this relationship is not well articulated nor adequately dealt with in empirical research. Multilevel analysis constitutes a theoretical and methodical approach which has been indicated for filling the gap between theoretical assumptions and their empirical tests (Jex & Bliese, 1999; Kozlowski & Klein, 2000). BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 Background Literature and Theoretical Model It deals with a cross-level model because it specifies relationships between constructs at different levels, looking to unite the company’s micro and macro perspectives (Kozlowski & Klein, 2000). BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar H. P. Brandão, J. E. Borges-Andrade, K. Puente-Palacios, J. A. Laros 6 Figure 1. Theoretical Model of the Expression of Management Competencies at Bank Branches. Criterion Variable Expression of management competencies at work Explanatory Variables Number of the T&D hours of the employee Learning strategies at work Perceived organizational support (individual’s perception deviation within the branch) Socio-professional data: gender, position, educational level and number of years of management experience Level 1 – Individuals (Branch Managers) Level 2 – Bank Branch Size of the branch (number of employees) Degree of the branch’s managerial complexity Percentage of profits distributed to managers Number of T&D hours (employees’ average) Explanatory Variables Control Variables Perceived organizational support (collective perception) Control Variables Socio-professional data: gender, position, educational level and number of years of management experience Figure 1. Theoretical Model of the Expression of Management Competencies at Bank Branche Method The study was done at the Bank of Brazil, a company partially owned by the Brazilian Government, whose mission is “to be the solution in financial services and intermediation, meet the expectations of customers and shareholders, strengthen the commitment between employees and the company and contribute towards the development of the country” (Banco do Brasil, 2008). When this study was carried out in 2008, the Bank of Brazil had more than US$300 billion in assets, about 50 million customers, 4,052 large, full-service branches in Brazil, 11,031 small, limited-service branches in Brazil, 42 branches abroad, 81,855 employees, and 9,119 interns (Banco do Brasil, 2008). Its branches are business units that account for the product and service sales, and are segmented according to bank customer markets (retail, private, small business, large companies, government, and other markets). Each branch is managed by an employee who holds the position of General Manager. Depending on the number of employees and customers and on the potential size of the customers market, a branch may have other management positions (accounts managers, for instance), which are subordinated to the general manager. In recent years, based on assumptions of management models and instruments such as Balanced Scorecard - BSC (Kaplan & Norton, 1997) and competence-based management (Durand, 2000; Heene & Sanchez, 1997), the Bank has managed the performance of its branches through the establishment of objectives, goals and competencies, aligned with its organizational strategy, in different performance perspectives: financial, customers, processes, organizational behavior and society (Brandão et al., 2008). BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar 7 7 Relationships between Learning, Context and Competency Relationships between Learning, Context and Competency Considering these characteristics of its management practices, the company has encouraged the accomplishment of applied research that may support and guide its strategies and management actions. This is the case of studies carried out by: (a) Brandão et al. (2008), describing the development and implementation of the competence-based management model adopted by the bank; (b) Leite and Porsse (2003), analyzing organizational learning processes in the company; (c) Brandão, Borges- Andrade, Freitas and Vieira (2010), which describes the development and validation process of a scale to measure management competencies at bank branches; and (d) Brandão and Borges-Andrade (2011), which describes the development and validation process of a scale to measure learning strategies used by bank managers for developing their competencies. Sample A total of 926 questionnaires were received, representing a return rate of 42%. Questionnaires were eliminated when they: (a) had more than 10% of missing answers; (b) received the same answer on the scale for all items (unvarying responses); (c) were multivariate outliers (identified by the Mahalanobis distance); (d) had respondents who worked for less than four months in their respective branches (the minimum amount of time needed to share perceptions); or (e) belonged to respondents who constituted the only reply from the branch where they worked (branches with only one respondent). With these exclusions, the adjusted sample totaled 775 participants grouped into 239 bank branches, with groups ranging from 2 to 8 members (an average of 3.25 managers per branch). On the whole, the respondents were males (69.3%), holding the position of team manager (69.6%), had a college degree or post-graduate degree (84%) and on average had around 8 years of experience in managerial functions. Method The measurement scales just listed were used for data collection in the present study, as described ahead. Primary and secondary data were used to test the hypothesized relationships in Figure 1. Primary data was collected through structured questionnaires – previously subjected to semantic validation(3) –, completed and returned by the internal mail system of the organization being studied, in March and April of 2008. The instruments were sent to all of the 2,205 managers working in 329 branches, which were randomly selected. Secondary data was obtained from Bank databases. BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar Measures As a result, the scores on perceived organizational support were separated into two distinct measurements: (a) one relative to the average of the workers’ perceptions at a branch, pertaining to level 2 of the analysis; (b) the other relative to the deviation variable within the branch (the individual score minus the group average), representing level 1 of the analysis; and e) Perceived organizational support (individual’s perception deviation within the branch): explanatory variable, pertaining to the individual level (level 1), which represents the relative position of the person within the group (the branch). It constitutes the deviation of the individual support score compared to the branch’s average (Hox, 2010; Kozlowski & Klein, 2000); that is, the difference between individual perception and collective perception of organizational support. Although the perceived support reflects certain beliefs held by members of a group regarding the quality of the treatment they receive from the company - conceived as a group level variable - such beliefs may result as much from the objective qualities of the support offered by the company as from the employee’s personal characteristics. Two employees from the same bank branch may report differing levels of perceived support, depending on their capacity to process information and on their interests, experiences and other personal attributes (Hox, 2010). As a result, the scores on perceived organizational support were separated into two distinct measurements: (a) one relative to the average of the workers’ perceptions at a branch, pertaining to level 2 of the analysis; (b) the other relative to the deviation variable within the branch (the individual score minus the group average), representing level 1 of the analysis; and f) Learning strategies at work: explanatory variable belonging to the individual level (level 1). Measure developed by Brandão and Borges-Andrade (2011), based on Holman et al. (2001), and Pantoja (2004), it contains 26 items grouped into five dimensions: extrinsic and intrinsic reflection (9 items, α = .92); interpersonal help seeking (5 items, α = .88); seeking help from written material (5 items, α = .79); reproduction (4 items, α = .79); practical application (3 items, α = .82). Participants responded on a 10-point scale, varying from 1 (I never do) to 10 (I always do). An example item is I ask for help from my co-workers when I need to learn something at work. Measures Besides these measures, the following were used as control variables: (a) on level 1 of analysis, gender, position held, education level, and years of management experience of the participants; and (b) on level 2, the percentage of profits distributed to managers, size and managerial complexity of the branch (secondary data). In this way, the intention was to isolate the eventual effects of these variables from the effects produced by the explanatory variables, which constitute the main object of interest of this study. The procedures adopted to develop and to evaluate the psychometrical quality of the three perceptual measures used (management competencies, learning strategies at work and perceived organizational support) are described in detail by Brandão et al. (2010), Brandão and Borges-Andrade (2011) and Abbad et al. (1999), respectively. All the dimensions of these three scales are valid and internally consistent(4), showing Cronbach's alphas equal to or above .79, as previously stated. Measures Laros 8 8 practices (8 items, α = .85), which were responded to on a 10-point agreement scale, ranging from 1 (I totally disagree) to 10 (I totally agree). An example item is In the organization where I work, the employees receive all orientation and information needed to efficiently execute their activities. The data were collected at the individual level (level 1), through the managers self- report, and later pooled together (by the average of responses from each branch) to represent the branch level (level 2); practices (8 items, α = .85), which were responded to on a 10-point agreement scale, ranging from 1 (I totally disagree) to 10 (I totally agree). An example item is In the organization where I work, the employees receive all orientation and information needed to efficiently execute their activities. The data were collected at the individual level (level 1), through the managers self- report, and later pooled together (by the average of responses from each branch) to represent the branch level (level 2); d) Number of T&D hours per employee: explanatory variable pertaining to the individual level (level 1), which represents the number of hours each employee spent on T&D activities in 2007 (secondary data); d) Number of T&D hours per employee: explanatory variable pertaining to the individual level (level 1), which represents the number of hours each employee spent on T&D activities in 2007 (secondary data); e) Perceived organizational support (individual’s perception deviation within the branch): explanatory variable, pertaining to the individual level (level 1), which represents the relative position of the person within the group (the branch). It constitutes the deviation of the individual support score compared to the branch’s average (Hox, 2010; Kozlowski & Klein, 2000); that is, the difference between individual perception and collective perception of organizational support. Although the perceived support reflects certain beliefs held by members of a group regarding the quality of the treatment they receive from the company - conceived as a group level variable - such beliefs may result as much from the objective qualities of the support offered by the company as from the employee’s personal characteristics. Two employees from the same bank branch may report differing levels of perceived support, depending on their capacity to process information and on their interests, experiences and other personal attributes (Hox, 2010). Measures The following measures were taken in order to test the model: a) Expression of management competencies: criterion variable, pertaining to the individual level (level 1), measured with fourteen items from the scale developed by Brandão et al. (2010), which described observable behaviors at work, representing three dimensions of management competencies: financial business management (4 items, α = .82); process management (4 items, α = .80); and socio-environmental management (6 items, α = .86). Participants responded on a 10- point scale anchored at the extremes by non-expressed competency (1) to fully expressed competency (10). An example item is Monitoring the quality of internal processes, in order to avoid having to replicate previous work efforts; b) Number of T&D hours per branch: explanatory variable at the branch level (level 2), which represents the average number of hours that branch managers dedicated to training and development activities (T&D) in 2007. It can be interpreted as an effort to develop competencies (Felix, 2005; Sonnentag et al., 2004). Data were collected from secondary sources (database from the organization being studied); b) Number of T&D hours per branch: explanatory variable at the branch level (level 2), which represents the average number of hours that branch managers dedicated to training and development activities (T&D) in 2007. It can be interpreted as an effort to develop competencies (Felix, 2005; Sonnentag et al., 2004). Data were collected from secondary sources (database from the organization being studied); c) Perceived organizational support (collective perception): explanatory variable, measured with a scale adapted from Abbad, Pilati and Borges-Andrade (1999). This measure has 28 items distributed throughout four dimensions: material support (6 items, α = .91); performance management practices (7 items, α = .87); workload (7 items, α = .85); and promotion and reward c) Perceived organizational support (collective perception): explanatory variable, measured with a scale adapted from Abbad, Pilati and Borges-Andrade (1999). This measure has 28 items distributed throughout four dimensions: material support (6 items, α = .91); performance management practices (7 items, α = .87); workload (7 items, α = .85); and promotion and reward BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar www.anpad.org.br/bar H. P. Brandão, J. E. Borges-Andrade, K. Puente-Palacios, J. A. Data analysis In multilevel regressions, the sample should contain more than 50 groups (level 2) and, to ensure greater accuracy of estimated parameters, a large number of groups is more important than a large number of individuals per group (Maas & Hox, 2004, 2005). Since in the present study the adjusted sample totaled 775 individuals grouped into 239 bank branches, it is safe to assume that this sample is large enough for both levels of analysis (individuals and groups) in order to generate reliable estimates. After testing the regression models, it was verified, following the recommendations of Rasbash, Steele, Browne and Prosser (2004), that these models met the assumptions of homoscedasticity and of a normal distribution of regression residuals, aspects which will be explained later when presenting the results. Another requirement which was examined refers to the intraclass correlation (ICC) of criterion variables, a coefficient that indicates the magnitude of differences between the groups (branches) or the answers consistency (Kreft & Leeuw, 1998). The calculations of this index are based on the comparison of the amount of variance between groups with the amount of variance within groups (Kozlowski & Klein, 2000). The intraclass correlation indicates which proportion of the variance can be attributed to differences among the units of the macrolevel (bank branches). Since the existence of significant differences at the level 2 of analysis, related to the criterion variables, constitutes an assumption for constructing and testing multilevel models (Van der Vegt et al., 2001), the ICC of these variables was calculated in order to confirm that the collected data justified adopting a multilevel approach. The three dimensions of competencies showed that they had intraclass correlations different from zero: financial business management (ICC = .05), process management (ICC = .04) and socio- environmental management (ICC = .11). This coefficient indicates that 5% of the variance of financial business management competencies, 4% of the variance in the process management competencies, and 11% of the variance in the socio-environmental competencies can be explained by differences between bank branches (level 2). Although it seems to be little substantial, intraclass correlations of this magnitude have justified the adoption of multilevel analysis (Snijders & Bosker, 1999), since they suggested the existence of certain dependence between individual observations, causing the so-called design effect (Hox, 2010). Data analysis After collecting the primary data, the statistical assumptions were verified in order to carry out a multilevel regression analysis (hierarchical linear model). Considering the recommendations of Miles and Shevlin (2001) and Maas and Hox (2005), it can be affirmed that: (a) there was sufficient linearity in the relationships between the explanatory variables and the three dimensions of management competencies (criterion variables), which was verified by visual inspection of scatter plots and observing the magnitude of the correlations between the variables; (b) no signs of multicollinearity BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar Relationships between Learning, Context and Competency Relationships between Learning, Context and Competency 9 were encountered since all of the correlation coefficients between the explanatory variables were less than .60; and (c) the sample size was large enough on both levels (individuals and branches) to diminish the eventual effects of small deviations from the normality. In single-level regressions, the sample should contain at least 50 individuals plus 8 respondents for each explanatory variable (Tabachnick & Fidell, 2007). In multilevel regressions, the sample should contain more than 50 groups (level 2) and, to ensure greater accuracy of estimated parameters, a large number of groups is more important than a large number of individuals per group (Maas & Hox, 2004, 2005). Since in the present study the adjusted sample totaled 775 individuals grouped into 239 bank branches, it is safe to assume that this sample is large enough for both levels of analysis (individuals and groups) in order to generate reliable estimates. After testing the regression models, it was verified, following the recommendations of Rasbash, Steele, Browne and Prosser (2004), that these models met the assumptions of homoscedasticity and of a normal distribution of regression residuals, aspects which will be explained later when presenting the results. were encountered since all of the correlation coefficients between the explanatory variables were less than .60; and (c) the sample size was large enough on both levels (individuals and branches) to diminish the eventual effects of small deviations from the normality. In single-level regressions, the sample should contain at least 50 individuals plus 8 respondents for each explanatory variable (Tabachnick & Fidell, 2007). Data analysis As the average size of the groups in this research is of 3.25 respondents, the design effect would reduce the effective size of the sample, according Hox (2010), to 696 (reduction of 10.2% in the adjusted sample) in the case of expression of financial management competencies, and 711 (8.3% reduction) and 621 (reduction of almost 20%) in relation to the process management competencies and to socio-environmental management competencies, respectively, if the data were analyzed using conventional regressions. Considering these parameters, using conventional regression models (ordinary least squares [OLS]) for data analysis could lead to an underestimation of regression standard errors, undermining the reliability of tests (Elovainio et al., 2000; Van Yperen et al., 1999) and increasing the likelihood of Type-1 Errors (Kreft & Leeuw, 1998). This is the reason why a multilevel regression model was considered more appropriate in the present research. Before proceeding with the model testing, the adequacy of the perceived organizational support measures (collective perception) was examined in order to represent group level attributes (bank branches). This was necessary because, even though perceived organizational support is theoretically defined as a group level construct (Abbad et al., 1999), the data regarding this variable were collected from individual responses from bank managers. In order to verify if the manager’s individual perceptions (level 1) could be aggregated (arithmetic mean) to represent the characteristics of the branches where they work (level 2), the intraclass correlation coefficient (ICC) and the interrater agreement index (rwg) were calculated for the four dimensions of perceived support, as James, Demaree, and Wolf (1993), and Kozlowski and Klein (2000) recommend. Such statistical indicators reveal the degree of similarity of the individual perceptions, which, if the magnitude is sufficient, www.anpad.org.br/bar BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 H. P. Brandão, J. E. Borges-Andrade, K. Puente-Palacios, J. A. Laros 10 justifies the adoption of the arithmetic mean as a level 2 measure. The following coefficients were obtained: material support (ICC = .24; rwg = .93); management performance practices (ICC = .12; rwg = .94); workload (ICC = .13; rwg = .81); and promotion and reward practices (ICC = .16; rwg = .86). Data analysis Although some dimensions of perceived organizational support had shown ICC values of only moderate magnitude (approximately .12), all the dimensions of this construct revealed rwg coefficients greater than .80, which indicate there was sufficient homogeneity of responses within groups (bank branches) to aggregate individual observations (Kozlowski & Klein, 2000; Kreft & Leeuw, 1998, Tucker, Sinclair, & Thomas, 2005). Together, these coefficients (ICC and rwg) attest to the construct validity of the aggregate measures (branches averages). After evaluating all of these requirements, the model was tested. Beforehand, all the explanatory and control variables were standardized, in order to allow for comparisons between the regression coefficients. The analyses were done in six steps, utilizing the deviance (-2Loglikelihood) to verify the adjustment of the models, as recommended by Hox (2010). In the first step, the called null or empty model was computed, to define a base of comparison in relation to the following steps (Hox, 2010; Van Yperen et al., 1999) and to decompose the variance of the expression of management competencies in the individual level variance (level 1) and in the branch level variance (level 2) (Van der Vegt et al., 2001). In the second step, control variables were inserted at the individual level (gender, position held, educational level and management experience) and at the branch level (size, degree of branch’s managerial complexity, and percentage of profit distribution). In the third step, explanatory variables at the individual level were added (number of T&D hours, the individual deviation for the four factors of perceived organizational support, and the five factors of learning strategies at work). In the fourth step, explanatory variables at the branch level were included (average of T&D hours and the four factors of collective perception of organizational support). In the fifth step, the existence of random effects was verified (Hox, 2010; Kozlowski & Klein, 2000) for level 1 variables, while the sixth step included interactions between explanatory variables from levels 1 and 2. Data analysis was performed with MLwiN, version 2.02, a software for analyzing hierarchically structured data (Rasbash, Steele, Browne, & Prosser, 2004; Snijders, 2008), using the Iterative Generalized Least Squares (IGLS) estimation method. BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 Results In relation to the criterion variable expression of financial business management competencies, the empty model was initially computed, which revealed an estimate of -2 Loglikelihood (deviance) equal to 2,673.67. This amount served as a parameter of comparison to verify the adjustment of the models in subsequent stages, once the deviance reflects the lack of adjustment between the data and the model. In step 2, level 1 (individual) and level 2 (branch) control variables were inserted as described in the previous Figure 1. In this stage, three variables revealed significant effects: degree of branch’s managerial complexity, position held, and education level of the participant; the first one pertaining to level 2 and the last two pertaining to level 1. The deviance was reduced to 2,642.91, and this reduction of 30.46 was significant at 5% level since the chi-square test (dividing the difference in deviance by the degrees of freedom) was above the critical value of 1.96 (Hox, 2010; Snijders, 2008). Level 1 explanatory variables were inserted in step 3. Significant effects were from: the frequency of use of extrinsic and intrinsic reflection and of seeking help from written material learning strategies and the individual’s perception (deviation within the branch) about the company’s performance management practices and the workload (factors of perceived organizational support). There was a substantial decrease in the deviance (182.32), which was significant (χ2 = 45.58), indicating an improvement in the adjustment of the model. Table 1 assists in visualizing and BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar Relationships between Learning, Context and Competency 11 comparing the estimated parameters in steps 1, 2 and 3 for the criterion variable expression of financial business management competencies. Given such parameters, it is worth mentioning that, in step 3, the regression coefficients of two control variables lost significance, but these were maintained in the model, for two reasons: (a) they showed significance in step 2, whose adjustment served as a parameter for comparison in relation to the other models; and (b) their removal damaged the adjustment of the model (elevating the deviance). Results Comparison between Models 1, 2 e 3 for the Expression of Financial Business Management Competencies Predictor Variables Model 1 (Empty) Model 2 (Reference): Inclusion of Control Variables Model 3: Inclusion of Level 1 Explanatory Variables Fixed Parameter Effect (β) and Standard Error t ratio Effect (β) and Standard Error t ratio Effect (β) and Standard Error t ratio (γ00) Intercept 8.02 (.05) --- 8.01 (.05) --- 8.01 (.05) --- (γ01) Degree of branch’s managerial complexity .11 (.05) 2.20* .09 (.05) 1.80 (γ10) Position held .14 (.05) 2.80* .03 (.05) .60 (γ20) Education level .16 (.05) 3.20* .15 (.05) 3.00* (γ30) Extrinsic and intrinsic reflection (learning strategy) .46 (.06) 7.67* (γ40) Seeking help from written material (learning strategy) .16 (.06) 2.67* (γ50) Performance management practices (perceived organizational support) .14 (.05) 2.80* (γ60) Workload (perceived support - individual deviation within the branch) .09 (.04) 2.25* Random Parameter Variance and Standard Error t ratio Variance and Standard Error t ratio Variance and Standard Error t ratio (σ2 u0) Level 2 Variance .09 (.06) 1.50* .08 (.06) 1.33 .08 (.05) 1.60* (σ2 e) Level 1 Variance 1.83 (.11) 16.64* 1.77 (.11) 16.10* 1.37 (.08) 17.13* Model Adjustment Deviance (-2Loglikelihood) 2,673.67 2,642.91 2,460.59 Number of estimated parameters 3 6 10 Difference between deviances 30.76 182.32 Difference between no. of parameters (d.f.) 3 4 Test χ2 10.25 45.58* Note p < 05 Comparison between Models 1, 2 e 3 for the Expression of Financial Business Management Competencies parison between Models 1, 2 e 3 for the Expression of Financial Business Managemen petencies Then in step 4, the explanatory variables from level 2 were inserted. Although only two variables (the number of T&D hours and the collective perception on material support) revealed significant effects, there was a new decrease in the deviance (15.79), which was significant (χ2 = 7.90), indicating an improvement in the model adjustment. In step 5, the random effects in level-1 variables were inserted. The seeking help from written material learning strategy and the individual deviance of the perceived performance management practices were shown to have random effects – also called aleatory – indicating that the magnitude of the effect exerted by these variables varies from branch to branch. One of these variables (seeking help from written material) revealed effects only www.anpad.org.br/bar BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 BAR, Rio de Janeiro, v. 9, n. BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar Results 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar H. P. Brandão, J. E. Borges-Andrade, K. Puente-Palacios, J. A. Laros 12 marginally significant at the .10 level, yet it was maintained in the model because its removal damaged the adjustment of the model (elevating the deviance). In this step, a new decrease in the deviance was observed, which shows an improvement in the model fit. According to parameters indicated by Hox (2010) and Snijders (2008), the reduction of 20.59 in the deviance was significant at 5% level, as shown in Table 2, since the chi-square test (χ2 = 4.12, calculated by dividing the difference in deviance by the number of degrees of freedom) was above the critical value of 1.96. Finally, in step 6, interaction terms were inserted between level 1 and 2 variables. Significant interaction effects were observed between the following variables: (a) the extrinsic and intrinsic reflection learning strategy and the individual deviation of perceived workload; and (b) the seeking help from written material and the individual deviation of perceived workload. This step revealed the model with the best adjustment, due to the fact that the inclusion of the previously stated interaction terms brought about a new reduction in the deviance (7.70), which was significant at 5% level (χ2 = 3.85). Employing the procedures recommended by Hox (2010) and Laros and Marciano (2008), the explained variance can be estimated at each level of analysis (approximate R2). After isolating the effects of the control variables (degree of bank branch’s managerial complexity, position held and educational level of the participant), at least 22.6% of level 1 variance and 37.5% of level 2 variance were explained by the other variables of this model. Table 2 shows the estimated parameters in steps 4, 5, and 6 for the criterion variable expression of financial business management competencies. Results Comparison between Models 4, 5, and 6 for the Expression of Financial Business Management Competences Continues Explanatory Variables Model 4: Inclusion of Level 2 Explanatory Variables Model 5: Inclusion of Random Effects Level 1 Variables Model 6: Inclusion of Interaction Terms Fixed Parameters Effect (β) and Standard Error t ratio Effect (β) and Standard Error t ratio Effect (β) and Standard Error t ratio (γ00) Intercept 8.01 (.05) --- 8.01 (.05) --- 8.01 (.05) --- (γ01) Degree of branch’s managerial complexity .08 (.05) 1.60 .08 (.05) 1.60 .09 (.05) 1.80 (γ10) Position held .03 (.05) .60 .03 (.05) .60 .03 (.05) .60 (γ20) Education level .14 (.05) 2.80 .13 (.05) 2.60* .12 (.05) 2.40* (γ30) Extrinsic and intrinsic reflection (learning strategy) .47 (.06) 7.83* .49 (.06) 8.17* .47 (.06) 7.83* (γ40) Seeking help from written material (learning strategy) .13 (.06) 2.17* .12 (.06) 2.00* .13 (.06) 2.17* (γ50) Performance management practices (perceived organizational support) .14 (.05) 2.80* .13 (.05) 2.60* .14 (.05) 2.80* (γ60) Workload (perceived support - individual deviation within the branch) .09 (.04) 2.25* .09 (.04) 2.25* .09 (.04) 2.25* (γ02) Number of T&D hours (branch) .15 (.05) 3.00* .15 (.05) 3.00* .15 (.05) 3.00* (γ03) Material support (collective perception on organizational support) .09 (.04) 2.25* .10 (.04) 2.50* .11 (.04) 2.75* (γ70) Interaction: reflection x workload – individual deviation -.16 (.06) 2.67* (γ80) Interaction: seeking material help x workload - individual deviation .17 (.06) 2.83* rison between Models 4, 5, and 6 for the Expression of Financial Business Management ences BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar www.anpad.org.br/bar 13 Relationships between Learning, Context and Competency Table 2 (continued) Note. p < .05. Results Explanatory Variables Model 4: Inclusion of Level 2 Explanatory Variables Model 5: Inclusion of Random Effects Level 1 Variables Model 6: Inclusion of Interaction Terms Random Parameter – Level 2 Variance and Standard Error t ratio Variance and Standard Error t ratio Variance and Standard Error t ratio (σ2 u0) Variance - intercept .05 (.05) 1.00 .10 (.05) 2.00 .11 (.05) 2.20* (σ2 u4) Slope variance - seeking help from written material .05 (.04) 1.25 .04 (.04) 1.00 (σ2 u5) Slope variance - performance management practices - deviation .08 (.04) 2.00* .09 (.04) 2.25* Random Parameter – Level 1 Variance and Standard Error t ratio Variance and Standard Error t ratio Variance and Standard Error t ratio (σ2 e) Level 1 Variance 1.37 (.08) 17.13* 1.18 (.08) 14.75* 1.17 (.08) 14.63* Model Adjustment Deviance (-2Loglikelihood) 2,444.80 2,424.21 2,416.51 Number of estimated parameters 12 17 19 Difference between deviances 15.79 20.59 7.70 Difference between no. of parameters (d.f.) 2 5 2 Test χ2 7.90 4.12* 3.85* Aiming to examine the nature of the associations between the variables, a plotting of identified interactions was done, using the procedures suggested by Aiken and West (1991). In Figure 2, the first graph shows the relationship between using the reflection learning strategy and the expression of financial business management competencies, as a function of the difference between individual and collective perceptions regarding workload (individual deviation within the branch). From the slopes of the regression lines, it is possible to see that when an individual’s perception regarding the workload is more positive than the branch’s collective perception (a standard deviation above the difference between individual and group perceptions), the relationship between the use of extrinsic and intrinsic reflection and the expression of financial business management competencies is weaker. On the other hand, when an individual perceives the workload as more inadequate compared to the group perception (a standard deviation below the difference between individual and collective perceptions), then the relationship between the reflection and the expression of financial business management competencies is revealed as stronger. This indicates that the positive individual deviation of the perceived workload (individual deviation within the branch) lessens the effect of the extrinsic and intrinsic reflection on the financial business management competencies. BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 Results The second graph in Figure 2 shows the relationship between using the strategy of seeking help from written material and the expression of financial business management competencies, as a function of the difference between individual and collective perceptions about workload (deviation within the branch). It can be noted that, when the individual’s perception regarding workload is more negative than that of the group (bank branch), the relationship between the use of seeking help from written material and the expression of financial business management competencies is practically null. On the other hand, when an individual’s perception regarding workload is more positive than the group perception, then there exists a reasonable relationship between seeking help from written material and the expression of these competencies. Better said, the positive individual deviation of the perceived workload (deviation within the branch) strengthens the effect of seeking help from written material over the financial business management competencies. www.anpad.org.br/bar BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 H. P. Brandão, J. E. Borges-Andrade, K. Puente-Palacios, J. A. Laros 14 Low frequency of reflection use High frequency of reflection use Financial management competencies Individual perception is more positive than collective perception regarding workload (individual deviation within the branch) Individual perception is more negative than collective perception regarding workload (individual deviation within the branch) High frequency of the use of seeking help from written material Low frequency of the use of seeking help from written material Where : Individual perception is more positive than collective perception regarding workload (individual deviation within the branch) Individual perception is more negative than collective perception regarding workload (individual deviation within the branch) Figure 2. Plot of the Moderating Effect Exerted by Workload on Relationships between Learning Strategies and the Expression of Financial Business Management Competencies Figure 2. Plot of the Moderating Effect Exerted by Workload on Relationships between Learning Strategies and the Expression of Financial Business Management Competencies Figure 2. Plot of the Moderating Effect Exerted by Workload on Relationships between Learning Strategies and the Expression of Financial Business Management Competencies Figure 3 presents the theoretical-empirical model for the expression of financial business management competencies, showing the predictor variables, the nature of the relationships (direct and moderating) and the magnitude of the effect (β) that they exert on the criterion variable. BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 Results Figure 3 presents the theoretical-empirical model for the expression of financial business management competencies, showing the predictor variables, the nature of the relationships (direct and moderating) and the magnitude of the effect (β) that they exert on the criterion variable. Finally, following orientations from Rasbash et al. (2004), and Laros and Marciano (2008), graphs on the dispersion of standardized residuals were produced, at levels 1 and 2 of analysis; which indicated that the assumptions of normality of the distribution of residuals, linearity of relationships, and homoscedasticity were not violated by the tested model. BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar Relationships between Learning, Context and Competency 15 Figure 3. Theoretical-empirical Model of the Expression of Financial Business Management Competencies. The previously stated steps were also adopted for testing the models relative to the other two criterion variables: the expression of process management and socio-environmental management competencies, whose final results are shown in Figures 4 and 5. After isolating the control variables’ effects, the explanatory variables presented in Figure 4, all together, explain 32.8% (approximate R2) of the variance of process management competencies at the level 1 of analysis (individuals); while those described in Figure 5, all together, explain 25.8% (approximate R2) of the variance of socio- environmental competencies in level 1 (individuals) and 29.2% (approximate R2) of the variance in level 2 (branches). Level 1 – Individual (Branch Managers) Level 2 – Bank Branch Criterion Variable Expression of financial business management competencies Explanatory Variables Degree of the branch’s managerial complexity Extrinsic and intrinsic reflection (learning strategy at work) Number of T&D hours (employees’ average) Seeking help from written material (learning strategy at work) Explanatory Variables Material support (factor of the collective perception of organizational support) Performance management practices (individual deviation of the perceived organizational support) Position held Education level (β = .09) (β = -.15) (β = .17) Workload (individual deviation of the perceived organizational support) (β = .09) (β = .15) (β = .11) (β = .47) (β = .13) (β = .14) (β = .03) (β = .12) Control Variable Control Variables Figure 3. Theoretical-empirical Model of the Expression of Financial Business Management Competencies. Results The previously stated steps were also adopted for testing the models relative to the other two criterion variables: the expression of process management and socio-environmental management competencies, whose final results are shown in Figures 4 and 5. After isolating the control variables’ effects, the explanatory variables presented in Figure 4, all together, explain 32.8% (approximate R2) of the variance of process management competencies at the level 1 of analysis (individuals); while those described in Figure 5, all together, explain 25.8% (approximate R2) of the variance of socio- environmental competencies in level 1 (individuals) and 29.2% (approximate R2) of the variance in level 2 (branches). The previously stated steps were also adopted for testing the models relative to the other two criterion variables: the expression of process management and socio-environmental management competencies, whose final results are shown in Figures 4 and 5. After isolating the control variables’ effects, the explanatory variables presented in Figure 4, all together, explain 32.8% (approximate R2) of the variance of process management competencies at the level 1 of analysis (individuals); while those described in Figure 5, all together, explain 25.8% (approximate R2) of the variance of socio- environmental competencies in level 1 (individuals) and 29.2% (approximate R2) of the variance in level 2 (branches). BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar H. P. Brandão, J. E. Borges-Andrade, K. Puente-Palacios, J. A. Laros 16 Figure 4. Theoretical-empirical Model of the Expression of Process Management Competencies. Note. p < .05; p < .01; p < .001. Level 1 – Individual (Branch Managers) Level 2 – Bank Branch Criterion Variable Expression of process management competencies Explanatory Variables Extrinsic and intrinsic reflection (learning strategy at work) Practical application (learning strategy at work) Performance management practices (individual deviation of the perceived organizational support) Gender (β = -.13) (β = .21*) (β = .08) (β = -.17*) (β = .32) (β = .24) (β = .10) (β = .14) Performance management practices (factor of the collective perception of organizational support) Control Variable Explanatory Variable Figure 4. Theoretical-empirical Model of the Expression of Process Management Competencies. Note. p < .05; p < .01; p < .001. gure 4. Theoretical-empirical Model of the Expression of Process Management Competencies. Figure 5. Theoretical-empirical Model of the Expression Socio-environmental Management Competencies. Note. p < .05; p < .01; p < .001. BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar Discussion This study aimed to examine, through a multilevel analysis, if the attributes of mangers of the Bank of Brazil (level 1 of analysis) and of the bank branches where they worked (level 2) predicted the expression of management competencies at work. Among the variables pertaining to level 2 (branch), the number of T&D hours revealed a significant effect on financial business management competencies (β = .15) and on socio-environmental management competencies (β = .16). The positive regression coefficients show that the greater the number of hours the branch dedicates to T&D activities, the higher the intensity of expression of competencies at work. This result suggests that formal learning occurred: planned and structured learning activities – whether an initiative of the organization or somehow supported by the organization – intended to promote knowledge, skills and attitudes acquisition (Sonnentag et al., 2004). In what is referred to as perceived organizational support (collective perception), the performance management practices factor revealed a significant effect on the expression of process management competencies (β = .08) and of socio-environmental management competencies (β = .15), while the material support factor affected the financial business management competencies (β = .11) and the socio-environmental management competencies (β = .13). The positive regression coefficients point out that, the better the group’s (branch) perception regarding those dimensions of organizational support, the greater the expression of the competencies of the branch managers. This relationship was expected because, when there are adequate working conditions, organizational support, and orientation, it is more probable that the employee manages to successfully apply knowledge, skills, and attitudes (KSAs) at work (Abbad et al., 2006; Rhoades & Eisenberger, 2002). In order to stimulate the expression of the managers’ competencies, such results suggest the importance of taking actions regarding individual components for the acquisition of KSAs and regarding organizational aspects, such as creating a context that offers psychosocial and material support to the employee. Among the variables pertaining to level 1 (individual), the learning strategy named extrinsic and intrinsic reflection showed the greatest contribution to explain the three dimensions of competencies: financial business management (β = .47), socio-environmental management (β = .45), and process management (β = .32). The more frequently this strategy is used, the greater the intensity of expression of competencies at work. Results Level 1 – Individual (Branch Managers) Level 2 – Bank Branch Criterion Variable Expression of socio- environmental management competencies Explanatory Variables Extrinsic and intrinsic reflection (learning strategy at work) Seeking help from written material (learning strategy at work) Explanatory Variables Material support (factor of the collective perception of organizational support) Performance management practices (individual deviation of perceived organizational support) Control Variables Gender Positon held (β = .16) (β = .24*) (β = .13) (β = .15) (β = .45) (β = .19) (β = .09) (β = .19*) Performance management practices (factor of the collective perception of organizational support) Degree of the branch’s managerial complexity Number of T&D hours (employees’ average) (β = .10) Control Variable Figure 5. Theoretical-empirical Model of the Expression Socio-environmental Management Level 1 – Individual (Branch Managers) Level 2 – Bank Branch Criterion Variable Expression of socio- environmental management competencies Explanatory Variables Extrinsic and intrinsic reflection (learning strategy at work) Seeking help from written material (learning strategy at work) Explanatory Variables Material support (factor of the collective perception of organizational support) Performance management practices (individual deviation of perceived organizational support) Control Variables Gender Positon held (β = .16) (β = .24) (β = .13) (β = .15) (β = .45) (β = .19) (β = .09) (β = .19*) Performance management practices (factor of the collective perception of organizational support) Degree of the branch’s managerial complexity Number of T&D hours (employees’ average) (β = .10) Control Variable Figure 5. Theoretical-empirical Model of the Expression Socio-environmental Management Competencies. Note. p < .05; p < .01; **p < .001. BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www. BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar 17 Relationships between Learning, Context and Competency BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 Discussion In dynamic sectors, such as the financial one, it is common for the manager to have to deal with unprecedented problems and situations, whose solutions are not pre- established. In these cases, it is up to the professional to rely on the act of reflecting upon the individual work and the organization, in order to find solutions to problems. This seems to induce the use of this learning strategy and its effects on the expression of competencies. Given the predictive relevance of this learning strategy, the Bank of Brazil could implement an instructional policy to develop such a strategy in its managerial training courses, making use, for example, of exercises and case studies that demand the participants’ reflection. Two other learning strategies revealed less expressive contributions. Seeking help from written material predicts the expression of financial business management competencies (β = .13) and of socio-environmental management competencies (β = .19), while practical application has a significant effect on the expression of process management competencies (β = .24). This positive effect seems to come from a peculiarity of the banking industry. Banks, in order to reduce being exposed to risks, commonly adopt rigid operational patterns (Banco do Brasil, 2008), usually in the form of written material (rules, regulations and internal proceedings). Such results represent an important contribution of this study to the deepening of research on the consequences of using learning strategies at work, for two reasons: (a) the predicting relationships identified here meet the theoretical supposition that the use of these strategies is positively associated with the efficacy of the learning process and its application at work (Warr & Downing, 2000); and (b) although some authors (Sonnentag et al., 2004) pointed out the need to examine whether learning BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar H. P. Brandão, J. E. Borges-Andrade, K. Puente-Palacios, J. A. Laros 18 strategies lead to competency development and expression, this appears to be the first study dedicated to examining these relationships in a work context. strategies lead to competency development and expression, this appears to be the first study dedicated to examining these relationships in a work context. Still on level 1 of analysis, the difference between individual and collective perception about organizational support (the individual deviation within the branch) exerts some effects. Discussion The individual deviation in relation to the company’s performance management practices, one of the factors of perceived organizational support, revealed significant effects on the expression of financial business management competencies (β = .14), process management competencies (β = .21), and socio- environmental management competencies (β = .24). The deviation regarding the workload factor, on the other hand, showed an influence (β = .09) on the expression of financial business management competencies. Such effects represent the influence of the individual’s relative standing within the branch – the difference between the individual score and the branch mean in the organizational support factors – on the expression of competencies at work, which some authors refer to as the frog- pond effect (Hox, 2010; Kozlowski & Klein, 2000). These results suggest the importance of providing adequate organizational support to the managers. Moderating effects exerted by factors of perceived organizational support were also identified. The manager’s perception regarding workload (individual deviation within the branch): (a) attenuates the effect of the reflection strategy over the expression of financial business management competencies; and (b) strengthens the influence exerted by the seeking help from written material strategy on the expression of those competencies. On the other hand, the collective perception regarding the company’s performance management practices: (a) attenuates the effect of the practical application strategy over the expression of process management competencies; and (b) strengthens the effect of the reflection learning strategy over the expression of those competencies. These last interactions indicate that the referred to dimension of organizational support moderates the relationship between the use of learning strategies at work and the expression of process management competencies. Furthermore, some level 1 variables (individual), like certain learning strategies employed by managers at work, revealed random effects (Hox, 2010): the magnitude of the influence they exert upon the expression of management competencies varies depending on which branch the individual works in. Differing from what was expected, some variables, such as the learning strategies of reproduction and interpersonal help seeking, did not reveal significant effects on any dimension of management competencies. This result suggests that the use of certain learning strategies does not necessarily result in the development and application of management competencies. Such strategies cannot be useful for all people, occupations and organizations, or for the development of any competency, as suggested by Warr and Downing (2000). o de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar Discussion Similarly, one of the dimensions of perceived support (promotion and reward practices) turned out to be a non-predictor of management competencies, suggesting that the positive perception regarding dimensions of organizational support do not always results in greater expression of competencies at work. The method used in this study imposes four main limitations on its results: . Self-report measures may have resulted in common variance impact. There is also no absolute guarantee that the judgments made by the participants represent reliable indicators of the expression of competencies, since estimates based on self-report could contain imprecision and halo errors (Van Yperen et al., 1999); . The use of a sample restricted to only one organization provided internal validation, but restricted the results to the company which was studied, preventing generalizations to other organizational contexts; . The use of a sample restricted to only one organization provided internal validation, but restricted the results to the company which was studied, preventing generalizations to other organizational contexts; . The ICC values of two criterion variables (.05 for financial business management competencies and .04 for process management competencies) are relatively small and may have arisen in the . The ICC values of two criterion variables (.05 for financial business management competencies and .04 for process management competencies) are relatively small and may have arisen in the BAR, Rio de Janeiro, v. 9, n. 1, art. 1, pp. 1-22, Jan./Mar. 2012 www.anpad.org.br/bar www.anpad.org.br/bar Relationships between Learning, Context and Competency 19 analyzed sample due to random errors. In this case, perhaps the use of multilevel analysis to test these models would not have been justified. analyzed sample due to random errors. In this case, perhaps the use of multilevel analysis to test these models would not have been justified. The cross-sectional nature of the study precludes causal statements about the relations between the studied variables (Van der Vegt et al., 2001), making it impossible to establish the causal order theoretically hypothesized in Figure 1. These possible limitations, although they might restrict the results, do not invalidate important contributions offered by this study. The main contribution refers to the explanation of the phenomenon studied by adopting a multilevel model, which had its appropriateness investigated through the use of multilevel analysis. Notes 1 This article was presented at XXXIV Encontro da Associação dos Programas de Pós-Graduação e Pesquisa em Administração – EnANPAD (the 34th Encounter of the Brazilian Academy of Management), September, 2010, under the title Relações entre aprendizagem, contexto e competência: um estudo multinível (Relationships between learning, context, and competency: a multilevel study). 2 In the literature about the subject, there exists a great variety of concepts for defining competency. This study does not intend to discuss the various theoretical currents and conceptual approaches which exist, since the subject has sufficiently been covered by many authors, such as McLagan (1997) and Gonczi (1999), among others. 3 Procedure that consists of previously submitting the questionnaire to some members of the population of interest and later to interview them, with the purpose of identifying and correcting eventual errors, suggestive items, and ambiguities in the instrument, in order to guarantee that the statements and scales are adequately understood by the research participants (Pasquali, L. (1998). Psicometria: teoria e aplicações. Brasília: Ed. Universidade de Brasília). 4 Besides, after collecting the primary data, all items representing the three perceptual measures (management competencies, learning strategies, and perceived organizational support) were subjected to a discriminating factor analysis, using procedures suggested by Hair, J. F., Jr., Anderson, R. E., Tatham, R. L., & Black, W. C. (1998). Multivariate data analysis. New Jersey: Prentice-Hall and Van Yperen et al. (1999), which provide evidence that the three scales represents empirically distinguishable constructs. Discussion Its utilization provided the identification of diverse types of effects: (a) direct ones exerted by level 1 variables; (b) cross-level direct ones (Kozlowski & Klein, 2000), exerted by level 2 variables; (c) frog-pond ones (Hox, 2010), exerted by the individual’s relative standing within the bank branch; (d) moderating ones exerted by dimensions of perceived organizational support over the relationship between learning strategies and management competencies; and (e) random ones (Kozlowski & Klein, 2000; Kreft & Leeuw, 1998) exerted by level 1 variables. The results indicate the appropriateness of using multilevel models, given the existing dependency between individual observations within the branches. Its empirical testing allowed more precise predictive estimates and a better examination of the relationships between variables that belong to different analysis levels (individual and branch). The use of this approach helped identify contextual factors that are directly or moderately associated with the expression of competencies at work, making it possible for this study to offer richer and more relevant contributions to the comprehension of the phenomenon under investigation. However, more studies are needed in order to confirm the consistency of these findings, especially in other professional areas, organizations and sectors of the economy. References Abbad, G., Freitas, I. A., & Pilati, R. (2006). Contexto de trabalho, desempenho competente e necessidades em TD&E. In J. E. Borges-Andrade, G. Abbad, & L. Mourão (Eds.), Treinamento, Abbad, G., Freitas, I. A., & Pilati, R. (2006). Contexto de trabalho, desempenho competente e necessidades em TD&E. In J. E. Borges-Andrade, G. 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https://openalex.org/W2807336302	https://peerj.com/articles/4901.pdf	English	null	In quest of contact: phylogeography of helmeted terrapins (<i>Pelomedusa galeata</i>,<i>P. subrufa</i>sensu stricto)	PeerJ	2,018	cc-by	10,737	ABSTRACT Based on rangewide sampling and three mitochondrial and two nuclear markers (together up to 1,850 bp and 1,840 bp, respectively), we examine the phylogeography of two helmeted terrapin species (Pelomedusa galeata and P. subrufa sensu stricto) and infer shifts of climatically suitable spaces since the Last Glacial Maximum using a modeling approach. Whilst P. galeata displays significant phylogeographic structuring across its range and consists of two deeply divergent lineages that could represent distinct species, P. subrufa shows no obvious phylogeographic differentiation. This seems to be related to historically stable or fluctuating ranges. One of the lineages within P. galeata appears to be confined to the westernmost, winter-rainfall region of South Africa and deserves special conservational attention due to the scarcity of surface water. The other lineage is distributed further east and is differentiated in three weakly supported subclades with parapatric distribution; one occurring inland, and two along the south and east coasts, respectively. As far as is known, P. subrufa occurs in South Africa only in the northeast of the country (Limpopo, Mpumalanga) and we report the species for the first time from the Lapalala Wilderness Area in the Waterberg region (Limpopo), approximately 350 km further west than previously recorded. We confirmed the occurrence of P. galeata only 80 km south of Lapalala. Thus, a sympatric occurrence of P. galeata and P. subrufa is possible. Another putative contact zone, for the two lineages within P. galeata, must be located in the Western Cape region, and further contact zones are likely for the eastern subclades within P. galeata. The nuclear loci provided no evidence for gene flow across taxa or genetic clusters within taxa. Future investigations should use denser sampling from putative contact zones and more nuclear markers to re-examine this situation. Despite few phylogeographic studies published for southern African biota, it seems likely that differentiation follows general rules, and that climate and physiographic barriers (e.g., the Great Escarpment) have shaped phylogeographic patterns. Submitted 26 October 2017 Accepted 15 May 2018 Published 5 June 2018 Corresponding author Uwe Fritz, uwe.fritz@senckenberg.de Academic editor Michael Wink Additional Information and Declarations can be found on page 17 DOI 10.7717/peerj.4901 Copyright 2018 Vamberger et al. ABSTRACT Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS Submitted 26 October 2017 Accepted 15 May 2018 Published 5 June 2018 Corresponding author Uwe Fritz, uwe.fritz@senckenberg.de Academic editor Michael Wink Additional Information and Declarations can be found on page 17 DOI 10.7717/peerj.4901 Subjects Biodiversity, Biogeography, Zoology Keywords Africa, Biogeography, Climatic niche modeling, Namibia, South Africa, Taxonomy Copyright 2018 Vamberger et al. How to cite this article Vamberger et al. (2018), In quest of contact: phylogeography of helmeted terrapins (Pelomedusa galeata, P. subr- ufa sensu stricto). PeerJ 6:e4901; DOI 10.7717/peerj.4901 In quest of contact: phylogeography of helmeted terrapins (Pelomedusa galeata, P. subrufa sensu stricto) Melita Vamberger1, Margaretha D. Hofmeyr2, Flora Ihlow1,3 and Uwe Fritz1 1 Museum of Zoology, Senckenberg Dresden, Dresden, Germany 2 Department of Biodiversity and Conservation Biology, University of the Western Cape, Bellville, South Africa 3 Zoological Research Museum Alexander Koenig, Bonn, Germany Subjects Biodiversity, Biogeography, Zoology Keywords Africa, Biogeography, Climatic niche modeling, Namibia, South Africa, Taxonomy INTRODUCTION Distributed under Creative Commons CC-BY 4.0 Distributed under Creative Commons CC-BY 4.0 Helmeted terrapins of the genus Pelomedusa are widely distributed across sub-Saharan Africa, the southwestern Arabian Peninsula and Madagascar. Together with the African hinged terrapins of the genus Pelusios, helmeted terrapins constitute the family Pelomedusidae, a group of side-necked terrapins endemic to Africa (TTWG, 2017). Pelomedusa was long assumed to be monotypic, with the single species P. subrufa (Bonnaterre, 1789) sensu lato. However, recent research revealed Pelomedusa as one of the most speciose turtle genera of the world. Currently Pelomedusa contains 10 formally recognized species and a minimum of five unnamed candidate species (Vargas-Ramírez et al., 2010; Fritz et al., 2011; Fritz et al., 2014; Fritz et al., 2015; Vargas-Ramírez, Petzold & Fritz, 2016; Wong, Fong & Papenfuss, 2010; Petzold et al., 2014; Nagy et al., 2015). Two Pelomedusa species have been recorded in South Africa (Petzold et al., 2014; Fritz et al., 2015). Most of the country is inhabited by P. galeata (Schoepff, 1792), which consists of two deeply divergent mitochondrial lineages. Each lineage can be considered as an unconfirmed candidate species sensu Padial et al. (2010), with pronounced genetic divergences resembling those of distinct turtle species (Petzold et al., 2014; Fritz et al., 2015). One of these candidate species, lineage I, is widely distributed across the central and eastern provinces of South Africa (Eastern Cape, Free State, Gauteng, KwaZulu-Natal, North West and eastern parts of the Northern and Western Cape). The other candidate species, lineage II, appears to be confined to the westernmost part of the country in the Western and Northern Cape Provinces (Petzold et al., 2014). In addition to the two lineages of P. galeata, another species (P. subrufa sensu stricto) has been recorded from the Kruger National Park region in the northeast of South Africa (Limpopo and Mpumalanga; Petzold et al., 2014; Fritz et al., 2015). Pelomedusa subrufa sensu stricto is distributed from southern Angola and Namibia through Botswana and the Democratic Republic of the Congo to Tanzania and Malawi (Petzold et al., 2014). Most likely, it occurs also in Zambia and Mozambique. Introduced populations live on Madagascar (Vargas-Ramírez et al., 2010; Wong, Fong & Papenfuss, 2010; Petzold et al., 2014). Pelomedusa galeata and P. Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 Sampling and laboratory procedures Sampling and laboratory procedures Fieldwork and sampling in South Africa were permitted by the Limpopo Provincial Gov- ernment (permit ZA/Lp/80202), Ezemvelo KwaZulu-Natal Wildlife (permit OP 139/2017), CapeNature (permit AAA007-00212-0056), the Department of Environmental Affairs, Eastern Cape (permit CRO117/13C & CRO 118/13CR), and Biodiversity Northern Cape Province (permit 245/2015). Fieldwork and sampling in Namibia were permitted by the Ministry of Environment and Tourism (permit 1910/2014). Terrapins were hand-collected or captured using baited traps, and blood and saliva samples were taken as approved by the Ethics Committee of the University of the Western Cape under ethical clearance number ScRiRC2008/39. Terrapins were released after sampling at the capture sites. Using alcohol-preserved blood or saliva samples and wet laboratory approaches for fresh material as described in Fritz et al. (2014), we generated sequences of the partial 12S rRNA gene (360 bp) for 36 Pelomedusa galeata and seven P. subrufa sensu stricto from South Africa and Namibia. Another sequenced mtDNA fragment of these samples comprised the partial ND4 gene plus adjacent DNA coding for tRNAs (816 bp), and a third mtDNA fragment corresponded to the partial cytochrome b (cyt b) gene (674 bp). In addition, we used homologous mtDNA sequences of helmeted terrapins from other investigations (Vargas-Ramírez et al., 2010; Wong, Fong & Papenfuss, 2010; Fritz et al., 2011; Fritz et al., 2014; Fritz et al., 2015; Petzold et al., 2014; Nagy et al., 2015). Including previously published material, we studied mtDNA sequences of 116 P. galeata and 41 P. subrufa sensu stricto and georeferenced their collection sites (Table S1). In addition, we sequenced two nuclear loci of samples representing all mitochondrial lineages and subclades and almost all sampling sites (Table S1). One locus, the intron 1 of the RNA fingerprint protein 35 gene (R35) has been previously shown to be species- diagnostic for P. galeata and P. subrufa (Vargas-Ramírez et al., 2010). The other locus, including coding and non-coding parts of the ornithine decarboxylase gene (ODC), is relatively variable in chelonians (Fritz et al., 2012; Praschag et al., 2017) and therefore looked promising to be also species-specific. Laboratory procedures for the nuclear loci followed Praschag et al. (2017) except that we applied newly designed internal primers for sequencing the R35 gene (forward: GCAAGGAAAAATGTTTG, reverse: ACGCTGACTCCATGCACA). The resulting R35 sequences were 1,101 bp long. The ODC sequences comprised a hardly readable simple-sequence-repeat (SSR) region, which could not be sequenced for all samples. INTRODUCTION subrufa are sister taxa and constitute together the sister clade to the remaining Pelomedusa species from more northerly regions of Africa and the Arabian Peninsula (Vargas-Ramírez et al., 2010; Fritz et al., 2011; Petzold et al., 2014). Even though a considerable number of mitochondrial DNA (mtDNA) sequences have been published by previous studies (Vargas-Ramírez et al., 2010; Vargas-Ramírez, Petzold & Fritz, 2016; Wong, Fong & Papenfuss, 2010; Petzold et al., 2014; Fritz et al., 2015), phylogeographic structuring within P. galeata and P. subrufa has not yet been examined. For the present study, we expanded the previous sampling considerably and collected 43 additional Pelomedusa samples to (1) investigate phylogeographic structure of the two species and (2) delimit the distributions of P. subrufa and the P. galeata lineages across South Africa. In doing so, we used the same three mitochondrial markers (together up to 1,850 bp) as in our previous studies and sequenced for crucial samples two nuclear loci (up to 1,840 bp). In addition, using genetically verified records, we calculated climatic niche models for each species and genetic lineage. 2/21 Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 Sampling and laboratory procedures We excluded this region from further analyses, yielding 739 bp length. Including some previously published data (Vargas-Ramírez et al., 2010), R35 sequences were available for 37 P. galeata and 16 P. subrufa. For the ODC gene, sequences were available for 31 P. galeata and 10 P. subrufa. Phylogenetic analyses and uncorrected p distances of mtDNA MATERIALS AND METHODS Sampling and laboratory procedures hylogenetic analyses and uncorrected p distances of mtDNA We concatenated individual mtDNA fragments for phylogenetic analyses and merged this dataset with previously published sequences, resulting in an alignment of 1,850 bp length that included 233 Pelomedusa sequences (also including sites outside South Africa and Namibia, and other species). Pelusios sinuatus served as the outgroup. For Pelomedusa Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 3/21 galeata, P. subrufa and the outgroup, accession numbers and collection sites are given in Table S1; for other species, see Petzold et al. (2014), Fritz et al. (2015), and Nagy et al. (2015). We assessed the best partitioning scheme using PARTITIONFINDER (Lanfear et al., 2012) and the Bayesian Information Criterion (BIC). Accordingly, we partitioned the dataset using each codon position of the protein-coding genes, the 12S gene and the lumped DNA coding for tRNAs as a distinct partition. We inferred phylogenetic relationships under Maximum Likelihood using RAxML 7.2.8 (Stamatakis, 2006) and the GTR + G substitution model across all partitions. We performed five independent ML searches using different starting conditions and the fast bootstrap algorithm to explore the robustness of the results by comparing the best trees. Then, we calculated 1,000 non-parametric thorough bootstrap replicates and plotted the values against the best tree. In addition, we calculated uncorrected p distances for each mtDNA fragment using MEGA 7.0.21 (Kumar, Stecher & Tamura, 2016) and the pairwise deletion option. Parsimony networks We calculated for concatenated mitochondrial sequences a parsimony network using POPART (http://popart.otago.ac.nz). Since the underlying algorithm is sensitive to missing data, we excluded all individuals with lacking genes. In addition, we removed all individual missing sites and homologous data, resulting in an alignment of 1,602 bp length, which contained 87 sequences of P. galeata and 18 sequences of P. subrufa. For network construction of nuclear data, we phased heterozygous R35 and ODC sequences using the PHASE algorithm in DNASP 5.10 (Librado & Rozas, 2009). For R35, we built two networks because the sequences of Vargas-Ramírez et al. (2010) were approximately 300 bp shorter than ours. One network comprised only our 86 phased sequences of 1,101 bp length, whereas the second also contained the previously published data (in total 106 phased sequences). It was based on an alignment trimmed to 699 bp to match the sequence lengths of Vargas-Ramírez et al. (2010). Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 Climatic niche models To assess whether historical climate fluctuations influenced the distribution of Pelomedusa galeata and P. subrufa, we computed climatic niche models for present conditions as well as for the Last Glacial Maximum (LGM) and the mid-Holocene using the machine-learning algorithm MAXENT 3.3.3k (Phillips, Anderson & Schapire, 2006; Phillips & Dudík, 2008). To remove spatial autocorrelation, we filtered the genetically verified occurrences for each species, as well as for subclades Ia-Ic and lineage II of P. galeata, retaining only one record per sampling site, and supplemented the dataset with unambiguously assignable records from VertNet (http://vertnet.org/). The resulting datasets contained 31 localities for P. subrufa (23 own and eight VertNet records) and 51 for P. galeata (47 own and four VertNet records). Of the latter, 14 localities corresponded to subclade Ia, 10 to Ib, 17 to Ic, and 10 to lineage II. We obtained eight uncorrelated bioclimatic predictors (R2 < 0.75) with a spatial resolution of 2.5 arc minutes (∼5 km at the equator) from WorldClim (http://www. worldclim.org/) for current and past climatic conditions (mid-Holocene, ∼6,000 BP; Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 4/21 LGM, ∼21,000 BP). For the mid-Holocene and LGM, we used datasets for three different general circulation model scenarios, namely the Community Climate System Model (CCSM4), the Model for Interdisciplinary Research on Climate (MIROC-ESM), and the Max-Planck-Institute Earth System Model P (MPI-ESM-P) that were statistically downscaled to a spatial resolution of 2.5 arc min. The following predictor variables were selected and clipped to a rectangular study extent: Bio 3 = isothermality (Bio2/Bio7)(*100), Bio 5 = maximum temperature of warmest month, Bio 7 = temperature annual range, Bio 8 = mean temperature of the wettest quarter, Bio 9 = mean temperature of the driest quarter, Bio 15 = precipitation of the wettest quarter, Bio 17 = precipitation of the driest month, and Bio 18 = precipitation of the warmest quarter. We trained models separately for P. subrufa, P. galeata, and for subclades Ia-Ic and lineage II of P. galeata using circular buffers of 200 km surrounding the respective records and projected the results onto the full study extent for current and past conditions. In MAXENT, we selected linear, quadratic and hinge features to reduce model complexity and applied a bootstrapping approach with 100 replicates randomly splitting the records into 80% used for training and 20% for model evaluation. RESULTS Phylogenetic analyses of mtDNA Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 Climatic niche models We performed a maximum number of 5,000 iterations and used the area under the curve AUC (Swets, 1988) for model evaluation. We used the average projection across 100 replicates for further processing, wherein we applied the ‘‘minimum training presence logistic threshold’’ as presence- absence threshold. Phylogenetic analyses of mtDNA The general branching pattern of our phylogenetic tree (Fig. 1) was in agreement with previous publications (Vargas-Ramírez et al., 2010; Wong, Fong & Papenfuss, 2010; Fritz et al., 2014; Fritz et al., 2015; Petzold et al., 2014) in that a well-supported major clade included all species and candidate species from the northern part of the distribution range of the genus Pelomedusa. This northern clade was sister to a weakly supported southern clade comprising Pelomedusa galeata and P. subrufa sensu stricto. Whilst the monophyly of P. subrufa was well supported, the monophyly of P. galeata received only weak bootstrap support of 61. Pelomedusa galeata showed clear genetic structuring, with two well-supported clades (clade I and clade II) corresponding to deeply divergent mitochondrial lineages. One of these mitochondrial lineages (clade I) comprised three weakly supported subclades; Ia, Ib, and Ic. Subclade Ia was sister to a weakly supported, more inclusive clade with subclades Ib and Ic. Localities for subclade Ia were from the interior of South Africa, at high elevations above the Great Escarpment, where summer-rainfall prevails (Fig. 2; records in the provinces of the Free State, Gauteng, Limpopo, North West, Northern Cape). Samples in subclade Ib approximated geographically to the subtropical (low-elevation) summer-rainfall region along the northeast coast of South Africa (Eastern Cape, KwaZulu- Natal). The samples of subclade Ic were from the south coast and adjacent inland regions, mostly below the Great Escarpment, with all-year (aseasonal) rain (Eastern Cape, Western Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 5/21 0.04 100 100 99 10 99 88 55 84 95 49 100 92 100 76 57 86 72 100 61 100 100 98 100 59 51 73 39 99 95 P. barbata P. kobe Northern Clade Southern Clade P. galeata Ia P. galeata Ib P. galeata Ic P. galeata II P. subrufa P. olivacea P. schweinfurthi P. variabilis 100 P. neumanni + P. cf. neumanni P. somalica 100 100 P. gehafie A B Figure 1 Maximum Likelihood tree for Pelomedusa using 1,850 bp of mtDNA with bootstrap values indicated. Terminal clades collapsed to cartoons; colors of cartoons correspond to Fig. 2 (open cartoons represent clades beyond the map sector). The northern clade contains several distinct candidate species (candidate species A and B; deeply divergent clades within species; see Petzold et al., 2014; Fritz et al., 2015; Nagy et al., 2015). Outgroup (Pelusios sinuatus) removed for clarity. Phylogenetic analyses of mtDNA For the complete tree displaying in- dividual samples, see Fig. S1. Inset: Pelomedusa galeata (subclade Ib), Nederland Farm, KwaZulu-Natal. Photo credit: M. Vamberger. Full-size DOI: 10.7717/peerj.4901/fig-1 P. kobe P. schweinfurthi B P. variabilis P. olivacea P. subrufa P. galeata Ib P. galeata Ic P. galeata II Figure 1 Maximum Likelihood tree for Pelomedusa using 1,850 bp of mtDNA with bootstrap values indicated. Terminal clades collapsed to cartoons; colors of cartoons correspond to Fig. 2 (open cartoons represent clades beyond the map sector). The northern clade contains several distinct candidate species (candidate species A and B; deeply divergent clades within species; see Petzold et al., 2014; Fritz et al., 2015; Nagy et al., 2015). Outgroup (Pelusios sinuatus) removed for clarity. For the complete tree displaying in- dividual samples, see Fig. S1. Inset: Pelomedusa galeata (subclade Ib), Nederland Farm, KwaZulu-Natal. Photo credit: M. Vamberger. Full size DOI: 10 7717/peerj 4901/fig 1 Cape). The other mitochondrial lineage (clade II) was from the western, winter-rainfall region of South Africa, represented by several samples from the southwestern Cape and the historical type specimen of Pelomedusa galeata devilliersi Hewitt (1935) from a site in the arid northwest of South Africa, close to the Namibian border. Even though P. subrufa also showed sequence variation, there was no obvious geographic pattern (Fig. 1 and Fig. S1). Considering the wide distribution of P. subrufa (Fig. 2), this is unexpected and contrasts with the phylogeographic structure found in P. galeata. Our new records for P. subrufa from the Lapalala Wilderness Area in the Waterberg region (Limpopo) currently represent the westernmost known occurrences for this species in South Africa. Before it was only known from the western border region of the Kruger Park. Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 Uncorrected p distances of mtDNA Mean uncorrected sequence divergences between Pelomedusa galeata and P. subrufa amounted to 5.6% for the 12S fragment and 10.3% for the cyt b fragment, two genes that have previously been used for species delimitation in Pelomedusa (Petzold et al., 2014). Within all P. galeata (lineages I and II together) and within P. subrufa, the sequence Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 6/21 P. subrufa P. galeata subclade Ia P. galeata subclade Ib P. galeata subclade Ic P. galeata clade II P. neumanni P. kobe P. cf. neumanni Figure 2 Genetically verified records of Pelomedusa galeata, P. subrufa sensu stricto and geograph- ically neighboring species in southern Africa and adjacent regions. Records are based on the present study and Petzold et al. (2014), Fritz et al. (2015) and Nagy et al. (2015); for details, see Table S1. Symbols with central black circles represent new records. For localities of samples with nuclear DNA data, see Fig. S2. Range of Pelomedusa spp. shaded (combined from Bates et al., 2014; TTWG, 2017). Inset: Pelomedusa subrufa, Lapalala Wilderness Area, Limpopo. Photo credit: F. Ihlow. Full-size DOI: 10.7717/peerj.4901/fig-2 P. subrufa P. galeata subclade Ia P. galeata subclade Ib P. galeata subclade Ic P. galeata clade II P. neumanni P. kobe P. cf. neumanni Figure 2 Genetically verified records of Pelomedusa galeata, P. subrufa sensu stricto and geograph- ically neighboring species in southern Africa and adjacent regions. Records are based on the present study and Petzold et al. (2014), Fritz et al. (2015) and Nagy et al. (2015); for details, see Table S1. Symbols with central black circles represent new records. For localities of samples with nuclear DNA data, see Fig. S2. Range of Pelomedusa spp. shaded (combined from Bates et al., 2014; TTWG, 2017). Inset: Pelomedusa subrufa, Lapalala Wilderness Area, Limpopo. Photo credit: F. Ihlow. F ll i DOI / j /fi divergence for 12S was 0.7% for each. For cyt b, the divergences were 2.0% for P. galeata and 1.3% for P. subrufa (Table 1). Between lineages I and II of P. galeata, the sequence divergence was 2.2% for 12S and 7.1% for cyt b. Additional values, and divergences for the mtDNA fragment comprised of the ND4 and tRNA genes, are summarized in Table 1. Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 Parsimony networks The concatenated mitochondrial sequences were grouped in five distinct clusters (Fig. 3). Among the four clusters corresponding to the clades and subclades of Pelomedusa galeata, a maximum of 100 mutation steps occurred; among the haplotypes of P. subrufa, a maximum of 38 steps. Clade II of P. galeata was separated from the most similar haplotype of clade I (subclade Ia) by a minimum of 73 steps; subclade Ia differed from subclade Ib by a minimum of 13 steps, and from subclade Ic by 14 steps. Subclades Ib and Ic diverged by a minimum of 11 steps from one another. The haplotype clusters of P. galeata and P. subrufa were connected in a loop, with subclades Ia and Ib differing from P. subrufa by a minimum of 119 mutation steps. Clade II of P. galeata was separated by a minimum of 126 mutations from P. subrufa. 7/21 Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 Table 1 Average uncorrected p distances (percentages) for the three studied mtDNA fragments. Between-group divergences below diagonal; within-group divergences in bold on the diagonal. 12S gal (all) gal I (all) gal II gal Ia gal Ib gal Ic sub ;galeata (all) 0.7 ;galeata I (all) – 0.4 ;galeata II – 2.2 0.3 ;galeata Ia – – 2.1 0 ;galeata Ib – – 2.2 0.4 0.1 ;galeata Ic – – 2.7 1.0 0.7 0.2 ;subrufa 5.6 5.7 4.8 5.6 5.7 6.1 0.7 cyt b gal (all) gal I (all) gal II gal Ia gal Ib gal Ic sub ;galeata (all) 2.0 ;galeata I (all) – 0.8 ;galeata II – 7.1 0.5 ;galeata Ia – – 6.8 0 ;galeata Ib – – 7.6 1.7 0.1 ;galeata Ic – – 7.5 1.2 1.6 0.3 ;subrufa 10.3 10.1 12.1 10.0 10.1 10.4 1.3 ND4 + tRNAs gal (all) gal I (all) gal II gal Ia gal Ib gal Ic sub ;galeata (all) 1.5 ;galeata I (all) – 0.6 ;galeata II – 5.0 0.6 ;galeata Ia – – 5.0 0.1 ;galeata Ib – – 5.0 1.0 0.1 ;galeata Ic – – 5.0 1.3 1.1 0.5 ;subrufa 7.1 7.1 7.6 7.0 7.2 7.1 0.8 Table 1 Average uncorrected p distances (percentages) for the three studied mtDNA fragments. Between-group divergences below diagonal; within-group divergences in bold on the diagonal. Between-group divergences below diagonal; within-group divergences in bold on the diagonal. Parsimony networks 12S gal (all) gal I (all) gal II gal Ia gal Ib gal Ic sub ;galeata (all) 0.7 ;galeata I (all) – 0.4 ;galeata II – 2.2 0.3 ;galeata Ia – – 2.1 0 ;galeata Ib – – 2.2 0.4 0.1 ;galeata Ic – – 2.7 1.0 0.7 0.2 ;subrufa 5.6 5.7 4.8 5.6 5.7 6.1 0.7 cyt b gal (all) gal I (all) gal II gal Ia gal Ib gal Ic sub ;galeata (all) 2.0 ;galeata I (all) – 0.8 ;galeata II – 7.1 0.5 ;galeata Ia – – 6.8 0 ;galeata Ib – – 7.6 1.7 0.1 ;galeata Ic – – 7.5 1.2 1.6 0.3 ;subrufa 10.3 10.1 12.1 10.0 10.1 10.4 1.3 ND4 + tRNAs gal (all) gal I (all) gal II gal Ia gal Ib gal Ic sub ;galeata (all) 1.5 ;galeata I (all) – 0.6 ;galeata II – 5.0 0.6 ;galeata Ia – – 5.0 0.1 ;galeata Ib – – 5.0 1.0 0.1 ;galeata Ic – – 5.0 1.3 1.1 0.5 ;subrufa 7.1 7.1 7.6 7.0 7.2 7.1 0.8 Even though much less variation occurred in the nuclear data, no haplotype sharing was observed between P. galeata and P. subrufa (Fig. 4). In the ODC network, haplotypes of P. galeata differed by a minimum of two mutations from haplotypes of P. subrufa. In the R35 network comprising the long sequences (1,101 bp), the two species differed by a minimum of seven mutations; in the R35 network with the short sequences (699 bp), the minimum was two steps. For the samples corresponding to the mitochondrial lineages within P. galeata, haplotype sharing was observed for the ODC gene between subclades Ia and clade II, and between subclades Ia, Ib, and Ic. However, unique haplotypes occurred in each subclade. Both R35 networks consisted of three haplotype clusters, one corresponding to P. subrufa, and the two others to lineages I and II of P. galeata, respectively. No haplotypes were shared between lineage I and lineage II. The number of mutations separating the two lineages of P. galeata resembled (1,101 bp network) or clearly exceeded (699 bp network) the divergence between P. subrufa and the two haplotype clusters of P. galeata. Haplotype sharing was observed for subclades Ia, Ib, and Ic of P. galeata, but to a lesser extent in the network based on the longer sequences. Unique haplotypes occurred for each subclade. Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 Parsimony networks S3), rather suggestive of ancestral l hi h f fl P. galeata Ic P. galeata Ib P. galeata Ia P. galeata II 5 8 P. subrufa Figure 3 Parsimony network for mitochondrial haplotypes of Pelomedusa galeata and P. subrufa. Symbol size corresponds to haplotype frequency; haplotypes colored according to mitochondrial lineages (Figs. 1 and 2). Lines connecting haplotypes represent one mutational step except when otherwise indi- cated by numbers. Missing node haplotypes, small black circles. Full-size DOI: 10.7717/peerj.4901/fig-3 Figure 3 Parsimony network for mitochondrial haplotypes of Pelomedusa galeata and P. subrufa. Symbol size corresponds to haplotype frequency; haplotypes colored according to mitochondrial lineages (Figs. 1 and 2). Lines connecting haplotypes represent one mutational step except when otherwise indi- cated by numbers. Missing node haplotypes, small black circles. Full-size DOI: 10.7717/peerj.4901/fig-3 P. galeata showed no correlation with geography (Fig. S3), rather suggestive of ancestral polymorphism than of gene flow. P. galeata showed no correlation with geography (Fig. S3), rather suggestive of ancestral polymorphism than of gene flow. Parsimony networks The occurrence of shared haplotypes of the two lineages or the individual subclades of Even though much less variation occurred in the nuclear data, no haplotype sharing was observed between P. galeata and P. subrufa (Fig. 4). In the ODC network, haplotypes of P. galeata differed by a minimum of two mutations from haplotypes of P. subrufa. In the R35 network comprising the long sequences (1,101 bp), the two species differed by a minimum of seven mutations; in the R35 network with the short sequences (699 bp), the minimum was two steps. For the samples corresponding to the mitochondrial lineages within P. galeata, haplotype sharing was observed for the ODC gene between subclades Ia and clade II, and between subclades Ia, Ib, and Ic. However, unique haplotypes occurred in each subclade. Both R35 networks consisted of three haplotype clusters, one corresponding to P. subrufa, and the two others to lineages I and II of P. galeata, respectively. No haplotypes were shared between lineage I and lineage II. The number of mutations separating the two lineages of P. galeata resembled (1,101 bp network) or clearly exceeded (699 bp network) the divergence between P. subrufa and the two haplotype clusters of P. galeata. Haplotype sharing was observed for subclades Ia, Ib, and Ic of P. galeata, but to a lesser extent in the network based on the longer sequences. Unique haplotypes occurred for each subclade. The occurrence of shared haplotypes of the two lineages or the individual subclades of Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 8/21 8 2 5 5 5 114 32 40 2 4 4 3 4 4 8 5 9 3 2 83 P. subrufa P. galeata II P. galeata Ia P. galeata Ib P. galeata Ic mtDNA (n = 105) 1602 bp 8 2 5 5 5 114 32 40 2 4 4 3 4 4 8 5 9 3 2 83 P. subrufa P. galeata II P. galeata Ia P. galeata Ib P. galeata Ic mtDNA (n = 105) 1602 bp Figure 3 Parsimony network for mitochondrial haplotypes of Pelomedusa galeata and P. subrufa. Symbol size corresponds to haplotype frequency; haplotypes colored according to mitochondrial lineages (Figs. 1 and 2). Lines connecting haplotypes represent one mutational step except when otherwise indi- cated by numbers. Missing node haplotypes, small black circles. Full-size DOI: 10.7717/peerj.4901/fig-3 P. galeata showed no correlation with geography (Fig. Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 Climatic niche models The models for current climatic conditions revealed distinct areas of climatically suitable space for the two Pelomedusa species (Fig. 5), with some overlap in north-eastern South Africa. The discrimination capability, expressed as AUCtest scores, of both models was high (P. subrufa = 0.79, P. galeata = 0.73), indicating a good discrimination of suitable and unsuitable areas. Across all 100 replicates, contributions of variables beyond 20% showed both models to be strongly affected by temperature and precipitation during the driest month and quarter (Table 2: Bio 9, Bio 17). However, the model for the widespread P. subrufa was also influenced by temperature during the wettest quarter (Bio 8 > 10%), Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 9/21 5 3 4 2 P. galeata subclade Ia P. galeata subclade Ib P. galeata subclade Ic P. galeata clade II (A) ODC (n = 82) 739 bp 6 3 4 3 3 P. subrufa 4 3 6 3 4 4 4 (B) R35 (n = 86) 1101 bp (C) R35 (n = 106) 699 bp Figure 4 Parsimony networks for nuclear haplotypes of Pelomedusa galeata and P. subrufa (A–C). Symbol size corresponds to haplotype frequency; haplotypes colored according to mitochondrial lineages (Figs. 1 and 2). Lines connecting haplotypes represent one mutational step except when otherwise indi- cated by numbers. Missing node haplotypes, small black circles. For geographic distribution of shared haplotypes, see Fig. S3. Full-size DOI: 10.7717/peerj.4901/fig-4 (B) R35 (n = 86) 1101 bp (A) ODC (n = 82) 739 bp Figure 4 Parsimony networks for nuclear haplotypes of Pelomedusa galeata and P. subrufa (A–C). Symbol size corresponds to haplotype frequency; haplotypes colored according to mitochondrial lineages (Figs. 1 and 2). Lines connecting haplotypes represent one mutational step except when otherwise indi- cated by numbers. Missing node haplotypes, small black circles. For geographic distribution of shared haplotypes, see Fig. S3. Full-size DOI: 10.7717/peerj.4901/fig-4 Figure 4 Parsimony networks for nuclear haplotypes of Pelomedusa galeata and P. subrufa (A–C). Symbol size corresponds to haplotype frequency; haplotypes colored according to mitochondrial lineages (Figs. 1 and 2). Lines connecting haplotypes represent one mutational step except when otherwise indi- cated by numbers. Missing node haplotypes, small black circles. For geographic distribution of shared haplotypes, see Fig. S3. Full-size DOI: 10.7717/peerj.4901/fig-4 whilst P. Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 Climatic niche models galeata was revealed to be strongly dependent on temperature annual range (Bio 7 >20%) and precipitation during the warmest quarter (Bio 18 > 10%), two variables less important for P. subrufa. Potentially suitable areas for the subclades of P. galeata were highly distinct (Fig. 5) and described by different predictors. For subclade Ia, inhabiting a highland summer-rainfall area, temperature annual range and mean temperature of the wettest and driest quarter contributed the most (Bio 7 and Bio 8 > 20%, Bio 9∼20%). However, the model was also impacted by precipitation during the driest month (Bio 17 > 10%). For subclade Ib, inhabiting a subtropical low elevation summer-rainfall area, the model was exclusively shaped by temperature-related variables (Bio 7∼40%; Bio 3 and Bio 9 >20%), whilst precipitation-related predictors contributed less than 5%. For subclade Ic, inhabiting the southern coast and adjacent inland regions characterized by aseasonal whilst P. galeata was revealed to be strongly dependent on temperature annual range (Bio 7 >20%) and precipitation during the warmest quarter (Bio 18 > 10%), two variables less important for P. subrufa. Potentially suitable areas for the subclades of P. galeata were highly distinct (Fig. 5) and described by different predictors. For subclade Ia, inhabiting a highland summer-rainfall area, temperature annual range and mean temperature of the wettest and driest quarter contributed the most (Bio 7 and Bio 8 > 20%, Bio 9∼20%). However, the model was also impacted by precipitation during the driest month (Bio 17 > 10%). For subclade Ib, inhabiting a subtropical low elevation summer-rainfall area, the model was exclusively shaped by temperature-related variables (Bio 7∼40%; Bio 3 and Bio 9 >20%), whilst precipitation-related predictors contributed less than 5%. For subclade Ic, inhabiting the southern coast and adjacent inland regions characterized by aseasonal Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 10/21 ! ! !! !!!! !! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !!!! ! ! ! ! ! ! !!! !! ! ! ! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! ! ! !! ! ! ! !!!! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 Climatic niche models 1000 km 500 km 250 km 500 km 500 km (A) Pelomedusa galeata (B) P. subrufa (D) P. galeata subclade Ib (E) P. galeata subclade Ic (F) P. galeata clade II (C) P. galeata subclade Ia ! ! ! ! ! ! !! ! ! !! !! ! !! own record VertNet record ! ! ! ! Current Figure 5 Climatic niche models for Pelomedusa galeata (A), P. subrufa (B) and genetic clusters within P. galeata (C–F) under current climatic conditions. Probabilities for habitat suitability range from low (blue) to high (orange). Full-size DOI: 10.7717/peerj.4901/fig-5 ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! 1000 km (B) P. subrufa ! ! !! ! ! !! !! ! ! ! !!! !! ! ! ! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! ! ! !! ! ! ! !!!! !! ! ! (A) Pelomedusa galeata ! ! ! ! ! !! own record VertNet record Current ! ! ! !! ! 500 km (D) P. galeata subclade Ib ! ! !! !!!! !! ! ! ! 500 km (C) P. galeata subclade Ia (D) P. galeata subclade Ib 500 km ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! 500 km (E) P. galeata subclade Ic ! (E) P. galeata subclade Ic ! ! ! !!!! ! ! ! 250 km (F) P. galeata clade II ! ! ! Figure 5 Climatic niche models for Pelomedusa galeata (A), P. subrufa (B) and genetic clusters within P. galeata (C–F) under current climatic conditions. Probabilities for habitat suitability range from low (blue) to high (orange). Full-size DOI: 10.7717/peerj.4901/fig-5 rainfall, variable contribution was highest for precipitation during the driest month (Bio 17 = 36%), precipitation seasonality, and temperature during the wettest quarter (Bio 15, Bio 8 > 10%). For subclade II from the western winter-rainfall area of South Africa, precipitation of the wettest quarter was the variable with the highest importance (Bio 16∼77%), whilst no other predictor exceeded 10%. All models for the subclades of P. galeata received excellent Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 11/21 Table 2 Contribution of selected bioclimatic predictor variables to current predictions as derived from MAXENT. Values exceeding 10% in bold. ID Variable contribution % current P. Climatic niche models subrufa P. galeata Ia Ib Ic II ;Bio 3 7.6 1.8 1.8 15.0 9.7 2.4 ;Bio 5 9.2 9.6 0.7 5.9 0.6 6.7 ;Bio 7 7.5 21.4 24.5 39.2 9.4 2.0 ;Bio 8 10.4 7.9 25.1 9.5 14.3 1.5 ;Bio 9 24.9 20.6 19.6 17.5 4.9 3.7 ;Bio 15 2.2 1.6 2.9 4.9 16.3 5.2 ;Bio 16 9.5 8.0 9.4 2.8 5.8 77.4 ;Bio 17 21.4 19.1 10.6 4.6 36.6 1.0 ;Bio 18 7.3 10.0 5.4 0.6 2.4 0.1 AUCtest scores (Ia = 0.81, Ib = 0.75, Ic = 0.78, II = 0.93) and suggest mutually exclusive climatic niches. Table 2 Contribution of selected bioclimatic predictor variables to current predictions as derived from MAXENT. Values exceeding 10% in bold. Table 2 Contribution of selected bioclimatic predictor variables to current predictions as derived from MAXENT. Values exceeding 10% in bold. AUCtest scores (Ia = 0.81, Ib = 0.75, Ic = 0.78, II = 0.93) and suggest mutually exclusive climatic niches. Projections onto past climatic conditions found similar patterns across all three scenarios (Figs. 6 and 7, Figs. S4–S7) with high discrimination abilities (MIROC-ESM: P. subrufa = 0.78, P. galeata = 0.73, Ia = 0.84, Ib = 0.77, Ic = 0.78, II = 0.95; CCSM4: P. subrufa = 0.73, P. galeata = 0.80, Ia = 0.86, Ib = 0.77, Ic = 0.80, II = 0.91; MPI-ESM-P: P. subrufa = 0.71, P. galeata = 0.80, Ia = 0.85, Ib = 0.81, Ic = 0.80, II = 0.91). (Figs. 6 and 7, Figs. S4–S7) with high discrimination abilities (MIROC-ESM: P. subrufa = 0.78, P. galeata = 0.73, Ia = 0.84, Ib = 0.77, Ic = 0.78, II = 0.95; CCSM4: P. subrufa = 0.73, P. galeata = 0.80, Ia = 0.86, Ib = 0.77, Ic = 0.80, II = 0.91; MPI-ESM-P: P. subrufa = 0.71, P. galeata = 0.80, Ia = 0.85, Ib = 0.81, Ic = 0.80, II = 0.91). Projections onto climatic conditions of the LGM revealed for P. subrufa a suitable area resembling the present situation, whilst the space for P. galeata shifted considerably, excluding a large area in the center of the extant distribution range. When the models for subclades Ia-Ic and lineage II of P. galeata were inspected individually, the models for subclade Ic and lineage II matched well with extant conditions. Climatic niche models However, the models for subclades Ia and Ib shifted to the northeast, outside the current niche, but remained geographically mutually exclusive (Figs. 6 and 7). All projections onto LGM conditions yielded high AUCtest scores (MIROC-ESM: P. subrufa = 0.80, P. galeata = 0.71, Ia = 0.85, Ib = 0.77, Ic = 0.76, II = 0.91; CCSM4: P. subrufa = 0.77, P. galeata = 0.73, Ia = 0.83, Ib = 0.77, Ic = 0.78, II = 0.94; MPI-ESM-P: P. subrufa = 0.79, P. galeata = 0.72, Ia = 0.85, Ib = 0.77, Ic = 0.76, II = 0.94). Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 DISCUSSION According to our present investigation, Pelomedusa galeata and P. subrufa sensu stricto, as well as the genetic lineages and subclades within P. galeata, occur parapatrically. In South Africa, P. subrufa is restricted to the northeast of the country, but with a wider distribution ranging approximately 350 km further westward than previously known (Fritz et al., 2015). All known South African records for P. subrufa lie in Mpumalanga and Limpopo. Our new records from the Lapalala Wilderness Area (Limpopo) represent the westernmost localities in South Africa. These sites are only 80 km north of the nearest record of P. galeata (subclade Ia) at Mosdene Farm, Mookgopong (Limpopo). Thus, a sympatric occurrence of the two species seems possible, as first suggested by Vargas-Ramírez, Petzold & Fritz (2016) using a Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 12/21 ! ! !! !!!! !! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !!!! ! ! ! ! ! ! !!! !! ! ! ! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! ! ! !! ! ! ! !!!! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! !! ! ! ! 1000 km 500 km 250 km 500 km 500 km (A) Pelomedusa galeata (B) P. subrufa (D) P. galeata subclade Ib (E) P. galeata subclade Ic (F) P. galeata clade II (C) P. galeata subclade Ia ! !! own record VertNet record ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Mid-Holocene MIROC-ESM Figure 6 Predicted climatic niches for Pelomedusa galeata (A), P. subrufa (B) and genetic clusters within P. galeata (C–F) during the mid-Holocene (MIROC-ESM). Probabilities for habitat suitability range from low (blue) to high (orange). For CCSM4 and MPI-ESM-P models, see Figs. S4–S7. Full-size DOI: 10.7717/peerj.4901/fig-6 species distribution modeling approach and corroborated by the modeling results of the present study. Our results indicate an even wider area of potential overlap, embracing the ! ! ! !!! !! ! ! ! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! ! ! !! ! ! ! !!!! !! ! ! (A) Pelomedusa galeata ! !! Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 DISCUSSION own record VertNet record ! ! ! ! Mid-Holocene MIROC-ESM ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! !! ! ! ! 1000 km (B) P. subrufa ! ! ! ! !! ! ! ! ! ! ! !! ! 500 km (D) P. galeata subclade Ib ! ! !! !!!! !! ! ! ! 500 km (C) P. galeata subclade Ia (C) P. galeata subclade Ia (D) P. galeata subclade Ib 500 km ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! 500 km (E) P. galeata subclade Ic ! ! ! ! !!!! ! ! ! 250 km (F) P. galeata clade II ! ! ! Figure 6 Predicted climatic niches for Pelomedusa galeata (A), P. subrufa (B) and genetic clusters within P. galeata (C–F) during the mid-Holocene (MIROC-ESM). Probabilities for habitat suitability range from low (blue) to high (orange). For CCSM4 and MPI-ESM-P models, see Figs. S4–S7. Full-size DOI: 10.7717/peerj.4901/fig-6 species distribution modeling approach and corroborated by the modeling results of the present study. Our results indicate an even wider area of potential overlap, embracing the central and northeastern parts of South Africa (Fig. 5). Our study confirms that the taxon currently identified as P. galeata is composed of two deeply divergent genetic lineages (I and II; Figs. 1 and 3), as earlier revealed by Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 13/21 ! ! !! !!!! !! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !!!! ! ! ! ! ! !! !!!! !! ! ! ! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! ! ! !! ! ! ! !!!! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! !! ! ! ! 500 km 250 km 500 km 500 km (A) Pelomedusa galeata (B) P. subrufa (D) P. galeata subclade Ib (E) P. galeata subclade Ic (F) P. galeata clade II (C) P. galeata subclade Ia ! !! own record VertNet record ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! DISCUSSION 1000 km LGM MIROC-ESM Figure 7 Predicted climatic niches for Pelomedusa galeata (A), P. subrufa (B) and genetic clusters within P. galeata (C–F) during the Last Glacial Maximum (MIROC-ESM). Probabilities for habitat suit- ability range from low (blue) to high (orange). For CCSM4 and MPI-ESM-P models, see Figs. S4–S7. Full-size DOI: 10.7717/peerj.4901/fig-7 Petzold et al. (2014) using mtDNA data. In addition, we found that lineage I is differentiated in three weakly supported subclades (Ia-Ic). Our nuclear DNA data also confirmed the distinctness of lineages I and II (Fig. 4). We found lineage II restricted to westernmost ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! !! ! ! ! (B) P. subrufa ! ! ! ! !! ! ! ! 1000 km ! ! !! !!!! !! ! ! ! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! ! ! !! ! ! ! !!!! !! ! ! (A) Pelomedusa galeata ! !! own record VertNet record ! ! ! ! LGM MIROC-ESM own record VertNet record ! ! ! !! ! 500 km (D) P. galeata subclade Ib ! ! !! !!!! !! ! ! ! 500 km (C) P. galeata subclade Ia (D) P. galeata subclade Ib 500 km ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! 500 km (E) P. galeata subclade Ic ! ! ! ! !!!! ! ! ! 250 km (F) P. galeata clade II ! ! ! Figure 7 Predicted climatic niches for Pelomedusa galeata (A), P. subrufa (B) and genetic clusters within P. galeata (C–F) during the Last Glacial Maximum (MIROC-ESM). Probabilities for habitat suit- ability range from low (blue) to high (orange). For CCSM4 and MPI-ESM-P models, see Figs. S4–S7. Full-size DOI: 10.7717/peerj.4901/fig-7 Petzold et al. (2014) using mtDNA data. In addition, we found that lineage I is differentiated in three weakly supported subclades (Ia-Ic). Our nuclear DNA data also confirmed the distinctness of lineages I and II (Fig. 4). We found lineage II restricted to westernmost South Africa, with several new records in the southwestern part of the Western Cape (Fig. 2; Table S1). Our climatic niche modeling supports that lineage II occurs only Petzold et al. (2014) using mtDNA data. Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 DISCUSSION In addition, we found that lineage I is differentiated in three weakly supported subclades (Ia-Ic). Our nuclear DNA data also confirmed the distinctness of lineages I and II (Fig. 4). We found lineage II restricted to westernmost South Africa, with several new records in the southwestern part of the Western Cape (Fig. 2; Table S1). Our climatic niche modeling supports that lineage II occurs only Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 14/21 in the winter-rainfall region of South Africa. Besides the localities in the southwestern Western Cape, there is a single record in the Northern Cape. It corresponds to the collection site of the historical type specimen of Pelomedusa galeata devilliersi (Hewitt, 1935), a taxon considered to be synonymous with P. galeata (Fritz et al., 2014; Petzold et al., 2014). However, the mtDNA sequences of the type specimen (Fritz et al., 2014) differ from those of the southwestern terrapins. Moreover, the collection site of the type lies far beyond the predicted range of lineage II (Fig. 5). This situation requires further research, including fieldwork around the type locality of P. g. devilliersi, to confirm the occurrence of Pelomedusa there and to obtain fresh material. Pelomedusa is generally rare in northwestern South Africa, with a large distributional gap between the western and central localities (Fig. 2). Western South Africa displays a south to north aridity gradient, which may play a role for the genetic differentiation of several other reptile species, for example, Chersina angulata (Daniels et al., 2007), Trachylepis sulcata (Portik, Bauer & Jackman, 2011), and Bitis arietans (Barlow et al., 2013). The phylogeographic pattern of P. galeata roughly parallels that of the puff adder B. arietans, as described in Barlow et al. (2013). The westerly clade of B. arietans corresponds to lineage II of P. galeata, the southern-to-eastern coastal clade to subclade Ic, the northeastern subclade to subclade Ib, and the northwestern subclade to subclade Ia. Similar to our conclusions for Pelomedusa (see below), Barlow et al. (2013) ascribed the phylogeographic pattern of B. arietans to climatic oscillations during the Plio-Pleistocene, with populations retracting to coastal and northern refugia when interior regions became inhospitable during glacial maxima. These authors proposed that low temperatures in the interior rather than aridity made the habitat inhospitable. It is noteworthy for Pelomedusa, but also for clawed frogs (Xenopus laevis) living in similar habitats (Furman et al., 2015) and for B. Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 DISCUSSION arietans (Barlow et al., 2013) living in very different habitats, that phylogeographic breaks coincide in South Africa with the Great Escarpment, i.e., with lineages confined either to lowland or highland. This suggests that physiographic structures shaped in concert with climatic factors the current phylogeographic structures of South African biota. Furthermore, the phylogeographic similarities of very different taxa imply that general paradigms exist, like in the Western Palearctic or the Nearctic (Hewitt, 2000; Hewitt, 2011; Schmitt, 2007; Schmitt & Varga, 2012; Riddle, 2016). However, the understanding of the phylogeography of sub-Saharan biota is distinctly less advanced than for the Western Palearctic and the Nearctic (Hewitt, 2000; Hewitt, 2011; Schmitt, 2007), even in comparatively well-researched countries like South Africa and Namibia. For unveiling general differentiation patterns and understanding their causes, the description of species- specific phylogeographic patterns is the necessary prerequisite. This study contributes to this ultimate goal and presents basic phylogeographic data for two wide-ranging terrapin species from southern Africa. As expected for thermophilic semiaquatic species like terrapins, our niche modeling confirms that the phylogeographic structure of Pelomedusa has been significantly impacted by temperature and aridity. Yet, P. galeata has been much more affected by climatic fluctuations since the LGM than P. subrufa. This difference could explain the absence of phylogeographic structuring in P. subrufa (Fig. 1 and Fig. S1). Moreover, our models Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 suggested for each of the four genetic clusters within P. galeata a distinct climatic niche (Fig. 5: subclades Ia-Ic, lineage II). Potentially suitable spaces for subclades Ia and Ib shifted northeastward during the LGM and moved southwestward in the Holocene (Figs. 5–7 and Figs. S4–S7). Since climatic instability and repeated shifts of grassland and semidesert biomes have been inferred for the past 140,000 years for the concerned regions (Huntley et al., 2016), it is likely that part of the range of P. galeata was highly dynamic for a long time, a situation contributing to phylogeographic divergence. In contrast, our models propose that the distributions of P. subrufa and of subclade Ic and lineage II of P. galeata remained largely stable since the LGM. The present study also has taxonomic and conservational implications. The genetic distances between clade I and clade II of P. DISCUSSION galeata (Table 1), with 2.2% mean divergence for the 12S gene and 7.1% for the cyt b gene, resemble divergences between currently recognized Pelomedusa species (2.6% to 12.2% for 12S, 5.6% to 18.6% for cyt b; Petzold et al., 2014). This supports the view that the genetic lineages represented by clade I and clade II should be treated as unconfirmed candidate species (Fritz et al., 2015), a category introduced by Padial et al. (2010) for groups of individuals within formally recognized species showing large genetic distances, but without further information. Unconfirmed candidate species deserve further study and additional characters may qualify them for the description as new species. Our present investigation revealed that lineages I and II also differ in one of the two nuclear markers (R35) to an extent resembling their divergence to P. subrufa sensu stricto. This supports that the two lineages represent distinct species and that a full taxonomic revision should be performed, involving morphological characters and resolving the nomenclatural issues described by Petzold et al. (2014). Within that work, sampling the putative contact zone between the westernmost records of lineage I and the easternmost records of lineage II will be mandatory. The two lineages are separated by a sampling gap of circa 220 km, corresponding to the region between the farms Chelance and Groenfontein near Worcester and Calitzdorp (Western Cape), respectively. The study of this contact zone and of other putative contact zones will provide insights in gene flow, possible hybridization and ongoing differentiation processes. Today, there is a broad array of mitochondrial and highly informative nuclear markers available, including SNPs, which are powerful tools for unravelling phylogeographic patterns and for understanding speciation processes beyond the description of the distribution of genetic lineages. Finally, the relatively small region revealed as potentially suitable for lineage II (Fig. 5) implies that the conservation of this candidate species requires more attention because surface water is scarce in this region. Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 CONCLUSIONS Nuclear markers confirm the distinctness of Pelomedusa galeata and P. subrufa sensu stricto. Pelomedusa galeata comprises two genetically deeply divergent lineages (I and II) that differ in mitochondrial and nuclear DNA. Lineage I shows considerable phylogeographic structure, with three distinct mitochondrial clades (Ia-Ic). Pelomedusa galeata and P. subrufa, as well as the four genetic clusters within P. galeata (Ia-Ic, II), seem to be Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 16/21 distributed parapatrically, each occupying a distinct climatic niche. However, some niche overlap was found for P. galeata and P. subrufa. One lineage of P. galeata (II) appears to be largely confined to a small region in southwestern South Africa and deserves more conservational attention due to the scarcity of surface water there. Further studies should focus on putative contact zones of the two species and of the genetic clusters within P. galeata and use informative nuclear markers to examine gene flow and hybridization. In South Africa, records of P. subrufa are restricted to the northeast of the country (Mpumalanga, Limpopo). The species ranges approximately 350 km further westward than previously known and a sympatric occurrence with P. galeata is possible. Unlike P. galeata, no phylogeographic structure was found for P. subrufa, which seems to be related to historically stable versus fluctuating distribution ranges. Our investigation is one of the rare studies describing the phylogeography of sub-Saharan biota, by thus laying the foundation for unravelling general phylogeographic patterns. Such paradigms are likely to exist, but the lack of individual case studies impedes their identification. According to the little information available, it seems likely that physiographic barriers (e.g., the Great Escarpment) generally correlate with phylogeographic breaklines and contributed together with climatic factors to the establishment of phylogeographic structuring. Funding Flora Ihlow was supported by a Margarethe Koenig Fellowship of the Zoological Research Museum Alexander Koenig, Bonn, Germany. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ACKNOWLEDGEMENTS Genetic investigations were conducted in the Senckenberg Dresden laboratory (SGN- SNSD-Mol-Lab). The authors thank Anja Rauh and Julia Pöschel for help with laboratory work and for management of samples. Special thanks go to our colleagues and friends Theunis Hofmeyr, Julia Pöschel, Anamarija Žagar, Marlene and Heinz Hinze from Nederlands Farm; Riaan and Corne Pohl and their children from Mosdene Farm; Annemieke and Hermann Müller from Lapalala Wilderness Area; Adrian Armstrong (Ezemvelo KwaZulu-Natal), Werner Conradie (Port Elizabeth Museum), and Krystal Tolley (SANBI) who supported us logistically during fieldwork and for donating samples. We thank the Western, Eastern and Northern Cape Nature, Limpopo Provincial Government and Ezemvelo KwaZulu-Natal Wildlife Permits Office for permits to collect biological material, and private landowners for allowing sampling on their properties. Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 Grant Disclosures The following grant information was disclosed by the authors: The following grant information was disclosed by the authors: Margarethe Koenig Fellowship of the Zoological Research Museum Alexander Koenig, Bonn, Germany. Margarethe Koenig Fellowship of the Zoological Research Museum Alexander Koenig, Bonn, Germany. 17/21 Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 Competing Interests Competing Interests The authors declare there are no competing interests. Animal Ethics The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): Terrapins were hand-collected or captured using baited traps, and blood and saliva samples were taken as approved by the Ethics Committee of the University of the Western Cape under ethical clearance number ScRiRC2008/39. Field Study Permissions Field Study Permissions The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers): Fieldwork and sampling in South Africa were permitted by Limpopo Provincial Government (permit ZA/Lp/80202), Ezemvelo KwaZulu-Natal Wildlife (permit OP 139/2017), CapeNature (permit AAA007-00212-0056), Department of Environmental Affairs, Eastern Cape (permit CRO117/13C & CRO 118/13CR), and Biodiversity Northern Cape Province (permit 245/2015). Fieldwork and sampling in Namibia were permitted by the Ministry of Environment and Tourism (permit 1910/2014). Author Contributions • Melita Vamberger and Flora Ihlow conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. • Margaretha D. Hofmeyr conceived and designed the experiments, authored or reviewed drafts of the paper, approved the final draft. • Uwe Fritz conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. • Uwe Fritz conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 DNA Deposition The following information was supplied regarding the deposition of DNA sequences: Sequences have been deposited at the European Nucleotide Archive (ENA) and accession numbers (LT992039–LT992252) are included in the Supplemental Information. Supplemental Information Vamberger et al. (2018), PeerJ, DOI 10.7717/peerj.4901 DOI 10.1093/molbev/msw054. 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https://openalex.org/W2795689009	https://kclpure.kcl.ac.uk/portal/files/93214481/Support_for_overweight_and_BICK_Publishedonline14March2018_GREEN_AAM.pdf	English	null	Abstracts	Maternal and child nutrition	2,018	public-domain	1,275	Citation for published version (APA): Bick, D., Glaria, A. A., Davie, P., & Chang, Y.-S. (2018). Support for overweight and obese breastfeeding women: A systematic review. In Maternal and Child Nutrition (Vol. 14 Supplement 2, pp. 9-10) https://doi.org/10.1111/mcn.12587 Citing this paper Pl h C t g t s pape Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections. Citation for published version (APA): Bick, D., Glaria, A. A., Davie, P., & Chang, Y.-S. (2018). Support for overweight and obese breastfeeding women: A systematic review. In Maternal and Child Nutrition (Vol. 14 Supplement 2, pp. 9-10) https://doi.org/10.1111/mcn.12587 Support for Overweight and Obese Breastfeeding Women: A Systematic Review Support for Overweight and Obese Breastfeeding Women: A Systematic Review Debra Bick¹ Amaia Artazcoz Glaria² Philippa Davie³ Yan-Shing Chang*¹ yan-shing.chang@kcl.ac.uk ¹ King’s College London, Florence Nightingale Faculty of Nursing and Midwifery, UK ² Médecins Sans Frontières, Nigeria ¹ King’s College London, Florence Nightingale Faculty of Nursing and Midwifery, UK ³ King’s College London, Institute of Psychiatry, Psychology & Neuroscience, UK The global epidemic of overweight and obesity constitutes a major public health problem, including among women of reproductive age and their infants. The World Health Organisation (WHO) target that at least 50% of all women breastfeed their infants exclusively during the first 6 months of life by 2025 (WHO, 2013) is likely to be threatened by the increased prevalence of obesity among women who give birth. Women who are overweight (Body Mass Index (BMI) ≥ 25) or obese (BMI ≥ 30) have poorer breastfeeding outcomes than their normal BMI counterparts, including lower levels of breastfeeding intention, initiation and early cessation (Babendure et al, 2015). Evidence of how to counter this situation is lacking. This systematic review aimed to identify (1) the perceptions and experiences of breastfeeding among women who were overweight and obese, (2) the type, content and effectiveness of support/interventions offered to breastfeeding women with higher BMI by healthcare professionals, peer supporters and family members, and (3) the perceptions and training needs of those who supported overweight and obese women to commence and continue to breastfeed. A systematic search of Medline, Maternity and Infant Care, CINAHL, SCOPUS, PsycInfo, Web of Science and Cochrane Library, grey literature and organisation websites was conducted. References from identified papers were hand searched. Studies published in English from January 1992 (following the launch of UNICEF’s Baby Friendly Initiative) to June 2016 from low, middle and high income countries were considered for inclusion. Three main search terms including obesity/overweight, breastfeeding and support, with subject MeSH terms were searched. The quality of included papers was assessed using a checklist designed for quantitative observational studies based on the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement (Barley et al, 2011) and appropriate Critical Appraisal Skills Programme checklists depending on methodology. Data were analysed using thematic analysis and narrative synthesis. Meta-analysis was not possible due to study heterogeneity. Eleven studies were included. 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Download date: 24. Oct. 2024 Please cite as: Bick, D., Glaria, A. A., Davie, P. and Chang, Y-S. (2018) Support for overweight and obese breastfeeding women: A systematic review. Maternal and Child Nutrition, 14 (Supplement 2), 9-10 . Please cite as: Bick, D., Glaria, A. A., Davie, P. and Chang, Y-S. (2018) Support for overweight and obese breastfeeding women: A systematic review. Maternal and Child Nutrition, 14 (Supplement 2), 9-10 . Support for Overweight and Obese Breastfeeding Women: A Systematic Review The problems faced by overweight/obese women fell into three categories: (1) physical difficulties presented by maternal physical weight when attempting to breastfeed including latching the baby onto the breast and finding a comfortable position. Delayed onset of lactogenesis, hormonal imbalance and mammary hypoplasia made breastfeeding more difficult; (2) psychosocial difficulties including body image and the negative impact on women’s self- esteem and confidence; and (3) additional difficulties faced by those providing support to these women especially because of the stigma attached to obesity. Healthcare professionals appeared to lack awareness or sympathy for women’s situations and failed to offer appropriate support. Some healthcare professionals appeared to resent having to provide additional support and time to help women, or seek extra resources, regarding them as unnecessary and unwarranted. There was a gap in the research on experiences and perspectives of partners and other relatives with only two small studies identified. Both highlighted that partners and relatives played an important role in helping women to breastfeed successfully and in their decisions to stop exclusive breastfeeding. No new studies on effectiveness of interventions were identified since the review by Babendure et al (2015). Influences on breastfeeding outcomes among overweight and obese women are multifactorial, with physical and psychosocial factors identified as barriers to successful breastfeeding. Research to address these as well as counter the negative body image these women also reported as a barrier to breastfeeding is urgently needed. Better clinical training and support is also key if national and international breastfeeding goals are to be achieved and negative impacts of obesity on women and their infants reduced. World Health Organization, (WHO) (2013) Essential nutrition actions: improving maternal, newborn, infant and young child health and nutrition. Geneva: WHO. References Babendure, J.B., Reifsnider, E., Mendias, E., Moramarco, M.W. and Davila, Y.R. (2015). Reduced breastfeeding rates among obese mothers: a review of contributing factors, clinical considerations and future directions. International breastfeeding journal, 10(1), p.1. Babendure, J.B., Reifsnider, E., Mendias, E., Moramarco, M.W. and Davila, Y.R. (2015). Reduced breastfeeding rates among obese mothers: a review of contributing factors, clinical considerations and future directions. International breastfeeding journal, 10(1), p.1. Barley, E. A., Murray, J., Walters, P., and Tylee, A. (2011). Managing depression in primary care: A meta‐synthesis of qualitative and quantitative research from the UK to identify barriers and facilitators. BMC Family Practice, 12(47). World Health Organization, (WHO) (2013) Essential nutrition actions: improving maternal, newborn, infant and young child health and nutrition. Geneva: WHO.
https://openalex.org/W4250494507	https://www.repository.cam.ac.uk/bitstreams/b345937c-aee3-41ef-b5b1-44a69cd00814/download	English	null	Single-Site Binding of Pyrene to Poly(ester-Imide)s Incorporating Long Spacer Units: Prediction of NMR Resonance-Patterns from a Fractal Model	null	2,020	cc-by	10,260	Single-site binding of pyrene to poly(ester-imide)s incorporating long spacer-units: prediction of NMR resonance-patterns from a fractal model† Marcus Knappert,a Tianqi Jin,a Scott D. Midgley,a Guanglu Wu, b Oren A. Scherman, b Ricardo Grau-Crespo a and Howard M. Colquhoun a Cite this: Chem. Sci., 2020, 11, 12165 All publication charges for this article have been paid for by the Royal Society of Chemistry Cite this: Chem. Sci., 2020, 11, 12165 All publication charges for this article have been paid for by the Royal Society of Chemistry Marcus Knappert,a Tianqi Jin,a Scott D. Midgley,a Guanglu Wu, b Oren A. Scherman, b Ricardo Grau-Crespo a and Howard M. Colquhoun Co-polycondensation of the diimide-based diols N,N0-bis(2-hydroxyethyl)hexaﬂuoroisopropylidene- diphthalimide, (HFDI), and N,N0-bis(2-hydroxy-ethyl)naphthalene-1,4,5,8-tetracarboxylic-diimide, (NDI), with aliphatic diacyl chlorides ClOC(CH2)xCOCl (x ¼ 5 to 8) aﬀords linear copoly(ester-imide)s. Such copolymers interact with pyrene via supramolecular binding of the polycyclic aromatic at NDI residues. This interaction results in upﬁeld complexation shifts and sequence-related splittings of the NDI 1H NMR resonances, but gives a very diﬀerent ﬁnal resonance-pattern from the copolymer where x ¼ 2. Computational modelling of the polymer with x ¼ 5 suggests that each pyrene molecule binds to just a single NDI residue rather than by intercalation between a pair of NDI's at a tight chain-fold, as was found for x ¼ 2. The new single-site binding model enables the pattern of 1H NMR resonances for copolymers with longer spacers (x ¼ 5 to 8) to be reproduced and assigned by simulation from sequence-speciﬁc shielding factors based on a type of fractal known as the last-fraction Cantor set. As this type of fractal also enables an understanding of pairwise binding systems, it evidently provides a general numerical framework for supramolecular sequence-analysis in binary copolymers. Received 7th July 2020 Accepted 9th October 2020 DOI: 10.1039/d0sc03730c rsc.li/chemical-science Using the latter technique, we recently showed that highly sequence-dependent 1H NMR complexation shis are produced in the spectra of copolyimides based on 1,4,5,8-naphthalene tetracarboxylic diimide (NDI) on complexation of an aromatic “probe” molecule such as pyrene or perylene.15,16 This phenomenon results from cumulative ring-current shielding17,18 of the central residue in an NDI-centred sequence by probe- molecules binding through complementary p–p-stacking.19–21 Such shielding results not only from the probe-molecule binding directly at the central “observed” NDI residue but, additionally, from complexation to NDI residues at neighbour- ing (and next-neighbouring, and next–next-neighbouring etc.) positions, viewed in both directions from the centre of the sequence. As separate resonances corresponding to “bound” and “unbound” NDI residues are not observed at sub- stoichiometric levels of pyrene, the system is clearly operating in the fast-exchange regime. aDepartment of Chemistry, University of Reading, Whiteknights, Reading, RG6 6AD, UK. E-mail: h.m.colquhoun@rdg.ac.uk bMelville Laboratory for Polymer Synthesis, Department of Chemistry, University of Cambridge, Lenseld Road, Cambridge, CB2 1EW, UK † Electronic supplementary information (ESI) available: Details of polymer synthesis and characterisation; titration data for all copolymers; details of the tting procedure for determination of fractal parameters from 1H NMR titration of the “x ¼ 5” copolymer with pyrene; details of titration simulation. Atomic coordinates for modelled oligomer complexes with pyrene, in mol2 format. See DOI: 10.1039/d0sc03730c This journal is © The Royal Society of Chemistry 2020 aDepartment of Chemistry, University of Reading, Whiteknights, Reading, RG6 6AD, UK. E-mail: h.m.colquhoun@rdg.ac.uk 1. Introduction Thus, NDI residues at the centres of diﬀerent sequences are inherently distinct and give diﬀerent complexation shis in the presence of pyrene, regardless of exchange between bound and unbound states. It must be emphasised that, under the conditions of the experi- ment, there is no exchange of monomer residues between diﬀerent sequences, and so an averaging of NDI resonances over all sequences is impossible. The concept of a central “observed” residue in a given sequence is important, because it greatly simplies sequence-analysis in high molecular weight copolymers. Even though a given copolymer sequence may contain many “observable” monomer residues (in the present context, NDI), each of these is also at the centre of its own sequence, overlapping with the original sequence but still, in NMR terms, representing a specic, intramolecular environment. Consequently, the central residue can be treated separately from other chemically equivalent residues in the same sequence. In NDI-based copolymers, ring-current shielding resulting from complexation of an aromatic molecule such as pyrene amplies the diﬀerences between magnetic envi- ronments in a copolyimide chain and thus enables the assignment of specic NMR resonances to diﬀerent como- nomer sequences.15 Remarkably, the patterns of 1H NMR resonances originating from NDI residues in random, binary copolyimide chains, in the presence of pyrene, exhibit a noticeable degree of self-simi- larity,34,35 i.e. the spectra consist of multiple copies of them- selves over a range of diﬀerent length-scales.15,16 These resonance-patterns have been shown to reect an underlying fractal distribution34–37 of ring-current shieldings generated by pyrene molecules binding to all the diﬀerent sequences within which a “central” NDI residue is embedded.15,16 For copoly(ether-imide)s15 and copoly(ester-imide)s16 studied previously, it was found that tight chain-folding22–24 allowed the aromatic “reader-molecule” (generally pyrene) to bind strongly to the polymer chain by intercalation between adjacent NDI residues (Fig. 1).25–27 In this context, the chain-fold may be The mathematical fractal underpinning the pattern of complexation shis in such systems was identied15 as a “last fraction” Cantor set. Interestingly, this was the earliest class of fractal to be discovered (by Smith in 1875).38–41 An atomistic model for copolymer–pyrene complexation was developed in which ring-current shielding falls oﬀ, empirically, by a factor of four as pyrene binds to NDI pairs successively more distant from the central, “observed” NDI residue (Fig. 1).15,16 This “factor of four” in turn dened the set of all predicted complexation shis, mathematically, as the fourth-quarter Cantor set. 1. Introduction 1. The storage, copying and processing of information in biolog- ical systems is achieved, universally and with high precision at the molecular level, by a group of sequence-dened, high molecular-weight, linear copolymers (DNA and/or RNA, and proteins).1–3 In principle, however, any copolymer sequence can represent information, because even the simplest AB copolymer is the equivalent of a binary string.4,5 Indeed, some very signif- icant progress in devising a synthetic “information chemistry” has been made in recent years, notably with the development of sequence-specic polymerisation (i.e. sequence-writing) meth- odologies and mass-spectrometric sequencing techniques,6–8 information-transfer protocols,9–11 and the use of small “reader- molecules” to recognise and report copolymer sequence- information.12–15 Multiple NDI signals are seen at high pyrene concentrations, even under fast-exchange conditions, because each “observed” NDI residue is at the centre of a diﬀerent but specic copolymer sequence. This sequence may in principle be of any length, although spectroscopic resolution generally limits the maximum “observable” length to a quintet or septet. This “surrounding sequence” denes the molecular and supramo- lecular environment of the central, observed NDI residue, because the distribution pattern of other NDI residues in the sequence determines the number and locations of all pyrene Chem. Sci., 2020, 11, 12165–12177 \| 12165 This journal is © The Royal Society of Chemistry 2020 Edge Article Edge Article Chemical Science viewed as a “half-closed bis-diimide macrocycle”, related to the true NDI-based macrocycles reported by Sanders et al. as components of donor–acceptor catenanes with dialkox- ynapthalenes as the donor groups.28–30 Somewhat related rotaxane complexes, comprising 4,4-bipyridinium macrocycles threaded onto a high-MW, chain-folding poly(- dialkoxynaphthalene), have been reported by Hodge et al.,31 and discrete oligomer-analogues of these, showing complex dynamic behaviour in solution and unusual end-group disorder (leading to pseudo-polymeric crystal structures) in the solid state were more recently described by Stoddart and co- workers.32,33 However, the systems described in ref. 31–33 are homopolymers (or homo-oligomers) rather than copolymers, so there is no “sequence-information” present. Moreover, the molecule involved in binding to the polymer or oligomer is a bipyridinium-based macrocycle (“Blue Box”) that is threaded onto the chain. This macrocycle, unlike the probe molecule – pyrene – used in the present work, does not dissociate from the chain on the timescale of the NMR experiment so that fast bound/unbound exchange is not observed. molecules binding to that sequence. This journal is © The Royal Society of Chemistry 2020 1. Introduction A complete mathematical description of this set is given in Section 3.3 and in the ESI† but, as with all one- dimensional Cantor sets, it can be visualised graphically by the iteration of an operation on a line. In this instance we divide a line of unit length into four equal parts, discard the fourth quarter, and repeat the procedure on the three remaining segments. The full set is obtained only aer an innity of iter- ations, but the group of “remaining segments” converges rapidly (in the absence of magnication) to a visually unchanging pattern. The rst three iterations of the construc- tion are shown in Fig. 2. Fig. 1 Schematic of pyrene binding to a chain-folding copoly(ester- imide) sequence.16 Key: purple ¼ pyrene; blue ¼ “observed” protons of the central NDI residue (red); black ¼ other NDI residues; gold ¼ non- binding (HFDI) residues. The aromatic diimide residues are linked by aliphatic diester units, [–CH2CH2OCO(CH2)2COOCH2CH2–], repre- sented as thin black lines. Studies of homopoly(ester-imide)s containing p-electron- poor NDI residues linked by aliphatic diester units (polymers 1 to 8 in Chart 1) have shown that the strength of supramo- lecular binding is highly dependent on the length of the diester spacer, with a sharp maximum in binding energy for homo- polymer 2, where x ¼ 2.16 Computational modelling suggested that the short, “x ¼ 2” diester spacer-unit forms a tight chain- Fig. 1 Schematic of pyrene binding to a chain-folding copoly(ester- imide) sequence.16 Key: purple ¼ pyrene; blue ¼ “observed” protons of the central NDI residue (red); black ¼ other NDI residues; gold ¼ non- binding (HFDI) residues. The aromatic diimide residues are linked by aliphatic diester units, [–CH2CH2OCO(CH2)2COOCH2CH2–], repre- sented as thin black lines. This journal is © The Royal Society of Chemistry 2020 12166 \| Chem. Sci., 2020, 11, 12165–12177 Edge Article Chemical Science Chemical Science work we nd that signicant upeld complexation shis of the NDI resonances (up to 0.5 ppm) may still be observed for long- spacer homopolymers and copolymers if suﬃciently high concentrations of pyrene (up to 10 equivalents per NDI residue) are used. Fig. 2 Graphical construction of the fourth-quarter Cantor set, showing the ﬁrst three iterations. This construction involves dividing a line of unit length into four equal parts, discarding the fourth quarter, and repeating the procedure on the remaining three segments, through an inﬁnity of iterations. In the limit, the construction converges to a maximum of 0.666., but as shown above, the limit is approached after a relatively small number of iterations. Sets deﬁned by a last-fraction construction of this type, though commonly described as “Cantor” sets, were actually discovered by Smith,38 and can be constructed using any number $3 (integral or non-integral) as the base. 2.1 Materials and instrumentation Starting materials, monomers, solvents and analytical instru- mentation were as described in a previous publication.16 2.2 Computational methods fold between two NDI residues that is especially favourable for pyrene binding by intercalation, with close (van der Waals) contact between the complexed aromatic and the two adjacent NDIs.16 Pyrene–NDI complexation energies were obtained using the self-consistent-charge density functional tight-binding (SCC- DFTB) approach, as implemented within the DFTB+ code.42 Parameters for all atoms and pairs including elements C, H, N, O were taken from the “mio” parameter set of the Slater– Koster library.43 Dispersion corrections based on a Lennard- Jones potential were applied in all simulations.44 Simula- tions of 1H NMR spectra were carried out using the “peak table to spectrum” script within Mnova (version 14.1, Mestrelab Research). Experimentally, the pattern of complexation shis seen for the corresponding 1 : 1 copoly(ester-imide), 9, (x ¼ 2), showed a close similarity to the fractal-based pattern observed previ- ously for the intercalating pyrene complex of a chain-folding NDI–HFDI copoly(ether-imide),15 conrming the dual-site binding model (Fig. 1) for this poly(ester-imide).16 In the present work we have investigated the complexation of pyrene to NDI/HFDI co-poly(ester-imide)s with longer diester-spacers (Chart 1, copolymers 10 to 13; x ¼ 5 to 8). The long-spacer NDI-based homopolymers 5 to 8 show very much smaller complexation shis, Dd, than the tightly chain-folding homopolymer 2, where x ¼ 2.16 Nevertheless, in the present 1. Introduction However, the pyrene complexes of copoly- mers 10 to 13 (x ¼ 5 to 8) show a very diﬀerent pattern of NDI signals from that observed for the homologous copolymer 9 (x ¼ 2), for which intercalative, dual-site binding was estab- lished.16 The new pattern is consistent with the long-spacer copolymers adopting chain-folds in which single-site binding is preferred but, despite the very diﬀerent binding mode, analysis of 1H NMR data shows that the “last-fraction” type of Cantor set still provides a logical basis for interpreting the observed complexation shis. Edge Article diimide (0.869 g, 2.45 mmol, dried at 100 C for 24 h), N,N0-bis(2- hydroxyethyl)hexauoroisopropylidene-diphthalimide (1.326 g, 2.50 mmol, dried at 100 C for 24 h) and 1,7-heptanedioyl dichloride (0.999 g, 5.07 mmol) were combined at room temper- ature and the reaction mixture was then heated at 120 C for 2 h under a slow nitrogen purge. Aer cooling to room temperature, the product mixture was dissolved in dichloromethane/ hexauoroisopropanol (4 : 1, v/v, 30 mL) and the solution was added dropwise with stirring into methanol (400 mL). The precipitated copolymer 10 was ltered oﬀ, dried at 80 C for 24 h, puried by three reprecipitations from the above solvent mixture into methanol, and nally ltered oﬀand dried again at 80 C for 24 h. Yield: 1.63 g, 56%. result from the presence of a chain-fold geometry between adjacent NDI residues that is particularly favourable for dual- site binding of pyrene.16 The very much smaller NDI complex- ation shis seen for homopoly(ester-imide)s having more extended spacers (Dd z 0.2 ppm for x ¼ 5 to 8) suggest that, with these polymers, pyrene no longer interacts strongly with the NDI residues via intercalative, dual-site binding (Dd z 0.75 ppm for x ¼ 2). In such systems, the lower the pyrene–NDI association constant, Ka, the smaller is the observed NDI complexation shiat a given concentration of pyrene, as the equilibrium position for pyrene-binding is shied further towards the unbound state. Resonances for monomer residues (HFDI) with no binding aﬃnity for pyrene show zero complex- ation shis, even at high concentrations of probe-molecule (see Section 3.5). Inherent viscosity (hinh, 25 C, CHCl3/TFE 6 : 1, v/v): 0.61 dL g1. Mn (from viscosity/GPC calibration: ESI, p. S6†) 22 000. Tg (DSC): 91 C. FTIR nmax ATR (cm1): 1780 (imide nC¼O), 1708 (ester nC¼O), 1389 (imide nC–N), 1190 (vs., nC–F), 1163 (ester nC–O–C). 1H NMR (400 MHz, CDCl3/TFA 9 : 1, v/v) d 8.75 (s, 4He), 7.93 (d, J ¼ 8.0 Hz, 2Hq/r), 7.84 (s, 2Ho), 7.77 (d, J ¼ 8.1 Hz, 2Hq/r), 4.58–4.36 (m, 8Ha/b), 4.37–4.21 (m, 4Hl), 4.08–3.86 (m, 4Hk), 2.38–2.11 (m, 8Hh/u), 1.68–1.42 (m, 8Hi/v), 1.42–1.11 (m, 6 Hj/w) ppm. Edge Article 13C NMR (100 MHz, CDCl3/TFE 6 : 1, v/v) d 174.65 (Cg/t), 167.32 (Cm), 163.14 (Cc), 139.00 (Cn/s), 136.01 (Cq/r), 132.61 (Cp), 132.25 (Cn/s), 131.28 (Ce), 126.78 (Cd/f), 126.41 (Cd/f), 124.89 (Co), 123.81 (Cq/r), 61.50 (Ca/k), 39.51 (Cb), 37.23 (Cl), 33.73 (Ch), 33.66 (Cu), 28.24 (Ci/v), 24.09 (Cj/w) ppm. A possible alternative to intercalation (1 : 2 binding) is single-site (1 : 1) binding of pyrene to each NDI residue. It is widely recognised – notably in molecular biology – that “multivalent” binding (where a small molecule binds simulta- neously to two or more sites on a macromolecule) is a key factor in producing a high association constant.45 This would account immediately for the high complexation shis seen for poly(ester-imide)s 2 and 9 (Chart 1) where 1 : 2 (“divalent”) binding at a tightly chain-folded NDI pair has been established. Conversely the much smaller complexation shis seen for homologous polymers 5 to 8 and 10 to 13 (Chart 1), with longer spacer-lengths, would be rationalised in terms of a lower asso- ciation constant for 1 : 1 (“univalent”) binding. Of course there are other factors that can inuence the relative magnitudes of association constants, specically changes in solvation and pre- organisation, but the supramolecular systems under discussion here involve just a single type of small molecule (pyrene) binding to a series of homologous poly(ester-imide)s in a single type of solvent. Solvation and/or pre-organisation contributions to binding should therefore be relatively unchanged between the diﬀerent systems. Synthetic and characterisation details for the other copoly- mers reported in this work (11, 12 and 13: x ¼ 6, 7 and 8 respectively) are given in the ESI.† The new copolymers were found to be soluble in mixed solvents containing chlorocarbons (CHCl3 or CH2Cl2) and proton-donor solvents such as tri- uoroethanol or hexauoropropan-2-ol, but were insoluble in standard GPC solvents such as THF or DMF. Thus, GPC analyses were not accessible, but a molecular weight calibration based on GPC data (in THF) and viscosity data (in CHCl3/triuoroethanol, 6 : 1 v/v) for the more soluble HFDI-homopoly(ester-imides)16 enabled inherent viscosities to be converted to number-average molecular weights (see Section 2.3 above and ESI†). 2.3 Synthesis of copolymer 10 (x ¼ 5) 1-Chloronaphthalene (2.5 mL, distilled from calcium hydride), N,N0-bis(2-hydroxyethyl)-1,4,5,8-naphthalenetetracarboxylic ter-imide homopolymers and copolymers based on NDI and HFDI. is © The Royal Society of Chemistry 2020 Chem Sci 2020 11 12165 12177 \| 12167 Chart 1 Ester-imide homopolymers and copolymers based on NDI and HFDI. Chart 1 Ester-imide homopolymers and copolymers based on NDI and HFDI. This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 12165–12177 \| 12167 Chemical Science Edge Article Chemical Science Chemical Science binding the central “I” residue is now always an allowed binding site and there are always two further potential binding positions, in each direction, viewed from the centre (Fig. 4b). In the case of dual-site binding, pyrene is only bound when there are two adjacent NDI residues, so that the number and posi- tions of binding sites are more restricted for a sequence of any given length. For example, in the sequence FIIFI, discussed above in a dual-site binding context, there is one central I residue, one adjacent I residue and one next-adjacent I residue, thus generating (for a single-site binding model) the three-digit shielding code 111 (Fig. 4b). performed a series of single-point energy calculations, in which the pyrene was moved stepwise across the chain-fold at 0.3 ˚A steps. The energy of the system fell initially, until the pyrene and NDI residue were just in van der Waals contact, but rose as these components moved apart, and then fell again as the pyrene approached the next NDI along the chain. The double-well potential energy curve (Fig. 3A) shows that single-site binding is indeed strongly favoured. Full minimisation of a seven- residue polymer model with pyrene bound at the central chain-fold conrmed this result, leading to a nal structure (Fig. 3B) in which pyrene is located in van der Waals contact with just one NDI residue (centroid–centroid distance ¼ 3.38 ˚A), and at more than twice this distance (7.24 ˚A) from the NDI at the other side of the chain-fold. Fig. 4 Alternative assignments of shielding codes for the quintet sequence based on (a) dual-site “intercalative” binding of pyr- ene at an NDI pair, “II”, or (b) single-site binding at every NDI residue. 3. Results and discussion 3.1 Dual-site or single-site binding? The proposal that single-site binding would be preferred at longer spacer-lengths between NDI residues was tested computationally by constructing an idealised, symmetrical, ester-imide chain-fold with x ¼ 5 and inserting a pyrene mole- cule at one side of the fold, parallel to, but at sub-van der Waals distance (2.92 ˚A) from, the adjacent NDI residue. To illustrate the potential energy curve as a function of pyrene position, we The NDI-based homopoly(ester-imide)s 1–8 (x ¼ 1 to 8) show a marked dependence of pyrene-induced complexation shion the value of x, with the complexation shishowing a sharp maximum at x ¼ 2 (Dd ¼ 0.73 ppm on addition of two equiva- lents of pyrene per NDI residue). This maximum was shown to 12168 \| Chem. Sci., 2020, 11, 12165–12177 This journal is © The Royal Society of Chemistry 2020 This journal is © The Royal Society of Chemistry 2020 Chemical Science Edge Article Edge Article central I and is not considered in the product), and di,j is the so- called Kronecker delta, dened as: central I and is not considered in the product), and di,j is the so- called Kronecker delta, dened as: The shielding code is simply a number whose successive digits represent diminishing contributions to the total ring- current shielding experienced by a central, “observed” NDI residue in a comonomer sequence, on complexation by an aromatic molecule. If the “fall-oﬀfactor” between digits were 10, the codes would be conventional decimal numbers but in previous work,15,16 this factor was found, empirically, to be close to a value of 4, so that the codes can be viewed, at least approximately, as quaternary numbers. di;j ¼ 0 if isj 1 if i ¼ j : The expression above simply means that the degeneracy doubles for each “1” in the code, because there are two ways of achieving an occupancy of 1, as seen in Table 1. The relative intensities of the peaks are then given by: All possible I-centred quintet sequences are enumerated in Table 1, together with their corresponding shielding codes assigned on the basis of single-site binding to “I” residues. Resolution of such sequences in the 1H NMR spectrum of a binary I/F copolymer, by complexation of a shielding molecule such as pyrene, should thus aﬀord nine resonances. In the present work, quintets are the longest sequences for which separate 1H NMR resonances can be resolved, even at high concentrations of pyrene. I Imax ¼ U Umax ¼ 1 Q k 21d1;Nk (1) (1) Clearly, the maximum intensity always corresponds to the “central” peak (111 for 3-digit codes – see Table 1) where Nk ¼ 1 for all values of k, therefore the degeneracy U ¼ Umax ¼ 4 and the relative intensity is 1. For all other peaks, the relative intensity is halved for each code-digit diﬀerent from 1. For example, the peak with code 101 has a relative intensity of 1/2 because it has one digit diﬀerent from 1, whereas the peak with code 100 has a relative intensity of 1/4 because it has two digits diﬀerent from 1. The relative intensities of these resonances correspond to the probability of each sequence occurring in the copolymer, and in a random, 1 : 1 copolymer all sequences of a given length have the same probability. 3.3 Numerical analysis of single-site binding The new single-site binding model was then analysed numeri- cally in terms of its predictions for ring-current shielding of the central NDI (“I”) in diﬀerent sequences within a binary NDI- HFDI (HFDI ¼ “F”) copolymer. This analysis shows that I- centred quintet sequences with single-site binding of pyrene generate three-digit “shielding codes”, rather than the two-digit codes found for dual-site binding. This is because, in single-site Fig. 4 Alternative assignments of shielding codes for the quintet sequence based on (a) dual-site “intercalative” binding of pyr- ene at an NDI pair, “II”, or (b) single-site binding at every NDI residue. Fig. 3 (A) Computed single-point (relative) energies for a model complex of pyrene with homopolymer 5 (x ¼ 5) as pyrene is moved across a symmetrical chain fold; (B) energy-minimised model for the same complex. Locating pyrene at the mid-point of the fold, considered for x ¼ 5 in an earlier study,16 can now be seen to represent only a metastable situation. See ESI† for structure ﬁles. Fig. 3 (A) Computed single-point (relative) energies for a model complex of pyrene with homopolymer 5 (x ¼ 5) as pyrene is moved across a symmetrical chain fold; (B) energy-minimised model for the same complex. Locating pyrene at the mid-point of the fold, considered for x ¼ 5 in an earlier study,16 can now be seen to represent only a metastable situation. See ESI† for structure ﬁles. Fig. 3 (A) Computed single-point (relative) energies for a model complex of pyrene with homopolymer 5 (x ¼ 5) as pyrene is moved across a symmetrical chain fold; (B) energy-minimised model for the same complex. Locating pyrene at the mid-point of the fold, considered for x ¼ 5 in an earlier study,16 can now be seen to represent only a metastable situation. See ESI† for structure ﬁles. Chem. Sci., 2020, 11, 12165–12177 \| 12169 Chemical Science In the present work, integrals measured for a well-resolved group of “triplet level” NDI reso- nances (copolymer 13, spectrum S9, ESI page S13†) show rela- tive values of 1.00 : 1.99 : 0.99 and are thus indeed consistent with the intensities predicted for a random copolymer. The relative intensity of a resonance can therefore be predicted directly from the number of diﬀerent sequences giving rise to the same shielding code, i.e. to the degeneracy of that code. Table 1 thus predicts a spectrum, in the NDI region, consisting of nine lines with relative intensities 1 : 2 : 1 : 2 : 4 : 2 : 1 : 2 : 1. 3.4 The 1H NMR spectrum as a Cantor set In a high-MW copolyimide, each NDI (“I”) residue may be regarded as being at the centre of a sequence which (in dilute solution) denes its molecular environment. Thus, in NMR terms, we need consider only the resonance arising from the central NDI residue in any sequence: other NDI residues in that sequence are themselves at the centres of other sequences and so are treated separately. The degeneracy U of a code can be formally expressed as a function of the code digits Nk (each digit corresponding, in molecular terms, to the occupancy by NDI residues of sites k steps away from the centre) as: In such systems, complexation of pyrene occurs under fast- exchange conditions on the NMR timescale, so that any atom- istic model for the complex can only represent a dynamic, time- averaged structure. Nevertheless, the “chain-folding with intercalation and shielding” model described above (Fig. 1) led directly15 to an expression (eqn (2)) that sums the shielding eﬀects, diminishing by a factor b, of pyrene binding at succes- sively more distant NDI pairs, up to a maximum value of k that is a function of the sequence-length being considered. This summation also takes account of how many pyrene molecules (Nk ¼ 0, 1 or 2) are bound at each type of position in the sequence, viewed from the central NDI residue. For example, the quintet sequence FIIFI has one II-pair adjacent to and including the central I residue, and zero II-pairs at the next- adjacent positions. This sequence thus has only two possible values for Nk (1 and 0) and is assigned the “shielding code” 10. Every possible quintet sequence (see Fig. 6 for experimental reasons why quintet sequences are exemplied here) can simi- larly be assigned a two-digit code using only the digits 0, 1 and 2, representing input values for Nk in eqn (2). Finally, the sum of shieldings may be scaled by a factor a that depends on the molar ratio of pyrene to NDI and on the concentration of NDI residues. Edge Article This procedure involves dividing a line of unit length into four equal parts, discarding the fourth quarter, and repeating these operations on the remaining three segments, through an inﬁnity of iterations. The construction is equivalent to eqn (2) when a ¼ 1, b ¼ 4, Nk ¼ 0, 1 or 2, and kmax ¼ N. In construction (A) the dual-site binding model results in prediction of a widely-spaced six-line spectrum (see ref. 15 for experimental conﬁrmation of this pattern) whereas in construction (B) the single-site model predicts a narrowly-spaced nine-line spectrum. In both cases the possible quintet sequences generate only a sub-set (codes shown in red) of the complete Cantor set, but these subsets diﬀer markedly as a result of the change in binding model. Note: in this ﬁgure, shielding codes are read vertically downwards. In the present system, relative complexation shis of the predicted resonances could be calculated using the fourth- quarter Cantor set (eqn (2) and Fig. 2). However, eqn (2) emerged specically from a tightly chain-folded, dual-site binding model,15 and it was by no means certain that it would remain valid for a single-site binding system. Specically, there was no obvious reason why the factor b, representing the fall-oﬀ factor of ring-current shielding with the numerical position k (adjacent ¼ 1, next-adjacent ¼ 2, next–next adjacent ¼ 3 etc.) of a pyrene binding site relative to the observed NDI residue, should remain close to a value of four when the positions of binding sites further out along the chain from the central NDI residue (previously dened as two-NDI intercalation sites) are now dened just as single NDI residues. However, to allow direct comparison of the predictions from dual-site and single- site binding models, eqn (2) (with a ¼ 1 and b ¼ 4) was applied to both models, with the results for quintet sequences shown in Fig. 5. Here the graphical construction of the fourth quarter Cantor set15,16 allows a prediction of the NDI resonance-patterns for both binding models. is (i) a constant, independent of pyrene concentration, and (ii) close to 4. In Fig. Edge Article Fig. 5 Graphical construction of the fourth-quarter Cantor set. This procedure involves dividing a line of unit length into four equal parts, discarding the fourth quarter, and repeating these operations on the remaining three segments, through an inﬁnity of iterations. The construction is equivalent to eqn (2) when a ¼ 1, b ¼ 4, Nk ¼ 0, 1 or 2, and kmax ¼ N. In construction (A) the dual-site binding model results in prediction of a widely-spaced six-line spectrum (see ref. 15 for experimental conﬁrmation of this pattern) whereas in construction (B) the single-site model predicts a narrowly-spaced nine-line spectrum. In both cases the possible quintet sequences generate only a sub-set (codes shown in red) of the complete Cantor set, but these subsets diﬀer markedly as a result of the change in binding model. Note: in this ﬁgure, shielding codes are read vertically downwards. of the copolymer/pyrene system, where a higher concentration tending to shithe binding equilibrium more towards the bound state, and/or (ii) an increasing molar ratio of pyrene to NDI residues, with a higher ratio leading to a higher proportion of NDI resides being in the bound state. The factor a has units of ppm and so enables the otherwise dimensionless total- shielding factor, T, to be expressed as a predicted complexa- tion shifor the central, “observed” NDI residue in each sequence. T ¼ a X kmax k ¼1 Nk bk (2) (2) Eqn (2), with a ¼ 1, b ¼ 4, Nk ¼ 0, 1 or 2, and kmax ¼ N, is the mathematical denition of the fourth-quarter Cantor set.15 It should however be noted that, whatever the value of b (>3, integral or non-integral), this type of equation represents a fractal set obtained by summing an exponential-decay series. Although a does change between spectra, due to the change in pyrene concentration, it is a constant for each individual spec- trum, and each spectrum can therefore be predicted from eqn (2). Since fractals are scale-invariant, the introduction of the factor a does not aﬀect the fractal nature of the system. The binding model shown in Fig. 1 thus predicts a fractal distribu- tion of ring-current shieldings for all possible sequences in a binary copolyimide. Fig. 5 Graphical construction of the fourth-quarter Cantor set. 3.4 The 1H NMR spectrum as a Cantor set The scaling factor reects an increasing level of ring- current shielding with (i) an increasing overall concentration U ¼ Y k 2d1;Nk where the symbol Q k denotes a product of the argument over values of k (the rst digit, which is always 1, corresponds to the where the symbol Q k denotes a product of the argument over values of k (the rst digit, which is always 1, corresponds to the Table 1 The sixteen possible “I”-centred quintet sequences in a 1 : 1, random, F/I copolymer. The three-digit “shielding” codes for these sequences are based on single-site binding to I residues. Codes are listed (top to bottom) in order of increasing predicted complexation shift. The degeneracies (U) are the number of diﬀerent sequences corresponding to the same code Code I-Centred quintet sequences U 100 FFIFF 1 101 FFIFI + IFIFF 2 102 IFIFI 1 110 FFIIF + FIIFF 2 111 FFIII + IIIFF + FIIFI + IFIIF 4 112 IFIII + IIIFI 2 120 FIIIF 1 121 FIIII + IIIIF 2 122 IIIII 1 Code I-Centred quintet sequences U This journal is © The Royal Society of Chemistry 2020 Chemical Science This journal is © The Royal Society of Chemistry 2020 Edge Article 5, construction (A), aer two iterations, generates all the possible two-digit shielding codes for I/F quintet sequences on the basis of dual-site (pairwise) binding of pyrene, and gives a predicted pattern for the corresponding 1H NMR resonances of NDI protons.16 Three potential shielding codes [01, 02 and 12, shown in black in construction (A)] are not generated by any I- centred quintet sequence on the basis of dual-site binding, and so no corresponding resonances are predicted for those codes. Construction (B) of Fig. 5 generates, aer three iterations, all possible three-digit codes for I/F quintets based on the single- site binding model. Here, only codes beginning with the digit 1 emerge from single-site binding to I-centred quintet sequences (Table 1), as the central I residue is now always a binding site for pyrene. Unused codes are again shown in black. Using an integral exponential fall-oﬀfactor (b ¼ 4) enables immediate visualisation of the type of fractal involved (a last- fraction Cantor set) via the graphical construction shown in Fig. 5, but there is no theoretical requirement for the fall-oﬀ factor b to be exactly 4. We have shown previously15 that b can take any value, including non-integral values, greater than 3 without aﬀecting the fractal character of eqn (2) (the corre- sponding fractal dimension is D ¼ ln(3)/ln(b): see Section 9 of the ESI† in ref. 15). However, the present analysis (see below) does suggest that the experimental value of b for copolymer 10 Although true mathematical fractals are valid across all length scales, in the physical world objects showing fractal character This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 12165–12177 \| 12171 Chemical Science Edge Article invariably display self-similarity over only a small number of diﬀerent length scales. Indeed, it is generally considered that real objects are “described as fractal if they contain parts that, at two or more smaller scales, appear in some way similar to the whole”.34 The experimental data (Section 3.5) do indeed provide evidence for self-similarity over only a small number of length scales, corresponding to three iterations of the mathematical construction (Fig. 5B and 6): copolymer systems showing more highly resolved NMR peak-separations are clearly needed to test the present fractal model further. In order to check that the nine 1H NMR lines observed at high pyrene : NDI ratios (Fig. Edge Article 6c) are indeed single NDI reso- nances resulting from resolution of the nine sequence-groups identied by shielding code in Table 1, and do not result from spin–spin coupling (which could potentially arise for ortho-related protons an NDI residue at the centre of an unsymmetrical sequence), we next carried out a 2D-JRES anal- ysis of the diimide region of the 1H NMR spectrum (Fig. 7). This type of spectrum plots J values against associated d values and enables the magnitudes of all J–J couplings in the spectrum to be determined. The JRES result is quite clear: ortho, meta and para couplings for the HFDI resonances are obviously identiable, having J- values (at 400 MHz) of 0.0170, 0.0040 and 0.0015 ppm respec- tively, and the maximum coupling in the NDI region of the JRES spectrum is ca. 0.0010 ppm. This latter J-value is negligible when compared to the separation of the various NDI resonances in Fig. 6c, for which the minimum value is ca. 0.02 ppm (a 20- fold diﬀerence): spin–spin coupling thus plays no part in generating the NDI resonance-pattern. 3.5 Experimental evidence for single-site binding The patterns of NDI resonances predicted in Fig. 5 are very diﬀerent for the two diﬀerent binding modes, even though exactly the same set of sixteen I-centred quintet sequences (Table 1) are involved. A 1H NMR titration of copolymer 10 against pyrene-d10 (0.5 to 10 equivalents per NDI residue) is shown in Fig. 6. The predictions of Table 1 and Fig. 5B are clearly vindicated, in that the nal spectrum (Fig. 6c) shows nine NDI resonances whose relative intensities agree, at least approximately, with the values (1 : 2 : 1 : 2 : 4 : 2 : 1 : 2 : 1) pre- dicted from single-site binding to a random 1 : 1 copolymer. Interestingly, as shown in Fig. 6, the 1H resonances associ- ated with non-binding HFDI residues are essentially unaﬀected by the presence of pyrene, even at the highest concentration. From the point of view of a “static” physical model it might be expected that neighbour-bound pyrenes would exert similar degrees of ring-current shielding on both NDI and HFDI resi- dues. The fact that they do not indicates that the dynamics of the system (fast exchange on the NMR timescale between diﬀerent copolymer chain-conformations; fast exchange between bound and unbound pyrene molecules; and the possibility of rapid diﬀusion of pyrene between binding sites) must be taken into account if a successful atomistic model is to Fig. 7 JRES spectrum showing homonuclear (1H–1H) couplings (vertical axis) in the diimide region for copolymer 10 in the presence of 8 mole equivalents of pyrene-d10 per NDI residue. The HFDI reso- nances are found between 7.7 and 8.0 ppm, and the (sequence- dependent) NDI resonances between 8.2 and 8.5 ppm. The 2D-JRES spectrum was run in (CDCl3/triﬂuoroethanol, 6 : 1 v/v) at a copolymer concentration of 4 mM in NDI residues. Fig. 6 1H NMR spectra of copolymer 10 (4 mM in NDI residues in CDCl3/triﬂuoroethanol, 6 : 1 v/v) in the presence of increasing levels of pyrene-d10. At low levels of pyrene (0 to 3 equivalents per NDI) only three NDI resonances can be identiﬁed. These are assigned to the I- centred triplets FIF, FII/IIF and III which, at these low pyrene concen- trations, are the only resolvable I-centred sequences. At higher pyrene concentrations however, nine resonances are resolved, and these are assigned to the nine groups of quintet sequences shown in Table 1. Edge Article T ¼ T0 þ a X 2 k ¼1 Nk bk (3) (3) titration data for these copolymers are given in the ESI.† The predicted pattern of intensities (1 : 2 : 1 : 2 : 4 : 2 : 1 : 2 : 1) at [P]/[I] ¼ 10 is again evident, but measurements of the line- spacings l1 and l2 (see ESI†) show that there is a small but consistent fall-oﬀin these as the spacer-length increases (Fig. 9). This is consistent with pyrene molecules being bound progres- sively further out from the central “observed” NDI residue in any particular sequence, with a consequent reduction in the long- range ring current shielding that, in NMR terms, diﬀerentiates one sequence from another. It may be noted, however, that the shielding fall-oﬀfactor b (eqn (3)), measured as the ratio of l1 to l2, remains close to a value of 4, as also found for other, related copolymer systems.15,16 Note however that b could, in principle, take any value $3 (including non-integer values) without aﬀecting the fractal character of eqn (3).15 We were then able to determine experimental values for the parameters a, b and T0 by tting complexation shis from the 1H NMR titration of copolymer 10 (x ¼ 5) to eqn (3) (see ESI† for full details of the tting procedure). This analysis showed that a and T0 both vary in an approximately linear way with the concentration of pyrene-d10, but that b is independent of pyrene concentration and remains constant, within experimental error, at a value close to 4 (Fig. 8). This result conrms that the observed pattern of NDI resonances seen in Fig. 5c does indeed reect the distribution of ring-current shieldings predicted from the fourth-quarter Cantor set. However, it is important to note that currently we can only verify the exponential fall-oﬀ with that value of b over the rst two steps of the model; contributions at longer distances cannot be resolved directly. We therefore cannot rule out that the fall-oﬀfactor b might change at higher k, which would require a more complicated model to describe the spectrum. Our fractal model (implying a constant b over all steps) is thus the simplest model that can be used to explain the current data. 3.7 Simulation of the 1H NMR titration between pyrene-d10 and copolymer 9 As a nal test of the validity of eqn (1) (for relative intensities) and eqn (3) (for complexation shis) across a range of pyrene concentration, the titration of copolymer 10 with pyrene was simulated for quintet sequences, at four diﬀerent [P]/[I] ratios, using the parameters a, b and T0 derived as shown in Fig. 8. The resulting simulation (4 Hz linewidth) is shown in Fig. 10B, where it is compared with the corresponding experimental data. The comparison is very close, although the simulation shows somewhat better signal-resolution at high [P]/[I] ratios. Edge Article Chemical Science Fig. 8 Variation of parameters a, b and T0 with the molar ratio of pyrene-d10 to NDI residues in copolymer 10. The value of b is essentially constant. The equations of ﬁt for a and T0 are: a ¼ 0.0175([P]/[I]) + 0.0776, and T0 ¼ 0.029([P]/[I]) + 1.268. Error bars in the T0 plot are not visible because they are smaller than the symbol size. be developed. This would go well beyond the scope of the present work, but a possible approach is suggested by a recent report that, in certain DNA molecules, the binding aﬃnity of a “central” recognition site for a specic protein is strongly amplied by DNA regions anking the recognition site. These regions contain long tracts of degenerate recognition-sites which appear to function as “antennae” that attract molecules of the protein to the central binding site through exchange among neighbouring binding sites.46,47 We next set out to evaluate the “fall-oﬀfactor”, b, for shielding under single-site binding conditions experimentally, rather than empirically. Eqn (2) was therefore generalised and expanded, taking the shielding by pyrene bound directly to the central NDI (T0) out of the summation, as this shielding is always present whatever the sequence under consideration (eqn (3)). This operation also has the eﬀect of transforming the summation term (for a ¼ 1 and k ¼ 1 to N: i.e. when the I- centred copolymer sequence is innitely long) from a subset of the fourth-quarter Cantor set (Fig. 5B) into the complete set. Even though spectroscopic resolution in the present system limits consideration to only quintet sequences, for which the resulting equation sums only over the range k ¼ 1 to 2, the rapid fall-oﬀin shielding as pyrene molecules are bound further out from the “observed” NDI residue leads to rapid convergence of the predicted resonance-pattern at physically- meaningful linewidths. Fig. 8 Variation of parameters a, b and T0 with the molar ratio of pyrene-d10 to NDI residues in copolymer 10. The value of b is essentially constant. The equations of ﬁt for a and T0 are: a ¼ 0.0175([P]/[I]) + 0.0776, and T0 ¼ 0.029([P]/[I]) + 1.268. Error bars in the T0 plot are not visible because they are smaller than the symbol size. 3.5 Experimental evidence for single-site binding Starred resonances represent residual pyrene protons in the (99.8%) deuterated pyrene. Further details of this titration, together with analogous titration data for copolymers 11, 12 and 13, are given in the ESI.† Fig. 6 1H NMR spectra of copolymer 10 (4 mM in NDI residues in CDCl3/triﬂuoroethanol, 6 : 1 v/v) in the presence of increasing levels of pyrene-d10. At low levels of pyrene (0 to 3 equivalents per NDI) only three NDI resonances can be identiﬁed. These are assigned to the I- centred triplets FIF, FII/IIF and III which, at these low pyrene concen- trations, are the only resolvable I-centred sequences. At higher pyrene concentrations however, nine resonances are resolved, and these are assigned to the nine groups of quintet sequences shown in Table 1. Starred resonances represent residual pyrene protons in the (99.8%) deuterated pyrene. Further details of this titration, together with analogous titration data for copolymers 11, 12 and 13, are given in the ESI.† Fig. 7 JRES spectrum showing homonuclear (1H–1H) couplings (vertical axis) in the diimide region for copolymer 10 in the presence of 8 mole equivalents of pyrene-d10 per NDI residue. The HFDI reso- nances are found between 7.7 and 8.0 ppm, and the (sequence- dependent) NDI resonances between 8.2 and 8.5 ppm. The 2D-JRES spectrum was run in (CDCl3/triﬂuoroethanol, 6 : 1 v/v) at a copolymer concentration of 4 mM in NDI residues. 12172 \| Chem. Sci., 2020, 11, 12165–12177 This journal is © The Royal Society of Chemistry 2020 This journal is © The Royal Society of Chemistry 2020 3.6 1H NMR titrations of pyrene-d10 with copolymers 10, 11 and 12 The pattern of NDI resonances observed for copolymer 10 (x ¼ 5) with increasing pyrene concentration (Fig. 6) was also found for the longer-spacer copolymers synthesised in this work (copoly- mers 11, 12 and 13, where x ¼ 6, 7 and 8 respectively). Full More rigorous simulations using longer sequence-lengths (septets and nonets) reproduced the experimental NMR This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 12165–12177 \| 12173 Edge Article Edge Article Chemical Science Fig. 9 Fall-oﬀin NDI peak separations l1 and l2 (see ESI†) in the 1H NMR spectra of copolymers 10, 11, 12 and 13 (x ¼ 5, 6, 7 and 8 respectively) at [P]/[I] ¼ 10. Note that, although l1 and l2 both diminish with increasing spacer-length x, the ratio l1/l2 is (within error) essentially constant. realistic linewidth of 4 Hz. Note that the “observed” pattern essentially converges beyond the septet level, because eqn (3) produces a rapid, exponential decay of calculated ring-current shielding with the distance of the binding site from the central NDI residue. It could be argued, a priori, that a quadratic decay of ring- current shielding with distance (analogous to the quadratic decay of a simple magnetic eld) might be expected. Indeed, if we consider just a quintet sequence, the distance of the “observed” NDI residue from the second-adjacent NDI is twice the distance from the rst-adjacent NDI, which on a quadratic- decay model would give a fall-oﬀin shielding by a factor of 22 ¼ 4, exactly as in the exponential (fractal) model. However, including longer sequence-lengths results in a much slower decay (1/4, 1/9, 1/25 etc.) than in the exponential model, where the corresponding values are (1/4, 1/16, 1/64 etc.). As a consequence, the higher-order resonances predicted by the quadratic model tend increasingly to “ll the gaps” between the lower-order signals, resulting in complete loss of the ne structure that is observed experimentally. This is illustrated in Fig. 11, where the “nonet” spectrum predicted from the exponential-decay model [11(f)] is compared to that from a quadratic model [11(g)], and both are compared to the experimental spectrum [11(h)]. Full details of these simulations are given in the ESI.† Fig. 9 Fall-oﬀin NDI peak separations l1 and l2 (see ESI†) in the 1H NMR spectra of copolymers 10, 11, 12 and 13 (x ¼ 5, 6, 7 and 8 respectively) at [P]/[I] ¼ 10. 3.6 1H NMR titrations of pyrene-d10 with copolymers 10, 11 and 12 Sci., 2020, 11, 12165–12177 This journal is © The Royal Society of Chemistry 2020 Edge Article Chemical Science Predicted NDI resonance pattern (0.5 Hz linewidth) for the 1H NMR spectrum of copolymer 10 at 10 mol equiv. of pyren -decay model (eqn (3)) for quintet sequences (kmax ¼ 2, nine resonances); (b) As (a) but simulated at 4 Hz linewidth; (c) ences (kmax ¼ 3, twenty-seven resonances); (d) As (c) but simulated at 4 Hz linewidth; (e) As (a) but for nonet sequenc resonances); (f) As (e) but simulated at 4 Hz linewidth; (g) As (f) but simulated from a quadratic-decay model; (h) e Fig. 11 (a) Predicted NDI resonance pattern (0.5 Hz linewidth) for the 1H NMR spectrum of copolymer 10 at 10 mol equiv. of pyrene, using the exponential-decay model (eqn (3)) for quintet sequences (kmax ¼ 2, nine resonances); (b) As (a) but simulated at 4 Hz linewidth; (c) As (a) but for septet sequences (kmax ¼ 3, twenty-seven resonances); (d) As (c) but simulated at 4 Hz linewidth; (e) As (a) but for nonet sequences (kmax ¼ 4, eighty-one resonances); (f) As (e) but simulated at 4 Hz linewidth; (g) As (f) but simulated from a quadratic-decay model; (h) experimental spectrum. 3.6 1H NMR titrations of pyrene-d10 with copolymers 10, 11 and 12 Note that, although l1 and l2 both diminish with increasing spacer-length x, the ratio l1/l2 is (within error) essentially constant. Fig. 9 Fall-oﬀin NDI peak separations l1 and l2 (see ESI†) in the 1H NMR spectra of copolymers 10, 11, 12 and 13 (x ¼ 5, 6, 7 and 8 respectively) at [P]/[I] ¼ 10. Note that, although l1 and l2 both diminish with increasing spacer-length x, the ratio l1/l2 is (within error) essentially constant. It also proved possible to test the quadratic model against the exponential model by reference to the “dual-site binding” data reported in ref. 15. Here the experimental spectrum is suﬃciently well-resolved to show the eﬀects of including septet, rather than just quintet sequences in the analysis. As shown in the ESI (Fig. S13†), comparisons of the predictions from both models with the experimental data strongly favour the original, exponential model. signals rather better than the quintet simulation, as a result of the emergence of additional (fractal) ne structure in these further iterations of the fourth-quarter Cantor set. The eﬀect is illustrated in Fig. 11 for [P]/[I] ¼ 10, where the “true” structures of the simulated spectra [(a), (c) and (e)] are evident at a line- width of 0.5 Hz (0.00125 ppm at 400 MHz), and the “observed” patterns [(b), (d) and (f)] are generated using a more physically- Fig. 10 (A): Experimental data for titration of copolymer 10 against pyrene-d10 in CDCl3/triﬂuoroethanol (6 : 1, v/v). [P]/[I] ¼ molar ratio of pyrene to NDI residues. Signals between 7.90 and 8.20 ppm correspond to residual protons in pyrene-d10. (B): simulated data from eqn (1) and (3), based on quintet sequences and using parameters a, b (¼ 4) and T0 as derived above, at a constant linewidth of 4 Hz. Fig. 10 (A): Experimental data for titration of copolymer 10 against pyrene-d10 in CDCl3/triﬂuoroethanol (6 : 1, v/v). [P]/[I] ¼ molar ratio of pyrene to NDI residues. Signals between 7.90 and 8.20 ppm correspond to residual protons in pyrene-d10. (B): simulated data from eqn (1) and (3), based on quintet sequences and using parameters a, b (¼ 4) and T0 as derived above, at a constant linewidth of 4 Hz. 12174 \| Chem. This journal is © The Royal Society of Chemistry 2020 4. Conclusions a sharp chain-fold, to “single-site” where each pyrene binds to just one NDI unit, with the polymer chain folding much more loosely. Nevertheless, the 1H NMR spectrum of the new 1 : 1 bound copolymer system retains the fractal character observed previously in the 1 : 2 binding system, although showing a diﬀerent resonance pattern. From this, and results reported in As the length of the diester spacer-unit between NDI residues in poly(ester-imide)s increases, the mode of supramolecular binding between pyrene and NDI changes from “dual-site”, i.e. intercalation between two adjacent diimide residues linked by Chem. Sci., 2020, 11, 12165–12177 \| 12175 This journal is © The Royal Society of Chemistry 2020 Edge Article Edge Article Chemical Science 5 H. M. Colquhoun and J.-F. Lutz, Information-containing macromolecules, Nat. Chem., 2014, 6, 455–456. an earlier paper,16 we show that a simple mathematical model, based on fractal geometry, describes well both scenarios. Computational modelling indicates that single-site binding is strongly preferred for at least one specic “long” spacer, and 1H NMR titrations of NDI/HFDI copolymers against pyrene show a pattern of NDI resonances emerging at high pyrene concen- trations that shows distinct fractal character in terms of chemical shi. A detailed analysis of the titration data shows that the underlying mathematical fractal is a last-fraction Cantor set, and that the relative intensities of the observed resonances correspond to the number of diﬀerent quintet sequences contributing to each resonance. Although the specic last-fraction involved (approximately one quarter) has been identied experimentally only from a resolution-limited range of NMR data, it has been shown by simulation that the observed NDI resonance-pattern is fully consistent with a mathematical model involving exponential decay of ring- current shielding by pyrene binding at neighbouring NDI binding sites. There are clear challenges for future research in developing a fully atomistic model that can account for this result, and in identifying novel copolymer/probe-molecule systems that aﬀord more highly resolved 1H NMR spectra. 6 M. G. T. A. Rutten, F. W. Vaandrager, J. A. A. W. Elemans and R. J. M. Nolte, Encoding information into polymers, Nat. Rev. Chem., 2018, 2, 367–381. 7 J.-F. Lutz, M. Ouchi, D. R. Liu and M. Sawamoto, Sequence- controlled polymers, Science, 2013, 341, 1238149. 8 J.-F. Lutz, J.-M. Lehn, E. W. Meijer and K. Matyjaszewski, From precision polymers to complex materials and systems, Nat. Rev. Chem., 2016, 1, 1–14. 9 D. Nunez-Villanueva, M. Ciaccia, G. Conﬂicts of interest 13 H. M. Colquhoun, Z. Zhu, C. J. Cardin, Y. Gan and M. G. B. Drew, Sterically controlled recognition of macromolecular sequence information by molecular tweezers, J. Am. Chem. Soc., 2007, 129, 16163–16174. There are no conicts of interest to declare. Acknowledgements 14 Z. Zhu, C. J. Cardin, Y. Gan and H. M. Colquhoun, Sequence- selective assembly of tweezer-molecules on linear templates enables frameshireading of sequence information, Nat. Chem., 2010, 2, 653–660. This work was sponsored by the H2020 program of the Euro- pean Union under the ITN project Euro-Sequences, H2020- MSCA-ITN-2014, grant No. 642083 (Marie Skłodowska-Curie PhD studentship to MK). Further support was provided by the Leverhulme Trust, (Emeritus Fellowship to HMC, grant No. EM- 2018-0161/4; and postdoctoral support to GW, grant No. RP- 2013-SL-008, Natural material innovation for sustainable living); by the ERC (Consolidator Grant to OAS, grant No. CAM-RIG 726470); and by EPSRC (programme grant No. EP/ L027151/1, to OAS). We thank Professors G. A. Morris and D. A. Leigh of the University of Manchester for assistance with 1H–1H homonuclear decoupling studies. We wish to thank a reviewer for much insight and assistance in clarifying some of the more complex issues discussed in this paper. 15 J. S. Shaw, R. Vaiyapuri, M. P. Parker, C. A. Murray, K. J. C. Lim, C. Pan, M. Knappert, C. J. Cardin, B. W. Greenland, R. Grau-Crespo and H. M. Colquhoun, Elements of fractal geometry in the 1H NMR spectrum of a copolymer intercalation-complex: identication of the underlying Cantor set, Chem. Sci., 2018, 9, 4052–4061. 16 M. Knappert, T. Jin, S. D. Midgley, G. Wu, O. A. 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https://openalex.org/W4361931077	https://aacr.figshare.com/ndownloader/files/39919743	English	null	Supplementary Figures 1-9 from AMG-176, an Mcl-1 Antagonist, Shows Preclinical Efficacy in Chronic Lymphocytic Leukemia	null	2,023	cc-by	1,122	Supplementary Figure 1 100 100 100 100 300 100 300 DMSO --- --- --- AMG-176[nM] Venetoclax[nM] 0 2 4 6 8 10 % Annexin V/PI +ve 100 100 100 100 300 100 300 DMSO --- --- --- AMG-176[nM] Venetoclax[nM] 0 2 4 6 8 10 % Annexin V/PI +ve Supplementary Figure 2 Supplementary Figure 2 0 20 40 60 80 100 % Annexin V/PI +ve 0 20 40 60 80 100 % Annexin V/PI +ve Rai Stage 0 I II,III,IV A B,C Binet Stage A B P=0.03 P=0.06 % Annexin V/PI +ve 0 20 40 60 80 100 WBC <30,000 ≥30,000 P=0.27 C D % Annexin V/PI +ve 0 20 40 60 80 100 WBC <25,000 ≥25,000 P=0.90 E % Annexin V/PI +ve 0 20 40 60 80 100 WBC <20,000 ≥20,000 P=0.62 B 0 20 40 60 80 100 % Annexin V/PI +ve A B,C Binet Stage B P=0.06 0 20 40 60 80 100 % Annexin V/PI +ve Rai Stage 0 I II,III,IV A P=0.03 A 0 D C Supplementary Figure 3 A B 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 % Annexin V/PI +ve % Annexin V/PI +ve 0 20 40 60 80 100 % Annexin V/PI +ve 0 20 40 60 80 100 % Annexin V/PI +ve % Annexin V/PI +ve IgHV ZAP-70 C E D Mutated Unmutated Positive Negative <3 ≥3 <40,000 ≥40,000 β ഺ M WBC NEG 11q T12 13q 17p Cytogenetics P=0.03 P=0.06 P=0.98 P=0.67 P=0.50 P=0.001 P=0.014 P=0.13 A B 0 20 40 60 80 100 0 20 40 60 80 100 % Annexin V/PI +ve % Annexin V/PI +ve IgHV ZAP-70 Mutated Unmutated Positive Negative P=0.03 P=0.67 B 0 20 40 60 80 100 % Annexin V/PI +ve ZAP-70 Positive Negative P=0.67 A 0 20 40 60 80 100 % Annexin V/PI +ve IgHV Mutated Unmutated P=0.03 B A 0 20 40 60 80 100 % Annexin V/PI +ve 0 20 40 60 80 100 % Annexin V/PI +ve C D <3 ≥3 <40,000 ≥40,000 β ഺ M WBC P=0.06 P=0.98 0 20 40 60 80 100 % Annexin V/PI +ve D <40,000 ≥40,000 WBC P=0.98 0 20 40 60 80 100 % Annexin V/PI +ve C <3 ≥3 β ഺ M P=0.06 D D C 0 20 40 60 80 100 % Annexin V/PI +ve E NEG 11q T12 13q 17p Cytogenetics P=0.50 P=0.001 P=0.014 P=0.13 Supplementary Figure 4 Supplementary Figure 4 0 20 40 60 80 100 % Annexin V/PI +ve 0 20 40 60 80 100 % Annexin V/PI +ve Rai Stage 0 I II,III,IV A B,C Binet Stage A B P=0.06 P=0.11 0 20 40 60 80 100 % Annexin V/PI +ve A B,C Binet Stage B P=0.11 0 20 40 60 80 100 % Annexin V/PI +ve Rai Stage 0 I II,III,IV A P=0.06 B A Binet Stage Supplementary Figure 5 DMSO 10 30 100 300 1000 V AMG-176 Mcl-1 GAPDH DMSO 30 100 300 1000 V AMG-176 Mcl-1 Vinculin DMSO 30 100 300 1000 V Mcl-1 Vinculin DMSO 30 100 300 1000 V AMG-176 DMSO 30 10 100 300 1000 AMG-176 Mcl-1 Vinculin Mcl-1 Vinculin DMSO 30 10 100 300 1000 AMG-176 Mcl-1 Vinculin DMSO 30 10 100 300 AMG-176 Mcl-1 Vinculin DMSO 30 100 300 1000 AMG-176 Mcl-1 Vinculin #306 #213 #137 #386 #463 #343 #194 #990 AMG-176 V V Supplementary Figure 5 #463 DMSO 30 100 300 1000 V AMG-176 Mcl-1 Vinculin #213 DMSO 30 10 100 300 1000 AMG-176 Mcl-1 Vinculin #137 DMSO 30 10 100 300 AMG-176 Mcl-1 Vinculin #990 V DMSO 30 100 300 1000 AMG-176 Mcl-1 Vinculin #386 V Supplementary Figure 6 AMG-176[nM] 0 100 30 10 300 1000 Bcl-2 Bcl-xL Mcl-1 Vinculin GAPDH 0 100 30 10 300 1000 AMG-176[nM] Bcl-2 Bcl-xL Mcl-1 Vinculin GAPDH 0 100 30 10 300 1000 AMG-176[nM] Bcl-2 Bcl-xL Mcl-1 Vinculin GAPDH AMG-176[nM] 0 100 30 10 300 1000 Bcl-2 Bcl-xL Mcl-1 Vinculin GAPDH 0 100 30 10 300 1000 AMG-176[nM] Bcl-2 Bcl-xL Mcl-1 Vinculin GAPDH 0 100 30 10 300 1000 AMG-176[nM] Bcl-2 Bcl-xL Mcl-1 Vinculin GAPDH 0 100 30 10 300 1000 AMG-176[nM] Bcl-2 Bcl-xL Mcl-1 Vinculin GAPDH A 0 50 100 150 200 250 % Mcl-1 Change B Supplementary Figure 7 P=0.03 10 100 300 30 0 1000 AMG-176[nM] 0 50 100 150 200 % Bcl-2 Change AMG-176[nM] 10 100 300 30 0 1000 C AMG-176[nM] 10 100 300 30 0 1000 0 50 100 150 200 % Bcl-xL Change Supplementary Figure 7 Supplementary Figure 7 A 0 50 100 150 200 250 % Mcl-1 Change B Supplementary Figure 7 P=0.03 10 100 300 30 0 1000 AMG-176[nM] 0 50 100 150 200 % Bcl-2 Change AMG-176[nM] 10 100 300 30 0 1000 C AMG-176[nM] 10 100 300 30 0 1000 0 50 100 150 200 % Bcl-xL Change A 0 50 100 150 200 250 % Mcl-1 Change P=0.03 10 100 300 30 0 1000 AMG-176[nM] B 0 50 100 150 200 % Bcl-2 Change AMG-176[nM] 10 100 300 30 0 1000 B C AMG-176[nM] 10 100 300 30 0 1000 0 50 100 150 200 % Bcl-xL Change Supplemantary Figure 8 A B AMG-176[nM] DMSO 30 100 300 Total Mitochondrial Cytosolic PARP Mcl-1 Bak Bax Cyto c Vinculin GAPDH Tom 20 PARP Mcl-1 Bak Bax Cyto c GAPDH Tom 20 1000 AMG-176[nM] DMSO 300 100 30 10 1000 DMSO 300 100 30 10 1000 DMSO 300 100 30 10 1000 Total Mitochondrial Cytosolic DMSO 30 100 300 1000 DMSO 30 100 300 1000 Supplemantary Figure 8 Supplemantary Figure 8 Supplemantary Figure 8 Supplemantary Figure 8 A PARP Mcl-1 Bak Bax Cyto c GAPDH Tom 20 AMG-176[nM] DMSO 300 100 30 10 1000 DMSO 300 100 30 10 1000 DMSO 300 100 30 10 1000 Total Mitochondrial Cytosolic A B B AMG-176[nM] DMSO 30 100 300 Total Mitochondrial Cytosolic PARP Mcl-1 Bak Bax Cyto c Vinculin GAPDH Tom 20 1000 DMSO 30 100 300 1000 DMSO 30 100 300 1000 Supplementary Figure 9 Supplementary Figure 9 Supplementary Figure 9 0 20 40 60 80 100 % Absolute cell death 0 20 40 60 80 100 % Absolute cell death % Bak % Bax A B 50 100 150 0 P=0.536 r²=0.026 50 100 150 0 P=0.436 r²=0.041 0 20 40 60 80 100 % Absolute cell death 0 20 40 60 80 100 % Absolute cell death % Bak % Bax A B 50 100 150 0 P=0.536 r²=0.026 50 100 150 0 P=0.436 r²=0.041 0 20 40 60 80 100 % Absolute cell death A 50 100 150 0 P=0.536 r²=0.026 0 20 40 60 80 100 % Absolute cell death B 50 100 150 0 P=0.436 r²=0.041 B
https://openalex.org/W2201982874	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0138422&type=printable	English	null	Conventional versus Analgesia-Oriented Combination Sedation on Recovery Profiles and Satisfaction after ERCP: A Randomized Trial	PloS one	2,015	cc-by	6,491	RESEARCH ARTICLE Background The importance of providing effective analgesia during sedation for complex endoscopic procedures has been widely recognized. However, repeated administration of opioids in order to achieve sufficient analgesia may carry the risk of delayed recovery after propofol based sedation. This study was done to compare recovery profiles and the satisfaction of the endoscopists and patients between conventional balanced propofol sedation and anal- gesia-oriented combination sedation for patients undergoing endoscopic retrograde cholan- giopancreatography (ERCP). Editor: Luigi Maria Cavallo, Universita' degli Studi di Napoli Federico II, ITALY Received: April 24, 2015 Accepted: August 27, 2015 Published: September 24, 2015 Conventional versus Analgesia-Oriented Combination Sedation on Recovery Profiles and Satisfaction after ERCP: A Randomized Trial Seokyung Shin1☯‡, Tak Geun Oh2☯‡, Moon Jae Chung2, Jeong Youp Park2, Seung Woo Park2, Jae Bok Chung2, Si Young Song2, Jooyoun Cho1, Sang-Hun Park1, Young Chul Yoo1, Seungmin Bang2 1 Department of Anesthesiology and Pain Medicine, Severance Hospital, Anesthesia and Pain Research Institute, Yonsei University College of Medicine, 50–1 Yonsei-ro, Seodaemun-gu, 03722, Seoul, Korea, 2 Division of Gastroenterology, Department of Internal Medicine, Severance Hospital, Institute of Gastroenterology, Yonsei University College of Medicine, 50–1 Yonsei-ro, Seodaemun-gu, 03722, Seoul, Korea a1111 a1111 ☯These authors contributed equally to this work. ‡ These authors are first authors on this work. * bang7028@yuhs.ac (SB); seaoyster@yuhs.ac (YCY) OPEN ACCESS Citation: Shin S, Oh TG, Chung MJ, Park JY, Park SW, Chung JB, et al. (2015) Conventional versus Analgesia-Oriented Combination Sedation on Recovery Profiles and Satisfaction after ERCP: A Randomized Trial. PLoS ONE 10(9): e0138422. doi:10.1371/journal.pone.0138422 Methods Two hundred and two adult patients scheduled for ERCP were sedated by either the Conven- tional (initial bolus of meperidine with propofol infusion) or Combination (repeated bolus doses of fentanyl with propofol infusion) method. Recovery profiles, satisfaction levels of the endos- copists and patients, drug requirements and complications were compared between groups. Copyright: © 2015 Shin 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. Results Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Patients of the Combination Group required significantly less propofol compared to the Con- ventional Group (135.0 ± 68.8 mg vs. 165.3 ± 81.7 mg, P = 0.005). Modified Aldrete scores were not different between groups throughout the recovery period, and recovery times were also comparable between groups. Satisfaction scores were not different between the two groups in both the endoscopists and patients (P = 0.868 and 0.890, respectively). Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. 1 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0138422 September 24, 2015 Recovery and Satisfaction after Sedation for ERCP Conclusions Considering the significant reduction in propofol dose, the non-inferiority of recovery profiles and satisfaction scores of the endoscopists and patients, analgesia oriented combination sedation may be a more safe yet effective sedative method compared to conventional bal- anced propofol sedation during ERCP. Introduction Sedation for gastrointestinal endoscopic procedures has become commonplace during the past decade, and many attempts have been made to find a safe and yet effective combination of sed- atives and analgesics. Despite considerable dispute, propofol has gained overall popularity as the sedative of choice by many endoscopists and anaesthesiologists, and has largely replaced the traditional use of benzodiazepines.[1–3] However, its lack of analgesic property, narrow therapeutic window and absence of a reversal agent have been recognized as the cause of adverse events related to oversedation.[4] In an attempt to reduce these complications, balanced propofol sedation (BPS) was pro- posed as a method that would provide safe and effective sedation by combining a low-dose of propofol with an opioid analgesic and/or benzodiazepine.[5] While the concept of BPS has been generally well received by the medical society, the issue of which and how much opioid analgesic should be combined with propofol is still under debate.[6,7] The conventionally used opioid was meperidine, and many clinicians administered this agent routinely for pain control during endoscopic procedures. However, meperidine is quickly losing its place as an analgesic for interventional procedures due to its many side effects and low efficacy.[8,9] The toxic potential of meperidine renders it inappropriate for use during endoscopic retrograde cholan- giopancreatography (ERCP), as such procedures are usually accompanied by severe pain and discomfort which may require repeated analgesics administration.[10] On the other hand, we found in a recent retrospective study that respiratory events were significantly reduced when effective analgesia was provided during ERCP by giving bolus doses of fentanyl as needed.[11] However, repeated doses of fentanyl may carry the risk of prolonged recovery time, which could be a problem in terms of patient safety and efficiency. This randomized, controlled trial was conducted to compare recovery profiles between con- ventional BPS using a single dose of meperidine and a combination method using repeated doses of fentanyl during propofol-based sedation for ERCP. We hypothesized that a combina- tion method would be able to offer satisfactory sedation without prolonging recovery times compared to conventional BPS. Satisfaction levels of the endoscopists and patients as well as drug requirements, cardiovascular and respiratory events were also assessed and compared between the two groups. Patient monitoring All endoscopic procedures were performed in a room dedicated to ERCP, fully equipped for advanced cardiac life support. Patients arrived at the endoscopy room with secure IV access, and nasal oxygen was supplied at 3 L min-1. Patient monitoring included non-invasive blood pressure measurement every 5 minutes, pulse oximetry (SpO2) and electrocardiography. Recovery score assessment using the modified Aldrete score[12] was initiated immediately after the procedure ended (0 minutes), and was followed at 5, 15 and 25 minutes post-proce- dure by a nurse that was blinded to the sedation regimen at the endoscopy recovery unit. Study population Adult patients of American Society of Anesthesiologists (ASA) physical status I ~ IV that were scheduled for elective ERCP between April 2013 and October 2013 were recruited for this study. All patients provided written informed consent. Pregnant or breastfeeding patients and those with known allergies to eggs, soy beans or sulfites were excluded from this study. Patients that had received sedation for another procedure within 24 hours prior to ERCP, were unable to provide informed consent or were under the age of 20 years were also excluded. Study design and sedation protocols The targeted level of sedation was MOAA/S (Modified Observer’s Assessment of Alertness/ Sedation Scale) score 3 to 4 in all patients. Sedation in the Conventional Group was done by administrating 25 mg of IV meperidine (Pethidine, Jeil Pharmaceutical Co. Lt., Daegu, Korea) and 1 mg kg-1 of propofol (Pofol, Dong Kook Pharmaceutical Co. Ltd., Seoul, Korea) initially, followed by propofol infusion at a maintenance rate of 60 μg min-1kg-1 using an automated pump (Terufusion1 Syringe Pump TE-331, Terumo Corporation, Tokyo, Japan). Additional doses of 10 mg of propofol were given when patients complained of discomfort/pain, or when the endoscopist demanded deeper sedation. Patients of the Combination Group were given 1 μg kg-1 of IV fentanyl (Hana pharmaceutical, Korea) and 0.4 mg kg-1 of propofol initially, fol- lowed by propofol infusion at a maintenance rate of 30 μg min-1 kg-1. Additional doses of 10 mg of propofol were given during insufficient sedation, while bolus doses of 0.5 μg kg-1 of fen- tanyl were given when additional analgesia was needed. The need for additional sedation and analgesia was judged by the attending anesthesiologist based on the visual observation of patient irritability or excessive movement. Benzodiazepines were not used in our study. Enrolled patients were randomly assigned to either the Conventional Group (n = 100) or the Combination Group (n = 102) by means of a table of random numbers generated by 4 block design with the SAS program (SAS institute Inc., Cary, NC, USA). The allocation sequence was assigned to each patient in sealed envelopes with equal randomization (1:1 for the two groups) at the point of sedation induction. The patients, all study investigators and medical personnel except the attending anesthetist providing sedation were blinded to group assignment. All ERCP procedures were performed by 5 endoscopists of similar experience (who performed more than 300 ERCPs annually for more than 3 years) and sedation was done by a qualified anesthesiology staff member trained for procedural sedation outside the operat- ing theater that was not involved in the study. Ethics statement The study protocol was approved by the Institutional Review Board and Hospital Research Ethics Committee of Severance Hospital, and registered at http://clinicaltrials.gov (registration number NCT01840371). 2 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0138422 September 24, 2015 Recovery and Satisfaction after Sedation for ERCP Recovery and Satisfaction after Sedation for ERCP time points, time required for full recovery was noted. Recovery time was defined as the time interval between scope withdrawal and the modified Aldrete score reaching 10 points. The endoscopists gave a satisfaction score at the end of the procedure, and patients were asked by a nurse that was blinded to the sedation regimen to give a score upon full recovery at the endoscopy recovery unit. Satisfaction scores were initially given on a verbally administered numerical rating scale of 0 to 100. Procedure duration was defined as the time between scope insertion and scope withdrawal. Adverse respiratory events such as desaturation and apnea were also recorded by the attending anesthetist and compared between the two groups. Desaturation was defined as SpO2 lower than 90% for more than 10 seconds that required either an increase in O2 supply, chin lift, jaw thrust maneuver or assisted mask ventilation at the discretion of the attending anesthetist. Apnea was defined as the cessation of voluntary respiration for more than 30 seconds under visual observation. Hypertension due to severe pain or undersedation was defined as more than a 20% increase of mean blood pressure (MBP) from baseline, and hypotension was defined as systolic blood pressure (SBP) < 90 mmHg or MBP < 60 mmHg. Bradycardia and tachycardia were each defined as heart rate (HR) < 60 bpm and > 100 bpm, respectively. d d d l l l ( AS) f Post-procedure pain scores were recorded on a visual analogue scale (VAS) of 0–10 at 6, 12, 18 and 24 hours. The number of times rescue analgesics or antiemetics were administrated were recorded during intervals of 0–12 hours and 12–24 hours after the procedure. Results The CONSORT flow diagram of this study is shown in Fig 1. Of the 232 patients that were assessed for eligibility, 12 patients that received propofol sedation for endoscopic ultrasound just before ERCP and another 5 patients in whom ERCP was cancelled were excluded from this study. The remaining 215 patients were randomly assigned to either the Conventional Group (n = 107) or the Combination Group (n = 108). After randomization, 7 patients in the Conven- tional Group and 6 patients in the Combination Group were dropped from analysis due to incomplete medical records or missing data at the time of analysis. The remaining 202 patients (100 and 102 patients of the Conventional Group and Combination Group, respectively) suc- cessfully completed the study and were included for analysis. Statistical analysis A Sample size was calculated based on the recovery times after propofol monosedation reported in a previous study.[7] To detect a 20% difference in recovery time with a non-inferi- ority design between groups, 100 patients per group would be required to obtain a power of 85%, assuming a type I error of 0.05. Continuous variables with normal distribution were ana- lyzed with the independent two sample t-test. Continuous variables that were not normally dis- tributed were analyzed with the Mann-Whitney U Test and expressed as median and interquartile range (IQR). Categorical variables were analyzed by the Chi-square or Fisher’s exact test. All statistical analyses were performed with IBM SPSS Statistics 20.0 (IBM Corp., Armonk, NY, USA). A P value of < 0.05 was considered statistically significant. Data are described as mean ± SD or number of subjects. Outcome measurements and definitions The primary outcome of this study was to compare recovery profiles between the two groups. In addition to assessing recovery based on the modified Aldrete score at the aforementioned 3 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0138422 September 24, 2015 Cardiovascular and respiratory parameters Cardiovascular and respiratory data were shown in Table 3. Baseline vital signs including mean blood pressure, heart rate and SpO2 were not different between the two groups. The number of patients that presented with significant hypertension or hypotension was not different between the two groups. Similarly, there was no difference in the incidence of tachycardia or bradycar- dia between groups. Two patients (2.0%) in the Conventional Group and 3 patients (2.9%) in the Combination Group experienced desaturation (P = 0.287), and all events were resolved with a temporary increase in oxygen flow without the need of scope removal. None of the patients required vasopressors, endotracheal intubation or premature termination of ERCP due to sedation-related complications. Patient characteristics Patient characteristics and procedure-related data are listed in Table 1. While there were no difference in patient characteristics with regard to gender, body mass index, history of smok- ing, alcohol intake or snoring and ASA classification, the patients in the Combination Group 4 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0138422 September 24, 2015 Recovery and Satisfaction after Sedation for ERCP were significantly older than those of the Conventional Group (P = 0.025). Reason for ERCP and type of performed ERCP were not significantly different between the two groups. Fig 1. CONSORT flowchart. doi:10.1371/journal.pone.0138422.g001 Fig 1. CONSORT flowchart. doi:10.1371/journal.pone.0138422.g001 were significantly older than those of the Conventional Group (P = 0.025). Reason for ERCP and type of performed ERCP were not significantly different between the two groups. Drug requirements and procedure duration Patients in the Combination Group required significantly less propofol compared to the Con- ventional Group (135.0 ± 68.8 mg vs. 165.3 ± 81.7 mg, P = 0.005). Procedure duration of the Conventional Group and the Combination Group were not significantly different (19.9 ± 13.4 and 21.4 ± 13.6 minutes, respectively, P = 0.445) (Table 2). Recovery and Satisfaction after Sedation for ERCP Pain scores and rescue drug administration Pain scores were significantly lower in the Combination Group compared to the Conventional Group at post-procedure 6 hours (2.5 ± 2.5 vs. 3.2 ± 2.4, P = 0.045) but not at 12, 18 or 24 Table 1. Patient characteristics. Values are mean ± SD or number (%) of patients. ERCP, endoscopic retrograde cholangiopancreatography. ASA, Ameri- can Society of Anesthesiologists Variables Conventional Group (n = 100) Combination Group (n = 102) P-value Age (years) 60.9 ± 14.6 65.3 ± 13.8 0.025• Gender (male/female) 52 / 48 60 / 42 0.329•• Body mass index (kg/m2) 22.8 ± 3.0 23.1 ± 3.2 0.454• Smoking History 0.387•• Yes 15 (15.0) 20 (19.6) No 85 (85.0) 82 (80.4) Alcoholic History 0.238•• Yes 16 (16.0) 23 (22.5) No 84 (84.0) 79 (77.5) Snoring History 0.259•• Yes 12 (12.0) 18 (17.6) No 88 (88.0) 84 (82.4) ASA class 0.427•• 1 11 (11.0) 13 (12.7) 2 45 (45.0) 35 (34.3) 3 40 (40.0) 47 (46.1) 4 4 (4.0) 7 (6.9) Reason for ERCP 0.840•• Bile duct cancer 21 (21.0) 25 (24.5) Pancreatic cancer 15 (15.5) 12 (11.8) Other malignancy 8 (8.0) 7 (6.9) Choledocholithiasis 44 (44.0) 49 (48.0) Miscellaneous 12 (12.0) 9 (8.8) Type of ERCP 0.63•• Diagnostic 13 (13.0) 9 (8.8) Diagnostic and therapeutic 81 (81.0) 87 (85.3) Failed cannulation 6 (6.0) 6 (5.9) •Analyzed with the independent two sample t-test, ••Analyzed with the Chi-square or Fisher’s exact test doi:10.1371/journal.pone.0138422.t001 Table 2. Drug requirements and procedure duration. Values are mean ± SD (range). Conventional Group (n = 100) Combination Group (n = 102) P-value• Drug requirements Propofol (mg) 165.3 ± 81.7 (40 to 390) 135.0 ± 68.8 (30 to 360) 0.005 Fentanyl (μg) - 107.2 ± 46.8 (40 to 300) - Propofol (μg/kg/min) 185.5 ± 134.5 129.6 ± 77.5 < 0.001 P d d i ( i ) 19 9 13 4 21 4 13 6 0 44 Table 1. Patient characteristics. Values are mean ± SD or number (%) of patients. ERCP, endoscopic retrograde cholangiopancreatography. Recovery profiles and satisfaction scores Modified Aldrete scores at 0, 5, 15 and 25 minutes after the procedure were not different between the two groups at any of the time points. Recovery times of the Conventional Group and the Combination Group were also comparable between groups [13 (5, 20) and 12 (5, 19) minutes, respectively, P = 0.662] (Table 4). Satisfaction scores of the endoscopists and patients were also not different between the two groups (P = 0.868 and 0.890, respectively) (Table 5). 5 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0138422 September 24, 2015 Recovery and Satisfaction after Sedation for ERCP Table 3. Cardiovascular and respiratory parameters. Values are mean ± SD or n (%) of patients. MBP, mean blood pressure. HR, heart rate Conventional Group (n = 100) Combination Group (n = 102) P-value Baseline MBP (mmHg) 87 ± 15 90 ± 16 0.160• Hypertension 22 (22.0) 21 (20.6) 0.806•• Hypotension 9 (9.0) 11 (10.8) 0.671•• Baseline HR (beats per minute) 89 ± 13 91 ± 19 0.283• Tachycardia 34 (34.0) 39 (38.2) 0.531•• Bradycardia 4 (4.0) 3 (2.9) 0.681•• Baseline SpO2 (%) 99.4 ± 1.6 99.6 ± 0.9 0.237• SpO2 < 90% 2 (2.0) 3 (2.9) 0.287•• •Analyzed with the independent two sample t-test, ••Analyzed with the Chi-square or Fisher’s exact test d i 10 1371/j l 0138422 t003 atory parameters. Values are mean ± SD or n (%) of patients. MBP, mean blood pressure. HR, heart rate doi:10.1371/journal.pone.0138422.t003 hours. The frequency of rescue analgesics administration was also significantly lower in the Combination group during the first 12 hours after the procedure (0.08 ± 0.9 vs. 0.5 ± 0.8, P = 0.023) but not between 12 and 24 hours. There was no difference between the two groups in the number of times rescue anti-emetics were given during 24 hours after the procedure (Table 6). ASA, Ameri- can Society of Anesthesiologists Variables Conventional Group (n = 100) Combination Group (n = 102) P-value Age (years) 60.9 ± 14.6 65.3 ± 13.8 0.025• Gender (male/female) 52 / 48 60 / 42 0.329•• Body mass index (kg/m2) 22.8 ± 3.0 23.1 ± 3.2 0.454• Smoking History 0.387•• Yes 15 (15.0) 20 (19.6) No 85 (85.0) 82 (80.4) Alcoholic History 0.238•• Yes 16 (16.0) 23 (22.5) No 84 (84.0) 79 (77.5) Snoring History 0.259•• Yes 12 (12.0) 18 (17.6) No 88 (88.0) 84 (82.4) ASA class 0.427•• 1 11 (11.0) 13 (12.7) 2 45 (45.0) 35 (34.3) 3 40 (40.0) 47 (46.1) 4 4 (4.0) 7 (6.9) Reason for ERCP 0.840•• Bile duct cancer 21 (21.0) 25 (24.5) Pancreatic cancer 15 (15.5) 12 (11.8) Other malignancy 8 (8.0) 7 (6.9) Choledocholithiasis 44 (44.0) 49 (48.0) Miscellaneous 12 (12.0) 9 (8.8) Type of ERCP 0.63•• Diagnostic 13 (13.0) 9 (8.8) Diagnostic and therapeutic 81 (81.0) 87 (85.3) Failed cannulation 6 (6.0) 6 (5.9) Table 1. Patient characteristics. Values are mean ± SD or number (%) of patients. ERCP, endoscopic retrograde cholangiopancreatography. ASA, Ameri- can Society of Anesthesiologists Pain scores and rescue drug administration Group at post procedure 6 hours (2.5 ± 2.5 vs. 3.2 ± 2.4, P 0.045) but not at 12, 18 or 24 Table 2. Drug requirements and procedure duration. Values are mean ± SD (range). Conventional Group (n = 100) Combination Group (n = 102) P-value• Drug requirements Propofol (mg) 165.3 ± 81.7 (40 to 390) 135.0 ± 68.8 (30 to 360) 0.005 Fentanyl (μg) - 107.2 ± 46.8 (40 to 300) - Propofol (μg/kg/min) 185.5 ± 134.5 129.6 ± 77.5 < 0.001 Procedure duration (min) 19.9 ± 13.4 21.4 ± 13.6 0.445 •Analyzed with the independent two sample t-test doi:10 1371/journal pone 0138422 t002 Table 2. Drug requirements and procedure duration. Values are mean ± SD (range). Conventional Group (n = 100) Combination Group (n = 102) P-value• Drug requirements Propofol (mg) 165.3 ± 81.7 (40 to 390) 135.0 ± 68.8 (30 to 360) 0.005 Fentanyl (μg) - 107.2 ± 46.8 (40 to 300) - Propofol (μg/kg/min) 185.5 ± 134.5 129.6 ± 77.5 < 0.001 Procedure duration (min) 19.9 ± 13.4 21.4 ± 13.6 0.445 •Analyzed with the independent two sample t-test doi:10.1371/journal.pone.0138422.t002 PLOS ONE \| DOI:10.1371/journal.pone.0138422 September 24, 2015 6 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0138422 September 24, 2015 6 / 11 • Analyzed with the Mann-Whitney U Test under conscious sedation experience discomfort and pain, implying that pain was often under- treated during and after these procedures. In the same vein, we found in a recent retrospective study that more opioids were administered during therapeutic ERCP compared to diagnostic procedures when performed under conscious sedation with propofol infusion.[11] The results of these studies point in the same direction—a need for more attention towards analgesia dur- ing ERCP, and especially during therapeutic procedures. The concept of BPS basically involves a single induction dose of opioid and/or benzodiaze- pine, followed by incremental doses of propofol in order to achieve a targeted level of moderate sedation.[14] However, it is important to recognize that propofol does not have any inherent analgesic properties and therefore requires an adjuvant drug that will complement this aspect. The main problem with BPS thus seems to be the ‘single induction dose of opioid’ that is used at the beginning of the procedure in a form of premedication. This overlooks the fact that pain levels and procedure times differ significantly between ERCP procedures, and may be one of the factors leading to oversedation due to excessive doses of propofol. By taking this aspect into consideration, the sedation regimen of the Combination Group of the present study incorpo- rated the repeated administration of an opioid according to the pain or discomfort of the patient during ERCP. Although meperidine has been most commonly used by many gastroen- terologists during endoscopy for a long time,[9] its toxic potential makes it inappropriate for repeated administrations, and doses as low as 46 mg have been reported to produce central ner- vous system symptoms.[15] The active metabolite, normeperidine, has a half-life ranging between 14 to 21 hours, and its accumulation may precipitate anxiety, hyperreflexia, myoclo- nus, seizures and mood changes.[8] In order to avoid such side effects, fentanyl was used as the opioid in the Combination Group. Fentanyl has a more rapid onset and clearance, lacks dan- gerous metabolites or allergenic properties and is therefore commonly recommended as the safe alternative to meperidine for procedural sedation.[9,16] Table 6. Pain scores and frequency of rescue drug administration. Values are mean ± SD. Discussion Since BPS was first described by Cohen et al.,[13] propofol-based sedation methods for endo- scopic procedures have gained much popularity. However, the debate on which regimen should be used is yet to be resolved. Sedation regimens cannot be standardized, but should be tailored according to the characteristics of the procedure such as the target site, duration, the general condition of the patient and nature of the intended procedure. Therefore, the relatively complex nature of ERCP procedures render it even more difficult to choose a safe and effective sedation regimen compared to simpler endoscopic procedures such as colonoscopy or esopha- gogastroduodenoscopy. We found in the present study that combining repeated doses of fenta- nyl for adequate pain control with continuous propofol infusion does not seem to prolong recovery times compared to a single dose of meperidine followed by continuous propofol infu- sion. Moreover, the proportion of endocopists and patients that felt ‘very unsatisfied’ with the overall sedation method were clearly smaller in the Combination Group. Appropriate analgesia has emerged as an important aspect to consider when deciding on a sedation regime. Jeurnink et al.[10] reported that more discomfort was experienced during and immediately after the procedure in patients that underwent therapeutic ERCP compared to diagnostic ERCP. They also concluded that one-third to one-half of patients undergoing ERCP Table 4. Recovery profiles. Values are median (IQR) Conventional Group (n = 100) Combination Group (n = 102) P-value• Aldrete score at 0 min 8 (7, 9) 8 (7, 9) 0.217 Aldrete score at 5 min 9 (8, 10) 9 (8, 10) 0.379 Aldrete score at 15 min 10 (9, 10) 10 (9, 10) 0.159 Aldrete score at 25 min 10 (10, 10) 10 (10, 10) - Recovery time (minutes) 13 (5, 20) 12 (5, 19) 0.662 • Analyzed with the Mann-Whitney U Test doi:10.1371/journal.pone.0138422.t004 PLOS ONE \| DOI:10.1371/journal.pone.0138422 September 24, 2015 7 / 11 Table 4. Recovery profiles. Values are median (IQR) 7 / 11 Recovery and Satisfaction after Sedation for ERCP under conscious sedation experience discomfort and pain, implying that pain was often under- treated during and after these procedures. In the same vein, we found in a recent retrospective study that more opioids were administered during therapeutic ERCP compared to diagnostic procedures when performed under conscious sedation with propofol infusion.[11] The results of these studies point in the same direction—a need for more attention towards analgesia dur- ing ERCP, and especially during therapeutic procedures. The concept of BPS basically involves a single induction dose of opioid and/or benzodiaze- pine, followed by incremental doses of propofol in order to achieve a targeted level of moderate sedation.[14] However, it is important to recognize that propofol does not have any inherent analgesic properties and therefore requires an adjuvant drug that will complement this aspect. The main problem with BPS thus seems to be the ‘single induction dose of opioid’ that is used at the beginning of the procedure in a form of premedication. This overlooks the fact that pain levels and procedure times differ significantly between ERCP procedures, and may be one of the factors leading to oversedation due to excessive doses of propofol. By taking this aspect into consideration, the sedation regimen of the Combination Group of the present study incorpo- rated the repeated administration of an opioid according to the pain or discomfort of the patient during ERCP. Although meperidine has been most commonly used by many gastroen- terologists during endoscopy for a long time,[9] its toxic potential makes it inappropriate for repeated administrations, and doses as low as 46 mg have been reported to produce central ner- vous system symptoms.[15] The active metabolite, normeperidine, has a half-life ranging between 14 to 21 hours, and its accumulation may precipitate anxiety, hyperreflexia, myoclo- nus, seizures and mood changes.[8] In order to avoid such side effects, fentanyl was used as the opioid in the Combination Group. Fentanyl has a more rapid onset and clearance, lacks dan- gerous metabolites or allergenic properties and is therefore commonly recommended as the safe alternative to meperidine for procedural sedation.[9,16] Table 5. Satisfaction scores of the endoscopist and patients. Values are median (IQR) Conventional Group (n = 100) Combination Group (n = 102) P-value• Endoscopist 90 (80, 100) 90 (80, 100) 0.868 Patient 90 (90, 100) 90 (80, 100) 0.890 • Analyzed with the Mann-Whitney U Test doi:10.1371/journal.pone.0138422.t005 Table 6. Pain scores and frequency of rescue drug administration. Values are mean ± SD. VAS, visual analogue scale Conventional Group (n = 100) Combination Group (n = 102) P-value• Pain score (VAS 0~10) Post-procedure 6hr 3.2 ± 2.4 2.5 ± 2.5 0.045 Post-procedure 12hr 1.4 ± 2.1 1.6 ± 2.1 0.615 Post-procedure 18hr 1.0 ± 1.6 1.0 ± 1.7 0.898 Post-procedure 24hr 0.9 ± 1.6 0.7 ± 1.4 0.332 Table 5. Satisfaction scores of the endoscopist and patients. Values are median (IQR) Conventional Group (n = 100) Combination Group (n = 102) P-value• Endoscopist 90 (80, 100) 90 (80, 100) 0.868 Patient 90 (90, 100) 90 (80, 100) 0.890 • Analyzed with the Mann-Whitney U Test Table 5. Satisfaction scores of the endoscopist and patients. Values are median (IQR) Recovery and Satisfaction after Sedation for ERCP We found in a recent retrospective analysis that providing analgesia by combining repeated doses of fentanyl with propofol could effectively reduce the risk of respiratory events when compared with propofol monosedation.[11] This result was attributed to the decreased risk of oversedation by reducing the overall dosage of propofol. However, it could not be concluded that repeated doses of fentanyl would not affect recovery profiles. We found in the present study that the fentanyl combination method did not prolong recovery times or worsen recov- ery profiles compared to conventional BPS. In other words, repeated administrations of fenta- nyl did not seem to affect patient recovery when used appropriately for procedural pain control. In addition, considering the suggested doses of propofol for sedation and maintenance of general anesthesia (25–75 μg/kg/min and 50–150 μg/kg/min, respectively),[17] the differ- ence in propofol requirements of roughly 55 μg/kg/min between the two groups seem to have clinical significance. The present study is notable in that patients were enrolled regardless of ASA class. While most previous studies done on sedation protocols enrolled patients of only ASA class 1–3,[18– 21] we also included patients of ASA class 4. However, the incidence of desaturation was very low in both groups, and all events were corrected with a temporary increase in oxygen supply without the need to interrupt the procedure. In terms of post-procedural pain, the Conventional Group showed significantly lower pain scores compared to the Combination Group at 6 hours after the procedure, and required less rescue analgesics during the first 12 hours. However, these results should be interpreted care- fully. The difference in mean VAS scores between the two groups was only 0.7 (3.2 ± 2.4 vs. 2.5 ± 2.5), which may have questionable significance in actual clinical practice. This also applies to the results of rescue analgesics administration during the first 12 hours, which is statistically significant, but does not seem to have much clinical significance (0.8 ± 0.9 vs. 0.5 ± 0.8). Whether the combination method for sedation during ERCP allows lesser post-procedural pain remains unclear in this study. This study has several limitations. First, this study was conducted in a single institution and enrolled only Asian patients that showed a tendency of a relatively low body mass index (22.9 ± 3.1 kg/m2) compared to the average Caucasian. PLOS ONE \| DOI:10.1371/journal.pone.0138422 September 24, 2015 • Analyzed with the Mann-Whitney U Test VAS, visual analogue scale Conventional Group (n = 100) Combination Group (n = 102) P-value• Pain score (VAS 0~10) Post-procedure 6hr 3.2 ± 2.4 2.5 ± 2.5 0.045 Post-procedure 12hr 1.4 ± 2.1 1.6 ± 2.1 0.615 Post-procedure 18hr 1.0 ± 1.6 1.0 ± 1.7 0.898 Post-procedure 24hr 0.9 ± 1.6 0.7 ± 1.4 0.332 Rescue analgesics (n) Post-procedure 0–12 hrs 0.8 ± 0.9 0.5 ± 0.8 0.023 Post-procedure 12–24 hrs 0.2 ± 0.6 0.2 ± 0.4 0.247 Rescue anti-emetics (n) Post-procedure 0–12 hrs 0.1 ± 0.5 0.0 ± 0.2 0.07 Post-procedure 12–24 hrs 0.0 ± 0.2 0.0 ± 0.2 0.72 •Analyzed with the independent two sample t-test doi:10.1371/journal.pone.0138422.t006 PLOS ONE \| DOI:10.1371/journal.pone.0138422 September 24, 2015 8 / 11 Table 6. Pain scores and frequency of rescue drug administration. Values are mean ± SD. VAS, visual analogue scale Conventional Group (n = 100) Combination Group (n = 102) P-value• res and frequency of rescue drug administration. Values are mean ± SD. VAS, visual analogue scale cue drug administration. Values are mean ± SD. VAS, visual analogue scale Table 6. Pain scores and frequency of rescue drug administration. Values are mean ± SD. VAS, visual analog Conventional Group (n = 100) Combination G 8 / 11 S1 Dataset. Clinical research data of all patients. (XLSX) S1 Dataset. Clinical research data of all patients. (XLSX) S1 Dataset. Clinical research data of all patients. (XLSX) Author Contributions Conceived and designed the experiments: SS TGO JC SHP SB YCY. Performed the experi- ments: SS TGO MJC JYP SWP JBC SYS SB YCY. Analyzed the data: SS TGO JC SHP YCY. Conceived and designed the experiments: SS TGO JC SHP SB YCY. Performed the experi- ments: SS TGO MJC JYP SWP JBC SYS SB YCY. Analyzed the data: SS TGO JC SHP YCY. Contributed reagents/materials/analysis tools: SS TGO JC SHP YCY. Wrote the paper: SS TGO SB YCY. ments: SS TGO MJC JYP SWP JBC SYS SB YCY. Analyzed the data: SS TGO JC SHP YCY. Contributed reagents/materials/analysis tools: SS TGO JC SHP YCY. Wrote the paper: SS TGO SB YCY. y Contributed reagents/materials/analysis tools: SS TGO JC SHP YCY. Wrote the paper: SS TGO SB YCY. Supporting Information S1 CONSORT Checklist. CONSORT 2010 checklist for reporting a randomiszed trial. (DOC) S1 Protocol. Clinical research protocol in original language (Korean). (DOCX) S1 Informed Consent Form. (DOCX) S1 Informed Consent Form. (DOCX) PLOS ONE \| DOI:10.1371/journal.pone.0138422 September 24, 2015 A study including a patient population of diverse ethnicity should provide more insight with regard to drug dosage and adverse events. Secondly, we were not able to detect a difference in respiratory events between the two groups despite the significant reduction in propofol dosage in the Combination Group. Moreover, the incidence of respiratory events observed in the present study was under 3% in both groups, which is much lower than the previously reported 6% in patients that received ERCP proce- dures under propofol monosedation in a retrospective study.[11] Lastly, patients of the Combi- nation Group were somewhat older that those of the Conventional Group with a mean age difference of 5 years. However, to what extent this difference may have affected the drug requirements and other outcomes are not clear. Overall, these results may imply that the most important factor in performing safe, yet effective sedation for therapeutic endoscopic proce- dures is not the sedative regimen, but the meticulous and careful monitoring of the sedation provider. In conclusion, combining repeated doses of fentanyl for adequate pain control with contin- uous propofol infusion does not seem to prolong recovery times compared to a single dose of meperidine followed by continuous propofol infusion. The resulting decrease in overall propo- fol dose may have more significance in patients with poor general conditions which are not uncommon with ERCP. Considering the pharmacologic characteristics of the two opioids and the trend of higher satisfaction levels of the endoscopists and patients, combination sedation using repeated administrations of fentanyl with propofol infusion may be a safer and more effective sedation method compared to conventional BPS with meperidine during ERCP. PLOS ONE \| DOI:10.1371/journal.pone.0138422 September 24, 2015 9 / 11 Recovery and Satisfaction after Sedation for ERCP Supporting Information S1 CONSORT Checklist. CONSORT 2010 checklist for reporting a randomiszed trial. (DOC) S1 Protocol. Clinical research protocol in original language (Korean). (DOCX) S2 Protocol. Clinical research protocol in English. (DOCX) S1 Case Report Form. (DOCX) S1 Informed Consent Form. (DOCX) S1 Dataset. Clinical research data of all patients. (XLSX) Supporting Information S1 CONSORT Checklist. CONSORT 2010 checklist for rep (DOC) S1 Protocol. Clinical research protocol in original languag (DOCX) S2 Protocol. Clinical research protocol in English. (DOCX) S1 Case Report Form. (DOCX) S1 Informed Consent Form. (DOCX) S1 Dataset. Clinical research data of all patients. (XLSX) References 1. Trummel J. Sedation for gastrointestinal endoscopy: the changing landscape. Curr Opin Anaesthesiol. 2007; 20: 359–364. PMID: 17620846 2. Garewal D, Powell S, Milan SJ, Nordmeyer J, Waikar P. Sedative techniques for endoscopic retrograde cholangiopancreatography. Cochrane Database Syst Rev. 2012; 6: CD007274. doi: 10.1002/ 14651858.CD007274.pub2 PMID: 22696368 3. Bo LL, Bai Y, Bian JJ, Wen PS, Li JB, Deng XM. Propofol vs traditional sedative agents for endoscopic retrograde cholangiopancreatography: a meta-analysis. World J Gastroenterol. 2011; 17: 3538–3543. doi: 10.3748/wjg.v17.i30.3538 PMID: 21941422 4. Garewal D, Waikar P. Propofol sedation for ERCP procedures: a dilemna? Observations from an anes- thesia perspective. Diagn Ther Endosc. 2012; 2012: 639190. doi: 10.1155/2012/639190 PMID: 22272061 5. Cohen LB, Hightower CD, Wood DA, Miller KM, Aisenberg J. Moderate level sedation during endos- copy: a prospective study using low-dose propofol, meperidine/fentanyl, and midazolam. Gastrointest Endosc. 2004; 59: 795–803. PMID: 15173791 6. Lee CK, Lee SH, Chung IK, Lee TH, Park SH, Kim EO, et al. Balanced propofol sedation for therapeutic GI endoscopic procedures: a prospective, randomized study. Gastrointest Endosc. 2011; 73: 206–214. doi: 10.1016/j.gie.2010.09.035 PMID: 21168838 7. Lee TH, Lee CK, Park SH, Lee SH, Chung IK, Choi HJ, et al. Balanced propofol sedation versus propo- fol monosedation in therapeutic pancreaticobiliary endoscopic procedures. Dig Dis Sci. 2012; 57: 2113–2121. doi: 10.1007/s10620-012-2234-0 PMID: 22615018 8. Latta KS, Ginsberg B, Barkin RL. Meperidine: a critical review. Am J Ther. 2002; 9: 53–68. PMID: 11782820 9. Daniel K, Schmelzer M. Why are we still using meperidine (demerol) for conscious sedation? Gastroen- terol Nurs. 2009; 32: 298–301. doi: 10.1097/SGA.0b013e3181b2c952 PMID: 19696609 10. Jeurnink SM, Steyerberg E, Kuipers E, Siersema P. The burden of endoscopic retrograde cholangio- pancreatography (ERCP) performed with the patient under conscious sedation. Surg Endosc. 2012; 26: 2213–2219. doi: 10.1007/s00464-012-2162-2 PMID: 22302536 11. Shin S, Lee SK, Min KT, Kim HJ, Park CH, Yoo YC. Sedation for interventional gastrointestinal endo- scopic procedures: are we overlooking the "pain"? Surg Endosc. 2013. doi: 10.1007/s00464-013-3133-y PLOS ONE \| DOI:10.1371/journal.pone.0138422 September 24, 2015 10 / 11 Recovery and Satisfaction after Sedation for ERCP 12. Aldrete JA. The post-anesthesia recovery score revisited. J Clin Anesth. 1995; 7: 89–91. PMID: 7772368 13. Cote GA, Hovis RM, Ansstas MA, Waldbaum L, Azar RR, Early DS, et al. Incidence of sedation-related complications with propofol use during advanced endoscopic procedures. Clin Gastroenterol Hepatol. 2010; 8: 137–142. doi: 10.1016/j.cgh.2009.07.008 PMID: 19607937 14. Vargo JJ, Cohen LB, Rex DK, Kwo PY. PLOS ONE \| DOI:10.1371/journal.pone.0138422 September 24, 2015 21. Mazanikov M, Udd M, Kylanpaa L, Mustonen H, Lindstrom O, Halttunen J, et al. Dexmedetomidine impairs success of patient-controlled sedation in alcoholics during ERCP: a randomized, double-blind, placebo-controlled study. Surg Endosc. 2013; 27: 2163–2168. doi: 10.1007/s00464-012-2734-1 PMID: 23355148 References Position statement: Nonanesthesiologist administration of pro- pofol for GI endoscopy. Am J Gastroenterol. 2009; 104: 2886–2892. doi: 10.1038/ajg.2009.607 PMID: 19956113 15. Koczmara C, Perri D, Hyland S, Rousseaux L. Meperidine (Demerol) safety issues. Dynamics. 2005; 16: 8–12. 16. Thomson A, Andrew G, Jones DB. Optimal sedation for gastrointestinal endoscopy: review and recom- mendations. J Gastroenterol Hepatol. 2010; 25: 469–478. doi: 10.1111/j.1440-1746.2009.06174.x PMID: 20370725 17. Reves JG, Glass PSA, Lubarsky DA, McEvoy MD, Martines-Ruiz R. Intravenous Anesthetics. In: Miller RD, editor. Miller's anesthesia. Philadephia, PA: Churchill Livingstone/Elsevier; 2010. 18. Angsuwatcharakon P, Rerknimitr R, Ridtitid W, Kongkam P, Poonyathawon S, Ponauthai Y, et al. Cocktail sedation containing propofol versus conventional sedation for ERCP: a prospective, random- ized controlled study. BMC Anesthesiol. 2012; 12: 20. doi: 10.1186/1471-2253-12-20 PMID: 22873637 19. Chawla S, Katz A, Attar BM, Go B. Endoscopic retrograde cholangiopancreatography under moderate sedation and factors predicting need for anesthesiologist directed sedation: A county hospital experi- ence. World J Gastrointest Endosc. 2013; 5: 160–164. doi: 10.4253/wjge.v5.i4.160 PMID: 23596538 20. Mazanikov M, Udd M, Kylanpaa L, Mustonen H, Lindstrom O, Halttunen J, et al. Patient-controlled sedation for ERCP: a randomized double-blind comparison of alfentanil and remifentanil. Endoscopy. 2012; 44: 487–492. doi: 10.1055/s-0031-1291655 PMID: 22450724 21. Mazanikov M, Udd M, Kylanpaa L, Mustonen H, Lindstrom O, Halttunen J, et al. Dexmedetomidine impairs success of patient-controlled sedation in alcoholics during ERCP: a randomized, double-blind, placebo-controlled study. Surg Endosc. 2013; 27: 2163–2168. doi: 10.1007/s00464-012-2734-1 PMID: 23355148 11 / 11
https://openalex.org/W4225125676	https://zenodo.org/records/8042210/files/E35360611522.pdf	English	null	Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique	International journal of engineering and advanced technology	2,022	cc-by	9,764	Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Omar Mohamed, Mohamed Nour, Iman Elazizy, Mona A. Hagras can be defined as the inherent chances for either profit or loss associated with a particular endeavor which approved that risk occurs due to limited knowledge, and due to projects consequences versus targets (Rashid, 2009). Also, Business risk is the probability that an actual return of an investment will be less than the expected return. The continuing process to identify, analyze, evaluate and monitor risk to alleviate the adverse effect of loss (Saeed, 2018). Projects are exposed to both internal risks (financial, design, contractual, construction, personal, involved parties and operational risks) and external risks (economic, social, political, legal, public, logistical and environmental risks). All the risks may influence cost, or time of the project in both negative and positive ways (Choudhry et.al, 2018). Risk concerns the deviation of one or more results, or of one or more future events from expected value. Technically, the main purpose of RMP is solving problems that suffer due to deviation of project deliverables, as to create an alternatives to proper functioning under conditions (Pooworakulchai, 2018). can be defined as the inherent chances for either profit or loss associated with a particular endeavor which approved that risk occurs due to limited knowledge, and due to projects consequences versus targets (Rashid, 2009). Also, Business risk is the probability that an actual return of an investment will be less than the expected return. The continuing process to identify, analyze, evaluate and monitor risk to alleviate the adverse effect of loss (Saeed, 2018). Projects are exposed to both internal risks (financial, design, contractual, construction, personal, involved parties and operational risks) and external risks (economic, social, political, legal, public, logistical and environmental risks). All the risks may influence cost, or time of the project in both negative and positive ways (Choudhry et.al, 2018). Risk concerns the deviation of one or more results, or of one or more future events from expected value. Technically, the main purpose of RMP is solving problems that suffer due to deviation of project deliverables, as to create an alternatives to proper functioning under conditions (Pooworakulchai, 2018). Abstract: Construction and rehabilitation of water control structures in Egypt considered as an important project, as it influences the usage of water resources in Egypt which become limited resources due to water scarcity existed in the last decades. International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Also these projects helps in the optimization of water resources, Moreover it helps in the growth of agricultural and industrial sector. Project Risk Management (RMP) is considered as a vital and important tool in decision making, thus RMP used as a planning management system to detect risks affecting project deliverables; such as cost and time target. This research shows how to optimize the deliverables for construction of box culverts in Egypt, through a well-defined risk management framework and real case study for a certain project executed in the last decade. Finally, this study shows how to calculate cost and time contingency for these projects through an integrated risk management technique. Finally this study shows hazard risk identification and assessment for these type of projects. The conclusion of this study show that the cost contingency needed to resolve different risk factors arise in the shown case study is to increase the estimated budget with average value 11.50 percent on the total estimated budget, as well as the time contingency is found with average value 16.00 percent to be added over the total original baseline schedule of the construction project. g The Egyptian water resources system is composed to many interacting components and intermingles with social, economic and environmental systems, which are also complex and uncertain. Fresh water resources include River Nile flow, precipitation and groundwater from both renewable and non-renewable aquifers (Gunidy, 2015). There is a large number of hydraulic control structures in the Nile valley and the Nile Delta, which play an important role for controlling, distribution and allocation of water, but these structures suffers different stages of degradation. The main problems in these structures is hydraulic inefficiency resulting from leakage and dysfunctional of gate operating. Also these structures suffer from structural instability resulting from the erosion of their foundations and differential settlement caused by high traffic loads. With the increasing trend of replacing barrages in Egypt, especially those built before the High Dam in 1971, there is a need to reduce their construction costs. Barrages/regulators and box culverts are structures used to control water levels along irrigation canals. The main elements of a these structures consist of a floor, abutments, piers, and a bridge. The cost of these structures depends primarily on its floor and secondarily on its abutments, piers, and bridge. Keywords: Risk, Risk Management, Cost Contingency, Time Contingency, Construction of Box Culverts in Egypt. * Correspondence Author Omar Mohamed, Ph.D. Student of Irrigation and Hydraulics Engineering Department, Faculty of Engineering – Ain Shams University, Cairo, Egypt. Email: omar_mohamed@gasco.com.eg Cairo, Egypt. Email: omar_mohamed@gasco.com.eg Prof. Dr. Mohamed Nour, Professor of Irrigation and Hydraulics Engineering Department, Faculty of Engineering – Ain Shams University, Cairo, Egypt. Prof. Dr. Mohamed Nour, Professor of Irrigation and Hydraulics Engineering Department, Faculty of Engineering – Ain Shams University, Cairo, Egypt. Prof. Dr. Mohamed Nour, Professor of Irrigation and Hydraulics Engineering Department, Faculty of Engineering – Ain Shams University, Cairo, Egypt. gyp Email: mhmdour2@gmail.com Email: mhmdour2@gmail.com Prof. Dr. Iman Elazizy, Professor of Hydraulics and Water Resources, Irrigation and Hydraulics Department, Faculty of Engineering – Ain Shams University, Cairo, Egypt, Vice –President of Student Affairs at 6th of October University, Egypt. Prof. Dr. Iman Elazizy, Professor of Hydraulics and Water Resources, Irrigation and Hydraulics Department, Faculty of Engineering – Ain Shams University, Cairo, Egypt, Vice –President of Student Affairs at 6th of October University, Egypt. Prof. Dr. Iman Elazizy, Professor of Hydraulics and Water Resources, Irrigation and Hydraulics Department, Faculty of Engineering – Ain Shams University, Cairo, Egypt, Vice –President of Student Affairs at 6th of October University, Egypt. Email: VPSA@O6U.edu.eg Email: VPSA@O6U.edu.eg Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Dr. Mona A. Hagras, Associated Professor of Irrigation and Hydraulics Department, Faculty of Engineering – Ain Shams University, Cairo, Egypt. Email: mona_hagras@eng.asu.edu.eg © The Authors. Published by Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP). This is an open access article under the CC-BY-NC-ND license http://creativecommons.org/licenses/by-nc-nd/4.0/ Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Manuscript received on 20 April 2022. Manuscript received on 20 April 2022. Revised Manuscript received on 25 April 2022. I. INTRODUCTION R Risk is the state of uncertainty, as an event or condition that, if it occurs, has a positive or negative effect on projects deliverables. Managing risk is an integral part of sound management and risk management helps to achieve projects objectives (Omar et al., 2020). Moreover, Business Risk Manuscript received on 20 April 2022. Revised Manuscript received on 25 April 2022. Manuscript published on 30 June 2022. Correspondence Author Manuscript received on 20 April 2022. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org 40 ng Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Fig.1 Barrage and canal sections showing the hydraulic parameters (Ashour et al., 2009). Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Fig.1 Barrage and canal sections showing the hydraulic parameters (Ashour et al., 2009). A. Technical data about construction of box culvert Culvert is a hydraulic structure which may be fully or partially submerged in water stream. Culverts mostly change the natural flow of water. It can be used to divert, disrupt or completely stop water flow. II. PROBLEM STATEMENT AND RESEARCH SCOPE Project participants mostly take their judgment and control their project activates by their intuitions and experience and randomly data handled inside projects, not by performing systematic RMP. This leads to not achieve project deliverables (Zabaal, 2007). The aim of this research is to optimize the deliverables of box culvert construction project through a risk management framework and accurate calculation of cost and time contingency of these projects. This research depends on case study, and to show how to compute cost and time contingency using integrated risk management technique, as data is classified into the following: - Background information: The technical information about box culverts as water structure constructed in Egypt for the last decade. - Description of the real projects: As describing full analysis about the risk management process of a certain project, as real project is discussed; showing cost and time analysis for this project, also describing the different risks occurred, finally shows how to measure cost and time contingency needed. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Table – 1: Cost and Time Breakdown for a Box culvert construction project in Egypt: Project Phases (Milestones activities) Cost (1000 LE) Time (days) Conceptual and feasibility studies. (350) (179) Technical feasibility studies 150 30 Economic feasibility studies 100 30 Project decision (Bid or no-bid) & Revising Project Framework with Stakeholders 50 20 Issuing frame work of the project and it feature 50 100 Engineering phase (530) (115) Choose site location and conduct hydraulic studies /Site Survey analysis 30 5 Perform Soil analysis and Geotechnical analysis and issuing reports 200 10 Issuing Final detailed Drawing and Specification 300 100 Construction phase (I) (3550) (79) Mobilization and Site preparation 100 7 Construction of temporary Canal 200 9 Build buffers for Canal Diversion 125 10 Diversion of main stream 50 15 Construction of dewatering system 200 10 Excavation by using mechanical tools 125 5 Piling system 2000 5 Soil Test in Site and Reports 50 60 Foundation of pile Cap 700 10 Table – 1: Cost and Time Breakdown for a Box culvert construction project in Egypt: Moreover culverts can be built on any water stream for a specific function. Culverts are water structures that allows water to flow under a road, rail way road, or similar obstruction from one side to the other side. As may be typically embedded so as to be surrounded by soil (Nanni et al., 2001). And figure (3) shows how box culvert is used in the intersection between water stream (channel) and a highway road. Fig.3. Roadway cross culvert length Fig.3. Roadway cross culvert length Culverts design depends on hydraulic technical data, which should contain the culvert length, loading analysis, and other items that lead to the completed culvert plans and its dimension. Box culverts may be made of reinforced concrete frames as closed system which support vertical or lateral earth pressure to be passive or active and part of load may be vehicles load. Culverts either single or multi-cell based on the hydraulic requirements. Culverts life time depends on the material that used to resist corrosion, as culverts having high initial cost can have a longer service life and also can have lower total operation and maintains (O&M) cost. The (O&M) cost includes maintenance costs, operational cleaning cost and risks associated with flooding. Moreover, future maintenance requirements can also save money in the long run. Maintenance costs for culverts may result from channel erosion at the inlet of water stream and its outlet, also erosion, deterioration of the culvert invert, sedimentation and embankment repair in case of overtopping. 42 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. and udes isks ance run. nnel ion, and me time oject hree box uals oject with oject its d in and Continue Table – 1: Cost and Time Breakdown for a Box culvert construction project in Egypt: Project Phases (Milestones activities) Cost (1000 LE) Time (days) Construction phase (II) (4450) (126) Plain concrete execution for footings (Zone I) 200 20 Reinforced Concrete execution for foundation & Ret. Walls(Zone I) 1000 30 Plain concrete execution for footings (Zone II) 200 20 Reinforced Concrete execution for foundation & Ret. Walls(Zone II) 1000 30 R.C. Slabs 600 50 Concrete Tests in Site and Report 50 60 Pitching Work and Leveling and Backfilling 350 15 Side Walks and Bridges super-structures 250 25 hydraulic and technical checks 100 15 Erecting of Gates and Screening 500 15 Re-Divert to the main stream 100 20 Handover and Project Completion (100) (10) Continue Table – 1: Cost and Time Breakdown for a Box Continue Table – 1: Cost and Time Breakdown for a Box culvert construction project in Egypt: Project Phases (Milestones activities) Cost (1000 LE) Time (days) Construction phase (II) (4450) (126) Plain concrete execution for footings (Zone I) 200 20 Reinforced Concrete execution for foundation & Ret. Walls(Zone I) 1000 30 Plain concrete execution for footings (Zone II) 200 20 Reinforced Concrete execution for foundation & Ret. Walls(Zone II) 1000 30 R.C. Slabs 600 50 Concrete Tests in Site and Report 50 60 Pitching Work and Leveling and Backfilling 350 15 Side Walks and Bridges super-structures 250 25 hydraulic and technical checks 100 15 Erecting of Gates and Screening 500 15 Re-Divert to the main stream 100 20 Handover and Project Completion (100) (10) III. METHODLOGY 1 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. completely stop water flow. The main methodology of data formulation and its analysis is meeting with project participants, to provide benchmark for this study. The following flow chart figure (2) shows the study methodology: Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. 41 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org 41 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Finally the risk factors are ssified into: the magnitude of risk is from (15 to 25) for Red color, it ans that this RF is important high risk factors as those uld be avoided or to be controlled by some engineering or Table -3: Risk identification and qualitative assessment concerning cost target of Box culvert water structure project: Factors Q1 Q2 Q3 Q4 Q5 Q6  Unexpected changes in cash flow 10 10 12 8 12 8 10.00 Over-Design for components of the project 8 8 10 6 12 8 8.67 Assigning non-applicable constructability method 10 8 10 6 12 8 9.00 Site accessibility problems 15 15 17 13 15 17 15.33 Geotechnical and soil analysis troubles issues 12 12 14 10 12 14 12.33 Labor productivity lower than required 12 15 17 13 15 17 14.83 Material transportation delay 8 8 10 6 8 10 8.33 Change orders at construction phase 4 20 22 18 20 22 17.67 Bad weather conditions 12 15 17 13 15 17 14.83 Table - 4: Risk identification and qualitative assessment concerning time target of Box culvert water structure project: Factors Q1 Q2 Q3 Q4 Q5 Q6  Unexpected changes in cash flow 10 8 10 4 12 8 8.67 Over-Design for components of the project 8 8 8 4 12 8 8.00 Assigning non-applicable constructability method 12 8 12 12 16 8 11.33 Site accessibility problems 15 15 18 20 22 24 19.00 Continue Table -4: Risk identification and qualitative Severity obability Increase in budget and duration From 0 to 3% (1) From 3% to 10% (2) From 10% to 20% (3) From 20% to 30% (4) More than 30% (5) are (1) 1 2 3 4 5 nlikely (2) 2 4 6 8 10 ssible (3) 3 6 9 12 15 kely (4) 4 8 12 16 20 rtain (5) 5 10 15 20 25 Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Box Culverts Construction Projects using Integrated Risk Management Technique Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique A. Risk identification and qualitative assessment for a box culvert s project Risk identification is considered as a critical step in the risk management process, risk identification is an organized, through approach to finding real risks associated with a project. Risk identification determines which risks might affect the project and their characteristics at each project phase. In this study, Participants in risk identification and assessment activities are (6) personnel whom are: Project managers for (Owner reprehensive - Consultant & Contractor) and project site engineers for contractors. Risk probability assessment investigates the likelihood that each specific risk will occur, risk impact assessment investigates the potential effect on a project objective. Notice that the influence of risk factors (RF) on the matrices are assigned in table –2 Table-2: Numerical qualitative risk analysis matrix for the project cost. Where: “Red Color shows important high risk factors, Yellow Color shows high risk factors, Green Color shows medium risk factors”: Severity Probability Increase in budget and duration From 0 to 3% (1) From 3% to 10% (2) From 10% to 20% (3) From 20% to 30% (4) More than 30% (5) Rare (1) 1 2 3 4 5 Unlikely (2) 2 4 6 8 10 Possible (3) 3 6 9 12 15 Likely (4) 4 8 12 16 20 Certain (5) 5 10 15 20 25 Table - 4: Risk identification and qualitative assessment concerning time target of Box culvert water structure project: Table - 4: Risk identification and qualitative assessment concerning time target of Box culvert water structure project: Factors Q1 Q2 Q3 Q4 Q5 Q6  Unexpected changes in cash flow 10 8 10 4 12 8 8.67 Over-Design for components of the project 8 8 8 4 12 8 8.00 Assigning non-applicable constructability method 12 8 12 12 16 8 11.33 Site accessibility problems 15 15 18 20 22 24 19.00 Table- 2 shows the frequency of each RF occurs as: (Rare) from zero to one time all over the project, (Unlike hood) two times through the project, (Possible) three times through the project, (Likely) four times through the project, (Frequently) five or more through the project. Finally the risk factors are classified into: -If the magnitude of risk is from (15 to 25) for Red color, it means that this RF is important high risk factors as those should be avoided or to be controlled by some engineering or administrative control measures, also these risk factors should be subjected to frequent assessment. Background for the project and discussing cost and time breakdown The following Table-1 and Figure (4) shows cost and time breakdown allover project life cycle for Box culvert project with number of three vents, each vent width equals three meters and with height of vent is about 2.50m. This box culvert exists in Alexandria city , as canal width equals 12meters, This project executed in 2016 with total project budget 9 100 000 L.E. (9Million 100 Thousand L.E.) with duration about 294days (9.8 Months). Moreover, this realistic water structure construction project in Egypt began in 5/2016, as the project began in its conceptual and feasibility studies phase and finished in 3/2017 after rediverting water stream to the main stream and accomplish handover of the project. Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. 2 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org 42 Construction Projects using Integrated Risk Management ique Table -3: Risk identification and qualitative assessment concerning cost target of Box culvert water structure project: Factors Q1 Q2 Q3 Q4 Q5 Q6  Unexpected changes in cash flow 10 10 12 8 12 8 10.00 Over-Design for components of the project 8 8 10 6 12 8 8.67 Assigning non-applicable constructability method 10 8 10 6 12 8 9.00 Site accessibility problems 15 15 17 13 15 17 15.33 Geotechnical and soil analysis troubles issues 12 12 14 10 12 14 12.33 Labor productivity lower than required 12 15 17 13 15 17 14.83 Material transportation delay 8 8 10 6 8 10 8.33 Change orders at construction phase 4 20 22 18 20 22 17.67 Bad weather conditions 12 15 17 13 15 17 14.83 ncy for Box Culverts Construction Projects using Integrated Risk Management Technique SCUSSION ssessment for a box itical step in the risk on is an organized, s associated with a which risks might stics at each project sk identification and l whom are: Project - Consultant & r contractors. s the likelihood that impact assessment ect objective. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Background for the project and discussing cost and time breakdown Notice on the matrices are analysis matrix for r shows important s high risk factors, rs”: RF occurs as: (Rare) t, (Unlike hood) two Table -3: Risk identification and qualitative assessment concerning cost target of Box culvert water structure project: Factors Q1 Q2 Q3 Q4 Q5 Q6  Unexpected changes in cash flow 10 10 12 8 12 8 10.00 Over-Design for components of the project 8 8 10 6 12 8 8.67 Assigning non-applicable constructability method 10 8 10 6 12 8 9.00 Site accessibility problems 15 15 17 13 15 17 15.33 Geotechnical and soil analysis troubles issues 12 12 14 10 12 14 12.33 Labor productivity lower than required 12 15 17 13 15 17 14.83 Material transportation delay 8 8 10 6 8 10 8.33 Change orders at construction phase 4 20 22 18 20 22 17.67 Bad weather conditions 12 15 17 13 15 17 14.83 Table - 4: Risk identification and qualitative assessment concerning time target of Box culvert water structure project: Factors Q1 Q2 Q3 Q4 Q5 Q6  Unexpected changes in cash flow 10 8 10 4 12 8 8.67 Over-Design for components of the project 8 8 8 4 12 8 8.00 Assigning non applicable and duration From 20% to 30% (4) More than 30% (5) 4 5 8 10 12 15 16 20 20 25 Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique omputing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique IV. ANAYLSIS AND DISCUSSION Risk identification and qualitative assessment for a box vert s project k identification is considered as a critical step in the risk nagement process, risk identification is an organized, ough approach to finding real risks associated with a ject. Risk identification determines which risks might ect the project and their characteristics at each project se. In this study, Participants in risk identification and essment activities are (6) personnel whom are: Project nagers for (Owner reprehensive - Consultant & ntractor) and project site engineers for contractors. k probability assessment investigates the likelihood that h specific risk will occur, risk impact assessment estigates the potential effect on a project objective. Notice t the influence of risk factors (RF) on the matrices are gned in table –2 ble-2: Numerical qualitative risk analysis matrix for project cost. Where: “Red Color shows important h risk factors, Yellow Color shows high risk factors, een Color shows medium risk factors”: ble- 2 shows the frequency of each RF occurs as: (Rare) m zero to one time all over the project, (Unlike hood) two es through the project, (Possible) three times through the ject, (Likely) four times through the project, (Frequently) e or more through the project. B. Quantitative risk analysis (QRA) by AHP for a box culvert project The methodology of QRA is using Analytical hierarchy process (AHP) to measure cost and time contingency for the case study shown. AHP developed by Thomas L.Saaty based on pair wise compression as it is consider as a multi-criteria decision-making approach. These comparisons are used to obtain the weights of importance of the decision criteria, and the relative performance measures of the alternatives in terms of each individual decision criterion. The assigned scale in this case study from 1 to 9 is adopted. In that scale, a score of 1 indicates equal importance and a score of 9 indicates that an element is extremely important than the other. Meanwhile, reciprocals to these scores (i.e. 1/2, 1/3...1/9) represent the counter importance relationships for assigning numerical scale values, survey forms are designed. Geotechnical and soil analysis troubles issues is a medium influence risk factor for both cost and time target , as soil – analysis investigation reports and studies is one of the important studies that must be accomplished in the engineering phase where early duration of project cycle and continual updated , thus to define all the features and criteria of the soil and then design all the footings and sub-structure of the project executed , so any change in the expected feature of this soil would give change in the design of the structure, which may lead to higher cost or delaying in the project duration due to change in recommendation of the soil or change in the statically system of the structure. A. Risk identification and qualitative assessment for a box culvert s project Sensitivity analysis concerning the influence of both cost (in hatched) and time target (in solid) – box culvert construction project B Quantitative risk analysis (QRA) by AHP for a box International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 Fig .5. Sensitivity analysis concerning the influence of both cost (in hatched) and time target (in solid) – box culvert construction project B Quantitative risk analysis (QRA) by AHP for a box Material transportation delay 12 12 14 10 10 12 11.67 Change orders at construction phase 8 20 22 18 20 22 18.33 Bad weather conditions 12 20 22 20 22 24 20.00 Fig .5. Sensitivity analysis concerning the influence of both cost (in hatched) and time target (in solid) – box culvert construction project Thus the RF “Changes orders at construction phase “is highest influence with perspective to cost target, where this factor depends on number of change orders done in the project and if there is change orders effect on the critical path of the project or not, as handling this point is so important to not have conflicts when the project is executed. Moreover change order has a lot of other terminology; such as change proposal at engineering phase and variation order at construction phase. Fig .5. Sensitivity analysis concerning the influence of both cost (in hatched) and time target (in solid) – box culvert construction project B. Quantitative risk analysis (QRA) by AHP for a box culvert project A. Risk identification and qualitative assessment for a box culvert s project Continue Table -4: Risk identification and qualitative assessment concerning time target of Box culvert water structure project: Continue Table -4: Risk identification and qualitative assessment concerning time target of Box culvert water structure project: Factors Q1 Q2 Q3 Q4 Q5 Q6  Geotechnical and soil analysis troubles issues 12 12 14 10 10 12 11.67 Labor productivity lower than required 12 15 17 13 20 22 16.50 Continue Table -4: Risk identification and qualitative assessment concerning time target of Box culvert water structure project: Factors Q1 Q2 Q3 Q4 Q5 Q6  Geotechnical and soil analysis troubles issues 12 12 14 10 10 12 11.67 Labor productivity lower than required 12 15 17 13 20 22 16.50 -If the magnitude of risk is from (6 to 12) for Yellow color, it means that the risk is high risk factors, so it has to be monitored but less priority than important high risk factors. -If the magnitude of risk is from (1 to 6) for Green color, it means that the risk is medium so to be monitored and controlled by a lower cost engineering and administrative control. And the tables below (3) & (4) shows the risk factor identified and assessed through project participants as the risk factors are nine risk factors affecting on both cost and time targets. 43 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. or e d 43 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org International Jo Material transportation delay 12 12 14 10 10 12 11.67 Change orders at construction phase 8 20 22 18 20 22 18.33 Bad weather conditions 12 20 22 20 22 24 20.00 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 10 12 11.67 20 22 18.33 22 24 20.00 struction phase “is t target, where this orders done in the t on the critical path nt is so important to executed. Moreover gy; such as change variation order at hest influence with d hi f Fig .5. Moreover, for calculating cost contingency the methodology is formulated by the following steps: - The First step is calculating the effectiveness (Eff.) for each project phase , to use this Eff. factor in the AHP cycle , table (5) shows the breakdown of the project, as the project is simply classified into main three phases as shown (Conceptual &feasibility studies and Engineering phase Construction phase (I) - Construction phase (II) and handover. And the sensitivity analysis for the means values showing the influences concerning budget and duration of the project is shown in figure (5): - The second step is beginning with the AHP analysis model for the three phases of the project with the mathematical model shown in tables (6), (7), and (8). 44 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Table-5: Risk effectiveness in each project phase concerning cost target: Project phases Cost (LE) *1000 Eff. (Box- Culvert water) Conceptual and feasibility studies & Engineering phase 880 0.0979 Construction phase (I). 3550 0.3953 Construction phase (II) & handover 4550 0.5066 Total Project cost 8 980 Table-5: Risk effectiveness in each project phase concerning cost target: Table-5: Risk effectiveness in each project phase concerning cost target: Total Project cost 44 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. 44 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Cost contingency in (percent) = 0.80 (Sc) + (S.V.) (1) As the result shows Summation of Influences affecting cost target (Sc) in tables (7) , (8) and (9) equal to (10.57) and also standard deviation (S.V.) is equal (3.45) , so equation (1) gives a cost contingency equal (11.91perecnt) , as this contingency needed to be add on the original estimated budget of the project. Moreover, for calculating cost contingency the methodology is formulated by the following steps: Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 And the calculation of cost contingency is adopted by superposition of the three tables shown above, and by the following equation: International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 And the calculation of cost contingency is adopted by superposition of the three tables shown above, and by the following equation: Cost contingency in (percent) = 0.80 (Sc) + (S.V.) (1) Moreover, for calculating time contingency the methodology is formulated by the following steps: - The First step is calculating the effectiveness (Eff.) for each project phase , to use this Eff. factor in the AHP cycle , table (9) shows the time breakdown of the project , as the project is simply classified into main three phases as shown (Conceptual &feasibility studies and Engineering phase – Construction phase (I) - Construction phase (II) and handover. - The second step beginning the AHP analysis model for the three phases of the project with the mathematical model in shown in tables (10), (11), and (12). - The First step is calculating the effectiveness (Eff.) for each project phase , to use this Eff. factor in the AHP cycle , table (9) shows the time breakdown of the project , as the project is simply classified into main three phases as shown (Conceptual &feasibility studies and Engineering phase – Construction phase (I) - Construction phase (II) and handover. &feasibility studies and Engineering phase – Construction phase (I) - Construction phase (II) and handover. - The second step beginning the AHP analysis model for the three phases of the project with the mathematical model in shown in tables (10), (11), and (12). g g p p p beginning the AHP analysis model for the three phases of the project with the mathematical model in (11), and (12). Table-9: Risk effectiveness in each project phase concerning time target: Project phases Duration (Days) Eff. (Box- Culvert water) Conceptual and feasibility studies & Engineering phase 132 0.449 Construction phase (I). 84 0.2857 Construction phase (II) & handover 78 0.2653 Total Project duration 294 Table-9: Risk effectiveness in each project phase concerning time target: Table-9: Risk effectiveness in each project phase concerning time target: Project phases Duration (Days) Eff. (Box- Culvert water) Conceptual and feasibility studies & Engineering phase 132 0.449 Construction phase (I). Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Moreover, for calculating cost contingency the methodology is formulated by the following steps: Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique T bl 6 P i i i d AHP l i ( i ) f RF l &f ibili di Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Table -6: Pairwise compression and AHP analysis (concerning cost target) for RF at conceptual &feasibility studies and engineering phase: Table -7: Pairwise compression and AHP analysis (concerning cost target) for RF at Construction phase (I): Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Table -6: Pairwise compression and AHP analysis (concerning cost target) for RF at conceptual &feasibility studies and engineering phase: Table -7: Pairwise compression and AHP analysis (concerning cost target) for RF at Construction phase (I): Table -8: Pairwise compression and AHP analysis (concerning cost target) for RF at Construction phase (II) and handover: rwise compression and AHP analysis (concerning cost target) for RF at conceptual &feasibility stud and engineering phase: Table -7: Pairwise compression and AHP analysis (concerning cost target) for RF at Construction phase (I): Table -8: Pairwise compression and AHP analysis (concerning cost target) for RF at Construction phase (II) and handover: Table -7: Pairwise compression and AHP analysis (concerning cost target) for RF at Construction phase (I): Table -8: Pairwise compression and AHP analysis (concerning cost target) for RF at Construction phase (II) and handover: irwise compression and AHP analysis (concerning cost target) for RF at Construction phase (I): Table -7: Pairwise compression and AHP analysis (concerning cost target) for RF at Constru 45 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Table -8: Pairwise compression and AHP analysis (concerning cost target) for RF at Construction phase (II) and handover: Table -8: Pairwise compression and AHP analysis (concerning cost target) for RF at Construction phase (II) and handover: Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Time contingency in (percent) = 0.80 (St) + (S.V.) (2) As the result shows Summation of Influences affecting time target (St) in tables (10) , (11) and (12) is equal to (13.91) and also standard deviation (S.V.) is equal (4.86) , so equation (2) gives a time contingency equals (15.99 percent) , as this contingency needed to be added on the baseline time schedule of the project. Hazard Identification (HAZID): This is the process of examining each work area and work task for the purpose of identifying all the hazards which are “inherent in the job”. Work areas include but are not limited to machine workshops, laboratories, office areas, agricultural and horticultural environments, stores and transport, maintenance and grounds, reprographics, and lecture theatres and teaching spaces. (2) Time contingency in (percent) = 0.80 (St) + (S.V.) (2) Moreover, for calculating time contingency the methodology is formulated by the following steps: 84 0.2857 Construction phase (II) & handover 78 0.2653 Total Project duration 294 Table- 10: Pairwise compression and AHP analysis (concerning time target) for risk factors at Conceptual & feasibility studies and Engineering phase: Table- 11: Pairwise compression and AHP analysis (Concerning time target) for risk factors at Construction phase (I): Table- 10: Pairwise compression and AHP analysis (concerning time target) for risk factors at Conceptual & feasibility studies and Engineering phase: Table- 11: Pairwise compression and AHP analysis (Concerning time target) for risk factors at Construction phase (I): Table- 11: Pairwise compression and AHP analysis (Concerning time target) for risk factors at Construction phase (I): 46 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org 46 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Table- 12: Pairwise compression and AHP analysis (Concerning time target) for risk factors at Construction phase (II) & handover: And the calculation of cost contingency is adopted by superposition of the three tables shown above, and by the following equation: Time contingency in (percent) = 0.80 (St) + (S.V.) (2) there are some definitions concerning safety risk management issues as follows: Hazard: Anything (e.g. condition, situation, practice, behavior) that has the potential to cause harm, including injury, disease, death, environmental, property and i d A h d b hi i i and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Table- 12: Pairwise compression and AHP analysis (Concerning time target) for risk factors at Construction phase (II & handover: And the calculation of cost contingency is adopted by superposition of the three tables shown above, and by the following equation: And the calculation of cost contingency is adopted by superposition of the three tables shown above, and by the following equation: there are some definitions concerning safety risk management issues as follows: Hazard: Anything (e.g. Moreover, for calculating time contingency the methodology is formulated by the following steps: condition, situation, practice, behavior) that has the potential to cause harm, including injury, disease, death, environmental, property and equipment damage. A hazard can be a thing or a situation. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org V. HAZARD IDENTIFICATION AND RISK ASSESSMENT Flammable material. Failure of mechanical parts and may cause damage of equip. or human injury - Slipping from liquid. (1,4) Low Rotatory machine. Unsafe equipment or tools. (1,4) Low Water jet. Severity Probability Concerning Injury and asset loss A B C D E Rare (1) 1 2 3 4 5 Unlikely (2) 2 4 6 8 10 Possible (3) 3 6 9 12 15 Likely (4) 4 8 12 16 20 Certain (5) 5 10 15 20 25 HAZOP: is to conduct a meeting for complying the proposed design with operation and safe conditions The tables (14) & (15) and (16) show how to identify hazard risks in construction of box culverts as to show HAZID process and risk assessment in table (14), also they show how to control this risk using exiting and additional controls and how to calculate residuals influence value after controlling hazard risk arise in the project as these points shown in tables (15) & (16). Also, table (13) shows hazard risk matrix which adopted in this type of construction project in Egypt. Table-13: Numerical hazard qualitative risk analysis matrix Table-13: Numerical hazard qualitative risk analysis matrix Table-13: Numerical hazard qualitative risk analysis matrix Severity Probability Concerning Injury and asset loss A B C D E Rare (1) 1 2 3 4 5 Unlikely (2) 2 4 6 8 10 Possible (3) 3 6 9 12 15 Likely (4) 4 8 12 16 20 Certain (5) 5 10 15 20 25 Table-13: Numerical hazard qualitative risk analysis matrix Table-13: Numerical hazard qualitative risk analysis matrix Severity Probability Concerning Injury and asset loss A B C D E Rare (1) 1 2 3 4 5 Unlikely (2) 2 4 6 8 10 Possible (3) 3 6 9 12 15 Likely (4) 4 8 12 16 20 Certain (5) 5 10 15 20 25 Where: - A is the hazard severity leads to injury to be resolved by first aid or asset loss is less to 300K Egyptian pound. - B is the hazard severity leads to minor injury or asset loss is less than 400K Egyptian pound. - C is the hazard severity leads to major injury or asset loss is less than 500K Egyptian pound. - D is the hazard severity leads to single fatality or asset loss less than 600K Egyptian pound. V. HAZARD IDENTIFICATION AND RISK ASSESSMENT Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. and grounds, reprographics, and lecture theatres and teaching spaces. ng P) , . Hazard identification and risk assessment is an important issue affecting the project success. And should be integrated by the above RMP to achieve project objective. Moreover, Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org 47 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 ISSN: 2249-8958 (Online), Volume-11 Issue-5, June HAZOP: is to conduct a meeting for complying the proposed design with operation and safe conditions The tables (14) & (15) and (16) show how to identify hazard risks in construction of box culverts as to show HAZID pr and risk assessment in table (14), also they show how to control this risk using exiting and additional controls and h calculate residuals influence value after controlling hazard risk arise in the project as these points shown in tables (15) & Also, table (13) shows hazard risk matrix which adopted in this type of construction project in Egypt. Table-13: Numerical hazard qualitative risk analysis matrix Where: - A is the hazard severity leads to injury to be resolved by first aid or asset loss is less to 300K Egyptian pound. - B is the hazard severity leads to minor injury or asset loss is less than 400K Egyptian pound. - C is the hazard severity leads to major injury or asset loss is less than 500K Egyptian pound. - D is the hazard severity leads to single fatality or asset loss less than 600K Egyptian pound. - E is the hazard severity leads to Multi- fatality or asset loss more than 600K Egyptian pound. Table-14: Hazard risk identification and assessment for box culvert construction projects: Activates Source of Danger Hazard ID. Basic risk rank (BRR) & (SOD) (HAZID) (S,P) Sub-activates For construction phase: Mobilization. Heavy loads. Dropping heavy loads from height. - (3, 4) Med. Electrical sources. An electrical shock causes injury or fatality of workers. (2, 4) Med. Mechanical equipment. Vehicles collision with personnel or with other vehicles. Moving vehicles. Stay in pinch point or position leads to injury of a personnel. - (3, 4) Med. V. HAZARD IDENTIFICATION AND RISK ASSESSMENT - E is the hazard severity leads to Multi- fatality or asset loss more than 600K Egyptian pound. - A is the hazard severity leads to injury to be resolved by first aid or asset loss is less to 300K Egyptian pound. - B is the hazard severity leads to minor injury or asset loss is less than 400K Egyptian pound. - C is the hazard severity leads to major injury or asset loss is less than 500K Egyptian pound. - D is the hazard severity leads to single fatality or asset loss less than 600K Egyptian pound. - E is the hazard severity leads to Multi- fatality or asset loss more than 600K Egyptian pound. Table-14: Hazard risk identification and assessment for box culvert construction projects: Activates Source of Danger Hazard ID. Basic risk rank (BRR) & (SOD) (HAZID) (S,P) Sub-activates For construction phase: Mobilization. Heavy loads. Dropping heavy loads from height. - (3, 4) Med. Electrical sources. An electrical shock causes injury or fatality of workers. (2, 4) Med. Mechanical equipment. Vehicles collision with personnel or with other vehicles. Moving vehicles. Stay in pinch point or position leads to injury of a personnel. - (3, 4) Med. Flammable material. Failure of mechanical parts and may cause damage of equip. or human injury - Slipping from liquid. (1,4) Low Rotatory machine. Unsafe equipment or tools. (1,4) Low Water jet. Table-14: Hazard risk identification and assessment for box culvert construction projects: 48 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org ing SP) Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Continue Table-14: Hazard risk identification and assessment for box culvert construction projects: Dewatering system. Lack or appetence of oxygen. Failing soil or rocks from of excavation intend that from an excavator or loader may cause damage of equip. or human injury. (3, 4) Med. Negative heights. Failure in generator electrical insulation. Harmful H2S. Noise pollution form generator may harm workers for higher exposure. (2, 4) Med. Harmful insects. Confined Space entry. Free fall of material due to gravity. Soil failure. (3, 4) Med. Excavation and temporary path rerouting. Equipment noise pollution. Breathing H2S (3, 4) Med. Piles and foundation. V. HAZARD IDENTIFICATION AND RISK ASSESSMENT Sparks lead to fire ignition (fire hazard). (3,5) high Concrete work. Sources of ignition. Harms in respiratory system of personnel due to breathing cement fumes and vapor. (2, 5) med. Sharp Heavy physical objects. (3, 5) high. Sidewalks and bridge. Free fall of material due to gravity. (3, 3) med. Pitching and backfilling. Sharp edges. Backfill the temporary path and finishes. Cement breathing. d Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique Continue Table-14: Hazard risk identification and assessment for box culvert construction projects: Table-15: Hazard existing and additional control of box culvert construction project: Activates Existing Controls ( proactive “pr.” / reactive”re.”) - (Infrastructure “I.” / Equipment “Eq.” / human “H.”) - (Administrative “Ad.” / Engineering “Eng.”) Additional Controls &Sub-activates Mobilization. Issuing and monitor site layouting (I-pr.-Ad.). - Assigning more insulation for electrical parts. Issuing and monitor lifting Plan (I.-pr.-Ad.). -Wearing protective personnel equipment “PPE” for electrical hazard. Install warning signs (I.-pr.-Ad.). - Updating site layouting and concentrating to mitigate new pinch point according to project progress Issuing and monitor site traffic plans (I.-pr.-Ad.). Review equipment certificate (Eq.-pr.-Ad.). Asses escape plan from site (I.-re.-Ad.). Review Personnel and work competency (H.-pr.-Ad.). Training of first Aid (H.-pr.-Ad.). Dewatering system. Review all geotechnical analysis study and used approved shoring system (I.-pr.-Eng.). Continual update of lifting and emergency plans including escape plans and site injury mitigation. Use an appropriated excavation width for trenches with approved design (I.-pr.-Eng.). Discussion of loading analysis and design during construction of foundation and piling including vibration and lateral load for trucks and loaders and excavators. Excavation and temporary path rerouting. Appropriate maintenance workshop and available spare parts (Eq.-pr.-Ad.). More discussion of accident scenarios during construction to mitigate or eliminate these accidents. Piles and foundation. Wearing protective personnel equipment “PPE” & site clinic and Ambulance (H.-pr.-Ad.) Safe guards and handrail. Concrete work. Review workers and contractor and tool competency towards activates accomplish and complying with HSE (H.-pr.-Ad.). Install sparks arrestors. Safety induction and awareness (H.-pr.-Ad.). Decrease exposure time of noise for workers and use earmuffs for workers. Sidewalks and bridge. Ensuring Implementation of equipment preventive maintenance (Eq.-pr.-Ad.). Use gas detectors to avoid exposure to harmful gases Pitching and backfilling. Use ready mix concrete in site (Eq.-pr.-Eng.). Backfill the temporary path and finishes. VI. CONCLUSION 3. Nael Zabaal (2007), "Risk management of pipeline infrastructure projects in Egypt". Thesis (M.Sc.) Faculty of Engineering, Cairo University, Cairo, Egypt. This study underline the significance of accomplishing a successful risk management process for box culvert construction project. As these projects are having interrelated activities and specific risk factors. Also these specific risk factors are mostly affecting cost and time targets, thus give obligation of identifying these risk factors and showing their influences on both estimated budget and duration. 4. Omar Mohamed, Mohamed Nour, Iman Elazizy, Mona A. Hagras (2020), “A suggested module for risk framework of water Control Construction Projects using qualitative and quantitative techniques” International Journal of Engineering and Advanced Technology (IJEAT), ISSN: 2249 – 8958, Volume-9 Issue-3. 5. Pooworakulchai, C. (2018), "Applied risk management in construction industry: A Review ". (Research gate) International Journal of engineering technologies and management research, Vol.5 (Iss.3), ISSN: 2454-1907. And the main conclusion shows in this study is as follows: 6. Project Management Institute (PMI). A Guide to the Project management Body of Knowledge (PMBOK Guide), 2017 6thEdition, ANSI/PMI 99-001-2017, Newton Square, Pennsylvania. ✓ This study shows how to perform a RMP for box culvert construction project and also how to use data come out from this process to achieve project deliverables. q y 7. Rafiq M. Choudhry, Sana Rafiq, Anwar Ahmed, Tauqir Ahmed (2018), “Estimating Probabilistic Cost and Time Contingency for Residential Building Projects". (Research gate) Proceedings of the 3rd International Conference on Civil, Structural and Transportation Engineering (ICCSTE'18), Niagara Falls, Canada – June 10 – 12, 2018, Paper No. 134, DOI: 10.11159/iccste18.134. ✓ According to the case study shown , the cost contingency needed to resolve different risk factors arise in the shown case study is to increase the estimated budget by 11.50 percent on the total estimated budget of the project , and time contingency 16.00 percent to be added over the total original baseline schedule. 8. Saeed, S. "Cost and Time Risk Management in Construction Projects". (Research gate) Tikrit Journal of Engineering Sciences, ISSN: 1813-162X (Print); 2312-7589 (Online), Kirkuk University, Iraq. 9. Tarek Alkhrdaji and Antonio Nanni (2001), “Design, Construction, and Field-Testing of an RC Box Culvert Bridge Reinforced with GFRP Bars ". (Research gate) Center for Infrastructure Engineering Studies (CIES), University of Missouri-Rolla, USA. V. HAZARD IDENTIFICATION AND RISK ASSESSMENT Wearing protective personnel equipment “PPE” & site clinic and Ambulance (H.-pr.-Ad.). e-15: Hazard existing and additional control of box culvert construction project: Decrease exposure time of noise for workers and use earmuffs for workers. Use gas detectors to avoid exposure to harmful gases ing SP) 49 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249-8958 (Online), Volume-11 Issue-5, June 2022 Table -16: Hazard analysis and values of residual risk rank for box culvert construction project: Activates& Hazard ID. Residual risk rank (RRR) Sub-activates (HAZID) (S,P) Mobilization. - Dropping heavy loads from height. (2,4) Med. - An electrical shock cause injury or fatality of workers. (1,4) Med. - Vehicles collision with personnel or with other vehicles. (2,4) Med. - Stay in pinch point or position leads to injury of a personnel. (1,4) Low - Failure of mechanical parts and may cause damage of equip. or human injury (1,4) Low Dewatering system. Failing soil or rocks from of excavation intend that from an excavator or loader may cause damage of equip. or human injury. (3, 4) Med. Failure in generator electrical insulation. Noise pollution form generator may harm workers for higher exposure. (1, 4) Med. Confined Space entry. Excavation and temporary path rerouting. Soil failure. (2, 4) Med. Piles and foundation. Breathing H2S (2, 4) Med. Concrete work. Sparks lead to fire ignition (fire hazard). (2, 5) Med. Sidewalks and bridge. Harms in respiratory system of personnel due to breathing cement fumes and vapor. (2, 5) med. Pitching and backfilling. (3, 4) med. Backfill the temporary path and finishes. (3, 3) med. Table -16: Hazard analysis and values of residual risk rank for box culvert construction project: VI. CONCLUSION ✓ This study shows in a tubular from the hazard risk identification (HAZID) framework and its assessment, as to help to perform the project in safe condition without accidents, as health safety and environment (HSE) is an important issue in construction projects. 10. Thomas L.Saaty (2013), "Decision Making with the Analytic ". Network Process International Series in Operations Research & Management Science Volume 195, Stanford University, Stanford, CA, USA. 11. W. Gunidy, "Egypt is the gift of Nile ", National water research center, Ministry of Water resources and irrigation, 2015, Cairo, Egypt. REFERENCES 1. Ibrahim Abdel Rashid (2009), "Risk and Risk Management definitions” Lectures in construction management, Faculty of Engineering, Cairo University, 2009, Cairo, Egypt. 1. Ibrahim Abdel Rashid (2009), "Risk and Risk Management definitions” Lectures in construction management, Faculty of Engineering, Cairo University, 2009, Cairo, Egypt. Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. 2. M. A. Ashour , S. T. El Attar , Y. M. Rafaat and M. N. Mohamed (2009) , “Water resources management in Egypt” , Journal of Engineering Sciences, Assiut University, Vol. 37, No. 2, pp. 26979. Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org 50 Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique AUTHORS PROFILE Box Culverts Construction Projects using Integrated Risk Management Technique Computing Cost and Time Contingency for Box Culverts Construction Projects using Integrated Risk Management Technique AUTHORS PROFILE Eng. Omar Mohamed, studied Civil Engineering at faculty of Engineering / Ain Shams University from 1999 to 2004. And awarded Master of Science (M.Sc.) for Arab Academy for Science and Technology and Maritime (AAST) at April 2016 with thesis entitled (Risk management in construction of gas plants and gas pipelines in Egypt. I began my Ph.D. research work in Faculty of Engineering / Ain shams University at 2017 and I’m still a Ph.D. student in the topic of (Risk management in the construction of water control structures). I’m still working at in Cairo, Egypt as a civil Engineer. And also I attend a lot of technical conference and sessions as a Technical speaker, and I’m looking forward to attend a conference in Egypt known as EGYPS2020. Professor Dr. Mohamed Nour El-Din, is a Water Resources scientist with extensive long experience in applying integrated water resources management and development in arid and semi-arid regions, water supply, rural sanitation, water resources planning, monitoring and evaluation of irrigation projects and drainage systems. The main experience is in Egypt, as well as several assignments in other countries as well through cooperation with consultants and international organizations. The experience is acquired also through his career as a university professor and as a consultant in several water resources and irrigation projects. And mainly the current work is a Professor of hydraulics and irrigation in faculty of Engineering / Ain shams University. Prof. Dr. Iman Elazizy, is a Vice –president of student affairs at 6th of October University, and Professor of Hydraulics and water resources - Faculty of Engineering., Ain Shams University Cairo, Egypt. The research interest is; Water quality modeling, Water recourses management, natural treatment process, wetland, GIS application in water recourses, Open channel hydraulics. Environmental Impact Assessment. Works as consultant for Environmental water project for EEAA Egypt. Dr. Mona A. Hagras, is Associate Professor of Irrigation& Hydraulics Department, Faculty of Engineering, Ain Shams University, Cairo, Egypt. She received PhD and MSc from Ain Shams University. She supervised many theses and published many papers in referred national and international journals. She is a reviewer of ASEJ journals. Her area of interest is Water Resources Management, Hydrology, Hydraulics, and Groundwater modeling. 51 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org AUTHORS PROFILE umber: 100.1/ijeat.E35360611522 940/ijeat.E3536.0611522 bsite: www.ijeat.org 51 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. 51 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org Retrieval Number: 100.1/ijeat.E35360611522 DOI: 10.35940/ijeat.E3536.0611522 Journal Website: www.ijeat.org 51
https://openalex.org/W2953529840	https://scholarworks.rit.edu/cgi/viewcontent.cgi?article=3009&context=article	English	null	Eating Competent Parents of 4th Grade Youth from a Predominantly Non-Hispanic White Sample Demonstrate More Healthful Eating Behaviors than Non-Eating Competent Parents	Nutrients	2,019	cc-by	11,946	Eating Competent Parents of 4th Grade Youth from a Eating Competent Parents of 4th Grade Youth from a Predominantly Non-Hispanic White Sample Demonstrate More Predominantly Non-Hispanic White Sample Demonstrate More Healthful Eating Behaviors than Non-Eating Competent Parents Healthful Eating Behaviors than Non-Eating Competent Parents Follow this and additional works at: https://repository.rit.edu/article This Article is brought to you for free and open access by the RIT Libraries. For more information, please contact repository@rit.edu. Rochester Institute of Technology Rochester Institute of Technology RIT Digital Institutional Repository RIT Digital Institutional Repository Articles Faculty & Staff Scholarshi 6-30-2019 Eating Competent Parents of 4th Grade Youth from a Eating Competent Parents of 4th Grade Youth from a Predominantly Non-Hispanic White Sample Demonstrate More Predominantly Non-Hispanic White Sample Demonstrate More Healthful Eating Behaviors than Non-Eating Competent Parents Healthful Eating Behaviors than Non-Eating Competent Parents Barbara A. Lohse Rochester Institute of Technology Melissa Pflugh Prescott University of Illinois Urbana-Champagne Leslie Cunningham-Sabo Colorado State University Faculty & Staff Scholarship Received: 29 April 2019; Accepted: 28 June 2019; Published: 30 June 2019 Abstract: The purpose of this study was to determine if the associations between eating competence (EC) and eating behaviors that were found in a USA sample of predominantly Hispanic parents of 4th grade youth could be replicated in a USA sample of predominantly non-Hispanic white parents of 4th graders. Baseline responses from parents (n = 424; 94% white) of youth participating in a year-long educational intervention were collected using an online survey. Validated measures included the Satter Eating Competence Inventory (ecSI 2.0TM), in-home fruit/vegetable (FV) availability, healthful eating behavior modeling, and FV self-eﬃcacy/outcome expectancies (SE/OE). Data were analyzed with general linear modeling and cluster analyses. The ﬁndings replicated those from the primarily Hispanic sample. Of the 408 completing all ecSI 2.0TM items, 86% were female, 65% had a 4-year degree or higher, and 53% were EC (ecSI 2.0TM score ≥32). Compared with non-EC parents, EC modeled more healthful eating, higher FV SE/OE, and more in-home FV availability. Behaviors clustered into those striving toward more healthful practices (strivers; n = 151) and those achieving them (thrivers; n = 255). Striver ecSI 2.0TM scores were lower than those of thrivers (29.6 ± 7.8 vs. 33.7 ± 7.6; p < 0.001). More EC parents demonstrated eating behaviors associated with childhood obesity prevention than non-EC parents, encouraging education that fosters parent EC, especially in tandem with youth nutrition education. Keywords: eating competence; parent feeding behavior; school-age youth; Hispanic; replication Eating Competent Parents of 4th Grade Youth from a Predominantly Non-Hispanic White Sample Demonstrate More Healthful Eating Behaviors than Non-Eating Competent Parents Barbara Lohse 1,* , Melissa Pﬂugh Prescott 2 and Leslie Cunningham-Sabo 3 Barbara Lohse 1,* , Melissa Pﬂugh Prescott 2 and Leslie Cunningham-Sabo 3 1 Rochester Institute of Technology Wegmans School of Health and Nutrition, Rochester, NY 14623, USA 2 Department of Food Science and Human Nutrition, University of Illinois Urbana-Champagne, Urbana, IL 61801, USA g g 1 Rochester Institute of Technology Wegmans School of Health and Nutrition, Rochester, NY 14623, USA 2 Department of Food Science and Human Nutrition, University of Illinois Urbana-Champagne, Urbana, IL 61801, USA 3 Department of Food Science and Human Nutrition, Colorado State University, Fort Collins, CO 80523, USA * Correspondence: balihst@rit.edu; Tel.: +1-814-880-9977 3 Department of Food Science and Human Nutrition, Colorado State University, Fort Collins, CO 80523, USA * Correspondence: balihst@rit.edu; Tel.: +1-814-880-9977 nutrients nutrients Recommended Citation Recommended Citation Lohse, B.; Pflugh Prescott, M.; Cunningham-Sabo, L. Eating Competent Parents of 4th Grade Youth from a Predominantly Non-Hispanic White Sample Demonstrate More Healthful Eating Behaviors than Non- Eating Competent Parents. Nutrients 2019, 11, 1501. This Article is brought to you for free and open access by the RIT Libraries. For more information, please contact repository@rit.edu. 1. Introduction Parents and caregivers inform children’s food and eating habits by shaping home and social environmentsandthroughactions(intendedornot)thatconstructattitudesandbeliefs[1,2]. Parentpractices and attitudes associated with healthful eating and making healthy foods more available help promote intake by children [2,3]. One approach to eating that is strongly associated with these healthful practices is eating competence (EC). EC, characterized as an intra-individual approach to eating and food-related attitudes and behaviors that entrains positive biopsychosocial outcomes [4,5], has been associated with higher diet quality [6–9], more healthful eating behaviors [8,10–13] and other lifestyle practices (e.g., being more physically active) [14], better sleep hygiene [15], and food resource management skills [12,13]. Competent eaters give themselves permission to eat foods they like in amounts that are satisfying, but have the discipline to provide nourishing meals and snacks on a routine basis. They enjoy eating and do not Nutrients 2019, 11, 1501; doi:10.3390/nu11071501 www.mdpi.com/journal/nutrients www.mdpi.com/journal/nutrients 2 of 15 Nutrients 2019, 11, 1501 feel guilty about this enjoyment, experiment with and eat a variety of foods, plan meals, make time to eat, and shape an environment that helps them attend to eating [4]. In a sample of parents of 4th graders (n = 339), which was mostly Hispanic (78%) with 35% denoting Spanish as their primary language, the parents who were EC demonstrated more modeling of regular meals and fruit and vegetable (FV) intake, better parent self-efficacy for providing meals and snacks that include FV, and greater FV availability in the home [5]. Parents denoted by cluster analysis as achievers (i.e., they more frequently ate dinner and breakfast with their child; had higher scores on scales that measured healthful modeling behavior, self-efficacy, and availability) were also more EC than the strivers, who demonstrated fewer of these desired behaviors. Achiever EC score was 34.9 ± 7.8 versus the Striver score of 30.3 ± 8.9, p < 0.001) [5]. The ethnicity of the sample is important because Hispanic children have a higher prevalence of being overweight or obesity [16–18], and Hispanic parents underestimate their child’s weight status [19]. In Colorado, Hispanic children have nearly twice the rate of obesity as non-Hispanic white youth [20]. 1. Introduction In addition, Hispanic parent feeding behaviors and attitudes may differ from non-Hispanics in ways that conflict with obesity prevention recommendations [21–26], for example, a low demanding/high responsive feeding style [24], setting limits on what and how much their elementary school children ate, punishing a child who did not want to eat [23], or using high levels of restriction and pressure to eat [21,26]. Comparing Hispanic parents to African–American parents using video capture strategies, Hughes et al. [22] noted high levels of Hispanic parental encouragement to eat (which puts their children at risk for overweight), but reduced use of high control strategies for their obese children. Child obesity prevention messages were interpreted differently among Hispanic, white, and black mothers of children 3 to 10 years old [25]. Hispanic mothers did not connect messages about calories, eating more FV, or family meals to impact on child weight and, unlike non-Hispanic white mothers, Hispanic mothers catered to a child’s preference more frequently. To date, conclusions about the relationship of EC to parent eating behaviors is limited to the sample of Hispanic parents [5]; generalization to other ethnicities has not been investigated. The importance of reproducibilityofthesurveyresultsisnotanewphenomenon, butthecalltoattendtoithasbecomeurgentin psychology and the social sciences [27–32]. Concerns about these external validation issues align with recent calls to consider reproducibility in behaviorally focused studies [28,33], by encouraging replication [27] and especially publication of replication research across study groups [29]. Therefore, the purpose of this study was to determine if the relationships previously reported between parent eating and feeding behaviors and EC in a sample of predominantly Hispanic parents participating in a school and family-based nutrition education intervention [5] are replicated in a sample of predominately non-Hispanic white parents participating in the same intervention. Findings will inform the external validity of the associations between EC and parent eating and feeding behaviors. 2.1. Study Design and Participants This descriptive, cross-sectional study utilized baseline data from parents with 4th grade children enrolled in schools participating in the controlled, school level randomized trial of Fuel for Fun’s (FFF) [34] impact on FV preferences, attitudes, and self-eﬃcacy toward cooking and making foods in 83 classrooms across eight schools in Fort Collins, Colorado from 2012–2016. Study design and methods paralleled those utilized for study of the intervention in the predominantly Hispanic sample [5]. Institutional review boards at the Blinded for Review Universities approved this project; CSU#12-3278H. The study registration at ClinicalTrials.gov is 5NCT02491294. 2.2. Data Collection Parents of 4th grade youth from the participating schools were recruited uniformly using flyers brought home by children and school e-blasts that invited them to visit a website to read about the study. Eligible participants (i.e., they could read English and were parents or the primary caregiver for a 4th 3 of 15 Nutrients 2019, 11, 1501 grader in the participating schools) were directed to the informed consent, then clicked the box denoting agreement to participate. Phone and email contacts of study personnel were included in the consent document. Printing the consent was a suggested action. Consented parents were directed to the online survey (Qualtrics, Provo, UT, USA). Classroom rosters were compared against survey entries of child name, teacher, and school to assure that the survey responder was eligible. After eligibility was affirmed, responders were sent an e-gift card for a discount or online store. Reminders to parents to access the study were sent 2–4 weeks after the initial announcement and enrollment was closed just prior to the start of the classroom intervention. 2.3.2. Self-Eﬃcacy and Outcome Expectancy Perception of self-eﬃcacy and outcome expectancies (SE/OE) related to preparing and oﬀering FV that would be accepted by their children was assessed using a validated 12-item scale with ﬁve response options ranging from strongly disagree to strongly agree [37]. Scores could range from 12 to 60. Cronbach’s alpha was 0.95 for SE/OE. 2.3.3. Fruit and Vegetable Availability In-home FV availability over the past week was determined by previously tested parental report of the presence or absence of eight fruits, nine vegetables, and three types of 100% juice in fresh, frozen, canned, or dried form [38,39]. 2.3.4. Eating Competence EC was measured using the construct validated Satter Eating Competence Inventory (ecSI 2.0TM) [12], which consists of 16 items scored from 0 to 3 (never/rarely to always). ecSI 2.0TM scores can range from 0 to 48, and can be categorized into 4 ecSI 2.0TM subscales: internal regulation, food acceptance, eating attitudes, and contextual skills, with possible subscale scores ranging from 0 to 9 (internal regulation, food acceptance) or 0 to 15 (eating attitudes, contextual skills). ecSI 2.0TM scores of 32 or greater denote EC. Cronbach’s alpha was 0.90 for ecSI 2.0TM. 2.3.5. Physical Activity Physical activity of parents was assessed using the validated International Physical Activity Questionnaire (IPAQ), which measures physical activities over the previous seven days. Responses were converted to metabolic equivalent of task (MET) minutes/week and used to determine categories of low, moderate, and vigorous activity [40]. Weight and height were self-reported. 2.3.1. Modeling The online instrument included a tested, face valid scale with internal consistency that assessed the frequency of 11 modeling behaviors related to meals and FV over the past week, with four response options ranging from never (0 days) to always (7 days) [35,36]. Possible scores ranged from 0 to 33. Cronbach’s alpha was 0.78 for modeling, suggesting internal consistency. 2.3.6. Caregiver Feeding Styles Parent feeding style was assessed using the Caregiver’s Feeding Style Questionnaire [22], a validated measure that consists of 19 items with five response options. Scores are converted to four caregiver feeding styles: uninvolved, indulgent, authoritarian and authoritative. In this sample, Cronbach’s alpha was 0.88. 4 of 15 Nutrients 2019, 11, 1501 2.3.8. Income On the basis of recommendations made with the previous Hispanic sample [5], a proxy measure of parent income, previously tested for face and content validity, was added to the online survey [43]. Parents were considered low-income if they used ≥1 income-based assistance program or always or often worried about money for food. 2.4. Statistical Analysis Data were analyzed with SPSS (version 25.0, 2017, Chicago, IL, USA). For each scale, item responses were summed to create a scale score with higher scores on the parent scales indicating preferred practice; missing items abrogated a total scale score. Parent modeling, SE/OE, and in-home FV availability results were categorized as below the median and at or above the median. EC was defined as ecSI/LI score ≥ 32 [12]. 2.4.1. Psychometrics Internal consistency for all surveys was aﬃrmed with Cronbach’s alpha > 0.77; EC score distribution was aﬃrmed as normal with no kurtosis, absolute skewness < 0.25, and a straight line Q–Q plot. Individual SE/OE items were compared between EC and non-EC parents with a Mann–Whitney U test, because they were not normally distributed. Total SE/OE score was transformed using the square root of the reﬂectance to achieve a normal distribution. 2.3.7. Stress To measure parent stress using a validated measure, but with minimal respondent burden, stress was measured using the single item included in the Community Health Database of Southeast Pennsylvania [41], which has been administered every two to three years since 1991. Littman et al. [42] demonstrated that measuring stress with a single item was a reliable, valid, and practical choice to limit assessment length. 2.4.2. Statistics Differences between EC and non-EC parents, EC tertiles, and median based-categories of parent outcome measures were assessed with t-test, analysis of variance (ANOVA), Mann–Whitney U (continuous variables), andchisquare(categoricalvariables). AScheffetestwasusedtoexamineunplannedcomparisons among group means in ANOVA. Study power to detect a difference of either four points on self-efficacy scale, two points on modeling scale, or two types of FV between EC and non-EC parents was 0.9. Cluster analyses were employed to identify relatively homogeneous subgroups within the sample. Independence among variables (SE/OE, modeling, vegetable availability, and fruit availability) was conﬁrmed (r ≤0.32) before being entered into the analysis. Two-step cluster analysis used sequential and hierarchical agglomerative methods pre-clustering and then sub-clustering of the data. The log-likelihood measure was used as a distance measure. The number of clusters was determined based on the largest relative increase in distance between the two closest clusters deﬁned by the Schwartz Bayesian criterion [44]. All clustering variables were standardized and a listwise deletion approach to missing data was used. t-tests and chi-square tests were used to test for diﬀerences between clusters (independent variable) on parent demographic characteristics, attitudes, and behaviors. In addition, diﬀerences in parent eating competence status across cluster groups were assessed using a general linear model that included parent body mass index (BMI) as a covariate and income status as a cofactor. General linear model results are reported as estimated marginal means ± standard errors. Level of signiﬁcance was set at p < 0.05. 5 of 15 Nutrients 2019, 11, 1501 3.1. Relationship between Eating Competence and Demographics 3.1. Relationship between Eating Competence and Demographics 3.1. Relationship between Eating Competence and Demographics Mean ecSI 2.0TM scores and subscale scores did not diﬀer signiﬁcantly between males (32.5 ± 8.5, n = 55) and females (32.1 ± 7.9, n = 350). Parents denoted as low-income had signiﬁcantly lower ecSI 2.0TM scores compared with other parents (30.3 ± 8.3, n = 145; 33.1 ± 7.7, n = 263, respectively; p = 0.001). BMI was lower (p < 0.001) in EC (n = 212) than non-EC parents (n = 190) (24.5 ± 4.9 vs. 27.7 ± 6.2). Also, a lower proportion of EC parents were overweight or obese compared with those not EC (11% vs. 27%, respectively; p < 0.001). 3. Results 1 Values are n (%) or mean ± SD, total n = 424 although n varies throughout the tables because of missing or incomplete responses ranging from 408 to 422 when not 424. % may not add to 100 because of rounding. Race/ethnicity counts are aﬃrmed responses and reported as a percent of the total sample. 2 Income-based assistance program participant or always, often worries about money for food. 3 ecSI 2.0 TM possible score 0–48; possible subscale scores: eating attitudes and contextual skills, 0–15; food acceptance and internal regulation, 0–9 4 SE/OE survey; possible score 12–60. SE/OE summed score transformed to achieve normal distribution and are reported as means and 95% conﬁdence intervals. Table entries are back transformed values. 5 Survey of modeling mealtime and FV-related behaviors; possible score 0–33. 6 Number of fruits available in the home; possible range 0–8. 7 Number of vegetables available in the home; possible range 0–9. 8 Number of juices available in the home; possible range 0–3. 9 Fruits, vegetables, and fruit juices summed; possible range 0–20. 10 Visual analog scale from 0 (no stress) to 10 (extreme stress). Variable n (%) In-home fruit availability 6 4.4 ± 1.4 In-home vegetable availability 7 6.6 ± 1.7 In-home juice available 8 1.1 ± 0.9 Total in-home fruits/veg available 9 12.0 ± 2.7 Parent perceived stress 10 6.7 ± 2.1 GED, General Education Diploma; BMI, body mass index; IPAQ International Physical Activity Questionnaire; ecSI 2.0TM, Satter eating competence inventory; FV, fruits and vegetables; SE/OE, self-eﬃcacy/outcome expectancies for fruits and vegetables. 1 Values are n (%) or mean ± SD, total n = 424 although n varies throughout the tables because of missing or incomplete responses ranging from 408 to 422 when not 424. % may not add to 100 because of rounding. Race/ethnicity counts are aﬃrmed responses and reported as a percent of the total sample. 2 Income-based assistance program participant or always, often worries about money for food. 3 ecSI 2.0 TM possible score 0–48; possible subscale scores: eating attitudes and contextual skills, 0–15; food acceptance and internal regulation, 0–9. 4 SE/OE survey; possible score 12–60. SE/OE summed score transformed to achieve normal distribution and are reported as means and 95% conﬁdence intervals. Table entries are back transformed values. 5 Survey of modeling mealtime and FV-related behaviors; possible score 0–33. 6 Number of fruits available in the home; possible range 0–8. 3. Results 7 Number of vegetables available in the home; possible range 0–9. 8 Number of juices available in the home; possible range 0–3. 9 Fruits, vegetables, and fruit juices summed; possible range 0–20. 10 Visual analog scale from 0 (no stress) to 10 (extreme stress). 3. Results Of the 564 that accessed the online survey, 450 consented and 424 completed the survey. Survey completers were nearly all non-Hispanic white females with some post-high school education (Table 1) and a mean age of 39.1 ± 6.0 years. Hispanic and non-Hispanic parents did not differ on proportion that were EC or scores on the ecSI 2.0TM, modeling, SE/OE, and FV availability measures. Non-Hispanic ethnicity was associated with a higher education level (p = 0.001). Parents not completing the ecSI 2.0TM items in the survey set (n = 16) did not differ from ecSI 2.0TM completers on any demographic or primary outcome measures. Of the 408 parents completing all ecSI 2.0TM items, 53% (n = 214) were EC. Table 1. Characteristics of parents of 4th grade participants in Fuel for Fun 1. Table 1. Characteristics of parents of 4th grade participants in Fuel for Fun 1. Variable n (%) Gender Female 362 (86) Race White 372 (88) American Indian/Alaska Native 7 (2) Black 3 (<1) Asian 15 (4) Native Hawaiian/Paciﬁc Islander 2 (<1) Ethnicity Hispanic, White 32 (8) Hispanic, Black 1 (<1) Education Did not complete high school 2 (<1) High school graduate or GED 25 (6) Some college/2y college degree 123 (29) 4y college degree 147 (35) Post-graduate college 127 (30) Parent feeding style Uninvolved 80 (20) Indulgent 122 (29) Authoritarian 121 (29) Authoritative 89 (22) BMI Category Underweight 6 (1) Normal weight 211 (50) Overweight 121 (29) Obese 80 (19) IPAQ Category Low 97 (23) Moderate 122 (29) High 205 (48) Self-identify as physically activeYes 292 (69) Low-income 2 152 (36) ecSI 2.0 scoreTM 3 32.1 ± 8.0 Subscales Eating attitudes 10.6 ± 2.9 Food acceptance 5.1 ± 2.1 Internal regulation 6.3 ± 2.0 Contextual skills 10.2 ± 3.0 SE/OE score 4 55.0 95% CI 54.3–55.6 Modeling 5 15.2 ± 4.2 6 of 15 Nutrients 2019, 11, 1501 Table 1. Cont. Variable n (%) In-home fruit availability 6 4.4 ± 1.4 In-home vegetable availability 7 6.6 ± 1.7 In-home juice available 8 1.1 ± 0.9 Total in-home fruits/veg available 9 12.0 ± 2.7 Parent perceived stress 10 6.7 ± 2.1 GED, General Education Diploma; BMI, body mass index; IPAQ International Physical Activity Questionnaire; ecSI 2.0TM, Satter eating competence inventory; FV, fruits and vegetables; SE/OE, self-eﬃcacy/outcome expectancies for fruits and vegetables. 3.2. Comparisons between EC and Non-EC Parents EC/ Total SE/OE score 4 21 Survey items 5 I can prepare fruit that my child will eat 21 I can prepare vegetables that my child will eat 21 I can prepare fruit that my child will like 21 I can prepare vegetables that my child will like 21 I can prepare a recipe with my child 21 If I buy fruit, my child will eat it 21 If I buy vegetables, my child will eat them 21 If I give my child fruit for a snack, my child will eat the fruit 21 If I give my child vegetables for a snack, my child will eat the vegetables 21 If I include fruit as part of a meal, my child will eat the fruit 21 If I include vegetables as part of a meal, my child will eat the vegetables 21 If I prepare a meal together with my child, my child will eat the meal 21 1 Values are means ± SD, except for Total SE/OE score, which is mean diﬀerent from non-EC parents: p ≤0.01, * p ≤0.001. EC, eatin expectancies; ecSI 2.0TM, Satter Eating Competence Inventory 2.0. 2 D ecSI 2.0 TM score < 32. 4 Possible scores 12 (low SE/OE) to 60 (high (SE achieve normal distribution and reported as mean and 95% conﬁdence and may exceed 60. 5 For each survey item, possible response is 1 (str non-EC compared with Mann–Whitney U. Figure 1. Parent eating competence (EC) proportion mapped to response options for selected modeling items. Response option of selected modeling survey item by EC and non-EC parents of 4th grade children. Figure 1 shows percent of total responses. Described below for each panel are the total number of parents that responded, the total number of parents for the response option, and the total number of EC parents for the response option. (A) Total responses, n = 408; 0–3 days/week (total n = 26, EC n = 8); 4–6 days/week (total n = 219, EC n = 104); every day (total n = 163, EC n = 102). (B) Total responses, n = 408; never (total n = 103, EC n = 46); sometimes (total n = 223, EC n = 111); often (total n = 56, EC n = 40); always (total n = 26, EC n = 17). 3.2. Comparisons between EC and Non-EC Parents (C) Total responses, n = 407; never (total n = 56, EC n = 22); sometimes (total n = 308, EC n = 165); often (total n = 28, EC n = 14); always (total n = 15, EC n = 13). (D) Total responses, n = 407; never (total n = 44, EC n = 17); sometimes (total n = 244, EC n = 122); often (total n = 104, EC n = 66); always (total n = 15, EC n = 9). (E) Total responses, n = 407; sometimes (total n = 20, EC n = 6); often (total n = 132, EC n = 55), always (total n = 255, EC n = 153). (F) Total responses, n = 407; never/sometimes (total n = 67, EC n = 23); often (total n = 172, EC n = 79); always (total n = 168, EC n = 112). EC, eating competence. P values denote a test of whether or not response options for each question are independent of eating competence status as measured by Chi-square. Table 2. Self-eﬃcacy/outcome expectancies (SE/OE) score and item responses compared between EC and non-EC parents 1. Table 2. Self-eﬃcacy/outcome expectancies (SE/OE) score and item responses compared between EC and non-EC parents 1. Table 2. Self-eﬃcacy/outcome expectancies (SE/OE) score and item responses compared between EC and non-EC parents 1. 3.2. Comparisons between EC and Non-EC Parents (C) Total responses, n = 407; never (total n = 56, EC n = 22); sometimes (total n = 308, EC n = 165); often (total n = 28, EC n = 14); always (total n = 15, EC n = 13). (D) Total responses, n = 407; never (total n = 44, EC n = 17); sometimes (total n = 244, EC n = 122); Figure 1. Parent eating competence (EC) proportion mapped to response options for selected modeling items. Response option of selected modeling survey item by EC and non-EC parents of 4th grade children. Figure 1 shows percent of total responses. Described below for each panel are the total number of parents that responded, the total number of parents for the response option, and the total number of EC parents for the response option. (A) Total responses, n = 408; 0–3 days/week (total n = 26, EC n = 8); 4–6 days/week (total n = 219, EC n = 104); every day (total n = 163, EC n = 102). (B) Total responses, n = 408; never (total n = 103, EC n = 46); sometimes (total n = 223, EC n = 111); often (total n = 56, EC n = 40); always (total n = 26, EC n = 17). (C) Total responses, n = 407; never (total n = 56, EC n = 22); sometimes (total n = 308, EC n = 165); often (total n = 28, EC n = 14); always (total n = 15, EC n = 13). (D) Total responses, n = 407; never (total n = 44, EC n = 17); sometimes (total n = 244, EC n = 122); often (total n = 104, EC n = 66); always (total n = 15, EC n = 9). (E) Total responses, n = 407; sometimes (total n = 20, EC n = 6); often (total n = 132, EC n = 55), always (total n = 255, EC n = 153). (F) Total responses, n = 407; never/sometimes (total n = 67, EC n = 23); often (total n = 172, EC n = 79); always (total n = 168, EC n = 112). EC, eating competence. P values denote a test of whether or not response options for each question are independent of eating competence status as measured by Chi-square. Figure 1. 3.2. Comparisons between EC and Non-EC Parents Parent eating competence (EC) proportion mapped to response options for selected modeling items. Response option of selected modeling survey item by EC and non-EC parents of 4th grade children. Figure 1 shows percent of total responses. Described below for each panel are the total number of parents that responded, the total number of parents for the response option, and the total number of EC parents for the response option. (A) Total responses, n = 408; 0–3 days/week (total n = 26, EC n = 8); 4–6 days/week (total n = 219, EC n = 104); every day (total n = 163, EC n = 102). (B) Total responses, n = 408; never (total n = 103, EC n = 46); sometimes (total n = 223, EC n = 111); often (total n = 56, EC n = 40); always (total n = 26, EC n = 17). (C) Total responses, n = 407; never (total n = 56, EC n = 22); sometimes (total n = 308, EC n = 165); often (total n = 28, EC n = 14); always (total n = 15, EC n = 13). (D) Total responses, n = 407; never (total n = 44, EC n = 17); sometimes (total n = 244, EC n = 122); Figure 1. Parent eating competence (EC) proportion mapped to response options for selected modeling items. Response option of selected modeling survey item by EC and non-EC parents of 4th grade children. Figure 1 shows percent of total responses. Described below for each panel are the total number of parents that responded, the total number of parents for the response option, and the total number of EC parents for the response option. (A) Total responses, n = 408; 0–3 days/week (total n = 26, EC n = 8); 4–6 days/week (total n = 219, EC n = 104); every day (total n = 163, EC n = 102). (B) Total responses, n = 408; never (total n = 103, EC n = 46); sometimes (total n = 223, EC n = 111); often (total n = 56, EC n = 40); always (total n = 26, EC n = 17). 3.2. Comparisons between EC and Non-EC Parents (B) Total responses, tal n = 223, EC n = 111); often (total n = 56, EC ponses, n = 407; never (total n = 56, EC n = 22); 28, EC n = 14); always (total n = 15, EC n = 13). 17); sometimes (total n = 244, EC n = 122); often ). (E) Total responses, n = 407; sometimes (total s (total n = 255, EC n = 153). (F) Total responses, (total n = 172, EC n = 79); always (total n = 168, test of whether or not response options for each as measured by Chi-square. ore and item responses compared between EC EC/non-EC EC Parents 2 Non-EC Parents 3 n/n 212/189 56.3 55.6–57.0 * 53.2 52.0–54.4 214/194 4.8 ± 0.7 * 4.5 ± 1.0 213/194 4.7 ± 0.7 * 4.3 ± 1.1 213/194 4.8 ± 0.6 * 4.5 ± 0.9 214/194 4.6 ± 0.8 * 4.2 ± 1.1 213/192 4.6 ± 0.7 * 4.3 ± 1.0 214/192 4.6 ± 0.7 * 4.4 ± 1.0 214/194 4.4 ± 0.9 * 4.0 ± 1.1 214/193 4.6 ± 0.8 * 4.3 ± 1.0 s 214/193 4.1 ± 1.1 3.7 ± 1.3 214/194 4.7 ± 0.8 * 4.4 ± 0.9 s 214/194 4.4 ± 0.9 * 4.0 ± 1.1 214/193 4.5 ± 0.8 * 4.2 ± 0.9 is mean and conﬁdence intervals. Asterisks indicate EC, eating competent; SE/OE, self-eﬃcacy/outcome 2.0. 2 Deﬁned by ecSI 2.0 TM score ≥32. 3 Deﬁned by high (SE/OE). SE/OE summed scores transformed to nﬁdence intervals. Table values are back transformed is 1 (strongly disagree) to 5 (strongly agree). EC and 7 of 15 and 7 of 15 and Nutrients 2019, 11, 1501 parents more freq f i b kf Nutrients 2019, 11, 1501 p q y , g , fruit at breakfast with their child (Figure 1). Figure 1. Parent eating competence (EC) proportion map modeling items. Response option of selected modeling survey grade children. Figure 1 shows percent of total responses. Desc number of parents that responded, the total number of parent number of EC parents for the response option. (A) Total resp 26, EC n = 8); 4–6 days/week (total n = 219, EC n = 104); every responses, n = 408; never (total n = 103, EC n = 46); sometimes = 56, EC n = 40); always (total n = 26, EC n = 17). 3.2. Comparisons between EC and Non-EC Parents (C) Total resp = 22); sometimes (total n = 308, EC n = 165); often (total n = 28, 13). (D) Total responses, n = 407; never (total n = 44, EC n = 17 Figure 1. Parent eating competence (EC) proportion mapped t items. Response option of selected modeling survey item b children. Figure 1 shows percent of total responses. Described b of parents that responded, the total number of parents for the r EC parents for the response option. (A) Total responses, n = 40 4–6 days/week (total n = 219, EC n = 104); every day (total n n = 408; never (total n = 103, EC n = 46); sometimes (total n = n = 40); always (total n = 26, EC n = 17). (C) Total responses, sometimes (total n = 308, EC n = 165); often (total n = 28, EC (D) Total responses, n = 407; never (total n = 44, EC n = 17); som (total n = 104, EC n = 66); always (total n = 15, EC n = 9). (E) T n = 20, EC n = 6); often (total n = 132, EC n = 55), always (total n = 407; never/sometimes (total n = 67, EC n = 23); often (total n EC n = 112). EC, eating competence. P values denote a test of w question are independent of eating competence status as mea Table 2. Self-eﬃcacy/outcome expectancies (SE/OE) score and and non-EC parents 1. 3.2. Comparisons between EC and Non-EC Parents Total SE/OE score 4 Survey items 5 I can prepare fruit that my child will eat I can prepare vegetables that my child will I can prepare fruit that my child will like I can prepare vegetables that my child will l I can prepare a recipe with my child If I buy fruit, my child will eat it If I buy vegetables, my child will eat them If I give my child fruit for a snack, my child will ea If I give my child vegetables for a snack, my child will ea If I include fruit as part of a meal, my child will ea If I include vegetables as part of a meal, my child will ea If I prepare a meal together with my child, my child wi 1 Values are means ± SD, except for Total SE/OE diﬀerent from non-EC parents: p ≤0.01, * expectancies; ecSI 2.0TM, Satter Eating Competen ecSI 2.0 TM score < 32. 4 Possible scores 12 (low achieve normal distribution and reported as mea and may exceed 60. 5 For each survey item, poss non-EC compared with Mann–Whitney U. 7 of 15 nner, vegetables at lunch, fruit as a snack, and n mapped to response options for selected survey item by EC and non-EC parents of 4th es. Described below for each panel are the total f parents for the response option, and the total al responses, n = 408; 0–3 days/week (total n = every day (total n = 163, EC n = 102). (B) Total etimes (total n = 223, EC n = 111); often (total n tal responses, n = 407; never (total n = 56, EC n n = 28, EC n = 14); always (total n = 15, EC n = C n = 17); sometimes (total n = 244, EC n = 122); pped to response options for selected modeling item by EC and non-EC parents of 4th grade cribed below for each panel are the total number or the response option, and the total number of , n = 408; 0–3 days/week (total n = 26, EC n = 8); total n = 163, EC n = 102). 3.2. Comparisons between EC and Non-EC Parents EC, eating competent; SE/OE, self-eﬃcacy/outcome ng Competence Inventory 2.0. 2 Deﬁned by ecSI 2.0 TM score ≥32. 3 Deﬁned by ores 12 (low SE/OE) to 60 (high (SE/OE). SE/OE summed scores transformed to orted as mean and 95% conﬁdence intervals. Table values are back transformed ey item, possible response is 1 (strongly disagree) to 5 (strongly agree). EC and ney U. Nutrients 2019, 11, 1501 parents more frequently ate dinner, veget fruit at breakfast with their child (Figure 1) Figure 1. Parent eating competence (EC modeling items. Response option of select grade children. Figure 1 shows percent of t number of parents that responded, the tot number of EC parents for the response op 26, EC n = 8); 4–6 days/week (total n = 219, responses, n = 408; never (total n = 103, EC = 56, EC n = 40); always (total n = 26, EC n = 22); sometimes (total n = 308, EC n = 165 13). (D) Total responses, n = 407; never (to Figure 1. Parent eating competence (EC) pr items. Response option of selected mode children. Figure 1 shows percent of total res of parents that responded, the total number EC parents for the response option. (A) Tot 4–6 days/week (total n = 219, EC n = 104); n = 408; never (total n = 103, EC n = 46); so n = 40); always (total n = 26, EC n = 17). (C sometimes (total n = 308, EC n = 165); ofte (D) Total responses, n = 407; never (total n = (total n = 104, EC n = 66); always (total n = n = 20, EC n = 6); often (total n = 132, EC n n = 407; never/sometimes (total n = 67, EC n EC n = 112). EC, eating competence. P valu question are independent of eating compet Table 2. Self-eﬃcacy/outcome expectancie and non-EC parents 1. 3.2. Comparisons between EC and Non-EC Parents EC parents reported more frequent modeling behaviors related to meals and FV intake compared with non-EC parents (16.1 ± 4.2 and 14.2 ± 4.1, respectively; p < 0.001). In addition, EC parents more frequently ate dinner, vegetables at dinner, vegetables at lunch, fruit as a snack, and fruit at breakfast with their child (Figure 1). EC parents averaged higher SE/OE scores related to FV preparation and consumption compared with non-EC parents (p < 0.001). Diﬀerences were statistically signiﬁcant (all p ≤0.002) for each of the 12 SE/OE survey items (Table 2). Vegetables were more available in the homes of EC parents compared with non-EC parents (6.8 ± 1.5 vs. 6.3 ± 1.8, respectively, p = 0.002), and combined FV availability was also higher in the homes of EC parents (12.4 ± 2.6 vs. 11.6 ± 2.8, respectively, p = 0.002). Fruit or juice availability did not diﬀer according to EC status (p = 0.08 vs. p = 0.24, respectively). After controlling for education level, diﬀerences between EC and non-EC parents remained signiﬁcant for the modeling (p = 0.001), in-home FV availability (p = 0.006), and SE/OE measures (p = 0.015). 7 of 15 ner, vegetables at dinner, vegetables at lunch, fruit as a snack, and (Figure 1). petence (EC) proportion mapped to response options for selected on of selected modeling survey item by EC and non-EC parents of 4th percent of total responses. Described below for each panel are the total ded, the total number of parents for the response option, and the total esponse option. (A) Total responses, n = 408; 0–3 days/week (total n = otal n = 219, EC n = 104); every day (total n = 163, EC n = 102). (B) Total n = 103, EC n = 46); sometimes (total n = 223, EC n = 111); often (total n = 26, EC n = 17). (C) Total responses, n = 407; never (total n = 56, EC n EC n = 165); often (total n = 28, EC n = 14); always (total n = 15, EC n = 7; never (total n = 44, EC n = 17); sometimes (total n = 244, EC n = 122); nce (EC) proportion mapped to response options for selected modeling cted modeling survey item by EC and non-EC parents of 4th grade of total responses. 3.2. Comparisons between EC and Non-EC Parents Described below for each panel are the total number tal number of parents for the response option, and the total number of on. (A) Total responses, n = 408; 0–3 days/week (total n = 26, EC n = 8); C n = 104); every day (total n = 163, EC n = 102). (B) Total responses, n = 46); sometimes (total n = 223, EC n = 111); often (total n = 56, EC C n = 17). (C) Total responses, n = 407; never (total n = 56, EC n = 22); = 165); often (total n = 28, EC n = 14); always (total n = 15, EC n = 13). er (total n = 44, EC n = 17); sometimes (total n = 244, EC n = 122); often s (total n = 15, EC n = 9). (E) Total responses, n = 407; sometimes (total 132, EC n = 55), always (total n = 255, EC n = 153). (F) Total responses, n = 67, EC n = 23); often (total n = 172, EC n = 79); always (total n = 168, nce. P values denote a test of whether or not response options for each ing competence status as measured by Chi-square. xpectancies (SE/OE) score and item responses compared between EC EC/non-EC EC Parents 2 Non-EC Parents 3 n/n e 4 212/189 56.3 55.6–57.0 * 53.2 52.0–54.4 child will eat 214/194 4.8 ± 0.7 * 4.5 ± 1.0 my child will eat 213/194 4.7 ± 0.7 * 4.3 ± 1.1 hild will like 213/194 4.8 ± 0.6 * 4.5 ± 0.9 my child will like 214/194 4.6 ± 0.8 * 4.2 ± 1.1 h my child 213/192 4.6 ± 0.7 * 4.3 ± 1.0 will eat it 214/192 4.6 ± 0.7 * 4.4 ± 1.0 will eat them 214/194 4.4 ± 0.9 * 4.0 ± 1.1 y child will eat the fruit 214/193 4.6 ± 0.8 * 4.3 ± 1.0 y child will eat the vegetables 214/193 4.1 ± 1.1 3.7 ± 1.3 child will eat the fruit 214/194 4.7 ± 0.8 * 4.4 ± 0.9 child will eat the vegetables 214/194 4.4 ± 0.9 * 4.0 ± 1.1 , my child will eat the meal 214/193 4.5 ± 0.8 * 4.2 ± 0.9 Total SE/OE score, which is mean and conﬁdence intervals. Asterisks indicate p ≤0.01, * p ≤0.001. 3.3. Comparisons among Parents according to Eating Competence Tertile 3.3. Comparisons among Parents according to Eating Competence Tertile .3. Comparisons among Parents according to Eating Competence Tertile SE/OE, parent modeling, and in-home vegetable availability also diﬀered among ecSI/LI tertiles (data not shown, all p ≤0.007). Post hoc Scheﬀe tests for these analyses revealed that parents in the lowest ecSI 2.0TM tertile had signiﬁcantly lower modeling scores than parents in the high and middle ecSI 2.0TM tertiles, and in-home vegetable availability was greater in the high than in the low tertile. SE/OE scores were signiﬁcantly greater for parents in the high ecSI 2.0TM tertile than in the middle or lower tertiles. Parent education was associated with tertile of ecSI 2.0TM score (p = 0.004). The proportion of parents who attended graduate school was highest among those in the high tertile (38% vs. 20% in the low and 33% in the middle tertiles). After controlling for education level, diﬀerences among tertiles remained signiﬁcant for modeling and in-home vegetable availability (p < 0.02), with a trend toward diﬀerence (p = 0.08) for SE/OE. 3.2. Comparisons between EC and Non-EC Parents EC/non-EC EC Parents 2 Non-EC Parents 3 n/n Total SE/OE score 4 212/189 56.3 55.6–57.0 * 53.2 52.0–54.4 Survey items 5 I can prepare fruit that my child will eat 214/194 4.8 ± 0.7 * 4.5 ± 1.0 I can prepare vegetables that my child will eat 213/194 4.7 ± 0.7 * 4.3 ± 1.1 I can prepare fruit that my child will like 213/194 4.8 ± 0.6 * 4.5 ± 0.9 I can prepare vegetables that my child will like 214/194 4.6 ± 0.8 * 4.2 ± 1.1 I can prepare a recipe with my child 213/192 4.6 ± 0.7 * 4.3 ± 1.0 If I buy fruit, my child will eat it 214/192 4.6 ± 0.7 * 4.4 ± 1.0 If I buy vegetables, my child will eat them 214/194 4.4 ± 0.9 * 4.0 ± 1.1 If I give my child fruit for a snack, my child will eat the fruit 214/193 4.6 ± 0.8 * 4.3 ± 1.0 If I give my child vegetables for a snack, my child will eat the vegetables 214/193 4.1 ± 1.1 3.7 ± 1.3 If I include fruit as part of a meal, my child will eat the fruit 214/194 4.7 ± 0.8 * 4.4 ± 0.9 If I include vegetables as part of a meal, my child will eat the vegetables 214/194 4.4 ± 0.9 * 4.0 ± 1.1 If I prepare a meal together with my child, my child will eat the meal 214/193 4.5 ± 0.8 * 4.2 ± 0.9 1 Values are means ± SD, except for Total SE/OE score, which is mean and conﬁdence intervals. Asterisks indicate diﬀerent from non-EC parents: p ≤0.01, * p ≤0.001. EC, eating competent; SE/OE, self-eﬃcacy/outcome expectancies; ecSI 2.0TM, Satter Eating Competence Inventory 2.0. 2 Deﬁned by ecSI 2.0 TM score ≥32. 3 Deﬁned by ecSI 2.0 TM score < 32. 4 Possible scores 12 (low SE/OE) to 60 (high (SE/OE). SE/OE summed scores transformed to achieve normal distribution and reported as mean and 95% conﬁdence intervals. Table values are back transformed and may exceed 60. 5 For each survey item, possible response is 1 (strongly disagree) to 5 (strongly agree). EC and non-EC compared with Mann–Whitney U. 8 of 15 Nutrients 2019, 11, 1501 3.3. Comparisons among Parents according to Eating Competence Tertile 3.4. Median Analyses ecSI 2.0TM scores were signiﬁcantly higher for parents at or above the median for modeling, vegetable availability, and SE/OE (all p < 0.01) scores. All 4 ecSI 2.0TM subscales were signiﬁcantly higher for parents at or above the median modeling scores. Eating attitudes, food acceptance, and contextual skills subscale scores were higher for parents at or above the median vegetable availability score. Food acceptance, contextual skills, and internal regulation subscale scores were higher for parent at or above the median SE/OE scores (Table 3). 9 of 15 Nutrients 2019, 11, 1501 Table 3. ecSI 2.0TM scores compared between parents below and at/above median survey scores 1. 3.4. Median Analyses SE/OE 2 Modeling 3 In-Home FV Availability 4 In-Home Fruit Availability 5 In-Home Vegetable Availability 6 Eating Competence 7 <Median ≥Median <Median ≥Median <Median ≥Median <Median ≥Median <Median ≥Median ecSI 2.0TM score 30.5 ± 7.6 * n = 180 33.6 ± 8.0 n = 221 29.5 ± 8.1 * n = 169 34.1 ± 7.3 n = 232 31.2 ± 8.3 n = 168 32.7 ± 7.8 n = 240 32.0 ± 8.7 n = 92 32.1 ± 7.8 n = 316 30.7 ± 8.4 n = 172 33.2 ± 7.6 n = 236 ecSI 2.0TM subscales Eating attitudes 10.3 ± 2.7 n = 183 10.9 ± 3.1 n = 227 10.1 ± 3.2 * n = 172 11.0 ± 2.6 n = 236 10.4 ± 3.0 n = 173 10.8 ± 2.8 n = 245 10.6 ± 3.3 n = 96 10.6 ± 2.8 n = 322 10.3 ± 3.1 * n = 178 10.9 ± 2.8 n = 240 Food acceptance 4.6 ± 2.0 * n = 185 5.6 ± 1.9 n = 226 4.5 ± 2.0 * n = 174 5.6 ± 1.9 n = 236 4.9 ± 2.1 * n = 172 5.3 ± 2.0 n = 246 4.9 ± 2.2 n = 95 5.2 ± 2.0 n = 323 4.8 ± 2.1 n = 179 5.4 ± 2.0 n = 239 Internal regulation 5.9 ± 2.0 * n = 186 6.6 ± 2.0 n = 227 6.0 ± 2.2 * n = 174 6.4 ± 1.9 n = 238 6.2 ± 2.1 n = 174 6.3 ± 2.0 n = 247 6.5 ± 2.2 n = 97 6.2 ± 2.0 n = 324 6.1 ± 2.1 n = 180 6.4 ± 2.0 n = 241 Contextual skills 9.6 ± 2.9 * n = 185 10.7 ± 2.9 n = 228 9.0 ± 2.9 * n = 173 11.1 ± 2.7 n = 237 9.8 ± 3.1 n = 173 10.4 ± 2.9 n = 247 10.1 ± 3.2 n = 96 10.2 ± 2.9 n = 324 9.6 ± 3.2 *** n = 180 10.6 ± 2.7 n = 240 1 Values are means ± SD. Asterisks indicate diﬀerent from ≥median: * p < 0.05, p ≤0.01, * p ≤0.001. Eating competent, EC; Satter Eating Competence Inventory for Low-Income, ecSI 2.0 TM; self-eﬃcacy/outcome expectancies, SE/OE; fruit and vegetables, FV. 3.5. Cluster Analyses The cluster analysis of parental food and eating behaviors delineated two clusters depicting thrivers (n = 255) and strivers (n = 151). Compared to strivers, thrivers had more self-eﬃcacy for providing fruits and vegetables to their child(ren), modeled more healthful eating behaviors, and identiﬁed higher in-home fruit and vegetable availability. Clusters, which did not diﬀer in age, gender, BMI, or income level, were validated with signiﬁcantly higher ecSI 2.0TM (p < 0.001) and subscale scores (all p ≤0.003) in thrivers compared with strivers (Table 4). Compared to thrivers, more strivers used an authoritarian parenting style (23% vs. 40%, p < 0.001) and had lower levels of physical activity (18% vs. 32%, p = 0.004). Eating competence scores (ecSI 2.0TM) remained greater (p < 0.001) among thrivers (33.5 ± 0.5) than in strivers (29.5 ± 0.6) when BMI, income level, or education were included in the general linear model. The average silhouette was of suﬃcient quality at 0.4. The discriminant function as a whole was signiﬁcant (Wilks lambda = 0.32; p < 0.001). SE/OE and vegetable availability were the strongest predictors for cluster delineation. Derivation variables are shown in Table 4. Table 4. Behaviors compared between parents in clusters of thriving or striving for healthful and competent feeding behaviors 1. Measure Thriver/Striver Thrivers Strivers n/n Mean/95% CI Mean/95% CI Derivative Variables SE/OE score 2 255/151 58.4/58.1–58.6 46.1/44.7–47.5 Mean ± SD Modeling score 3 255/151 16.5 ± 3.8 12.8 ± 3.9 Fruit Availability 4 255/151 4.6 ± 1.2 3.8 ± 1.5 Vegetables Availability 5 255/151 7.2 ± 1.2 5.6 ± 1.8 Validation Variables ecSI 2.0TM score 6 250/144 33.7 ± 7.6 * 29.6 ± 7.8 ecSI 2.0TM Subscales Eating attitudes 253/147 11.0 ± 2.8 10.1 ± 2.9 Food acceptance 254/149 5.6 ± 1.9 * 4.3 ± 2.0 Internal regulation 254/150 6.5 ± 2.0 * 5.9 ± 2.1 Contextual skills 254/149 10.7 ± 2.8 * 9.3 ± 3.0 BMI 250/150 25.7 ± 5.9 26.5 ± 5.7 Age (years) 254/150 38.8 ± 5.9 39.7 ± 6.0 Frequency n (%) Parent Feeding Style * Uninvolved 49 (20) 29 (20) Indulgent 91 (36) 28 (19) Authoritarian 58 (23) 58 (40) Authoritative 53 (21) 30 (21) Gender Male 32 (13) 23 (15) Female 221 (87) 127 (85) IPAQ Category 7,** Low 46 (18) 49 (32) Moderate 76 (30) 40 (26) High 133 (52) 62 (41) Table 4. 3.4. Median Analyses 2 SE/OE, possible score 12–60; median = 56. 3 Modeling survey, possible score 0–33; median = 15. 4 In-home fruit and vegetable availability, possible range 0–20; median = 12. 5 In-home fruit availability, possible range 0–8; median = 4. 6 In-home vegetable availability, possible range 0–9; median = 7. 7 ecSI 2.0TM, possible score 0–48; possible subscale scores: Eating attitudes and contextual skills 0–15, food acceptance and internal regulation 0–9. Table 3. ecSI 2.0TM scores compared between parents below and at/above median survey scores 1. 1 Values are means ± SD. Asterisks indicate diﬀerent from ≥median: * p < 0.05, p ≤0.01, * p ≤0.001. Eating competent, EC; Satter Eating Competence Inventory for Low-Income, ecSI 2.0 TM; self-eﬃcacy/outcome expectancies, SE/OE; fruit and vegetables, FV. 2 SE/OE, possible score 12–60; median = 56. 3 Modeling survey, possible score 0–33; median = 15. 4 In-home fruit and vegetable availability, possible range 0–20; median = 12. 5 In-home fruit availability, possible range 0–8; median = 4. 6 In-home vegetable availability, possible range 0–9; median = 7. 7 ecSI 2.0TM, possible score 0–48; possible subscale scores: Eating attitudes and contextual skills 0–15, food acceptance and internal regulation 0–9. Nutrients 2019, 11, 1501 10 of 15 10 of 15 3.5. Cluster Analyses Behaviors compared between parents in clusters of thriving or striving for healthful and competent feeding behaviors 1. 11 of 15 Nutrients 2019, 11, 1501 Table 4. Cont. Measure Thriver/Striver Thrivers Strivers Low-Income Status Low-income 8 89 (35) 56 (37) Not low-income 166 (65) 95 (63) 1 Values are n (%) or mean ± SD, total n = 406, although n varies throughout the tables because of missing or incomplete responses ranging from 394 to 404 when not 406. Asterisks indicate diﬀerent from striver parents: p ≤0.01, * p ≤0.001. SE/OE, self-eﬃcacy/outcome expectancies; ecSI 2.0TM, Satter eating competence inventory. 2 SE/OE possible score 12–60; SE/OE summed scores transformed to achieve normal distribution and may exceed 60. Table entries are back transformed. 3 Modeling survey possible score 0–33. 4 Number of fruits available in the home possible range 0–8. 5 Number of vegetables available in the home, possible range 0–9. 6 ecSI 2.0TM possible score 0–48; possible subscale scores: eating attitudes and contextual skills, 0–15; food acceptance and internal regulation, 0–9. 7 IPAQ, International Physical Activity Questionnaire; responses converted to met min/week and identiﬁed as low, moderate, and vigorous activity categories. 8 Parents who reported often or always worrying about money or participation in an income-based program, for example, the Supplemental Nutrition Assistance Program or Women, Infants, and Children Supplemental Nutrition Program, were deﬁned as low income. 4.1. Summary The results from this relatively large sample of non-Hispanic white parents of 4th grade youth supported the tenets of EC [4]. Eating and feeding behaviors, physical activity level, weight status, and demographic characteristics were assessed and compared according to EC status. EC parents demonstrated stronger role modeling behaviors (e.g., eating dinner, eating vegetables at dinner, and cooking with their child). Parent behaviors clustered into two groups (thrivers and strivers) that did not diﬀer in age or gender. However, thrivers not only demonstrated more role modeling, SE/OE behaviors, and home FV availability, but they were more physically active, less likely to utilize an authoritarian parent feeding style and more EC even after adjusting for BMI and income. These ﬁndings corroborated those from the mostly Hispanic sample [5] with the exception that unlike the Hispanic sample, which had signiﬁcantly diﬀerent ecSI 2.0TM scores between those below or at and above the median for home FV availability (32.4 ± 8.2 vs. 34.8 ± 8.7; p < 0.05) [5], ecSI 2.0TM scores in the current study were not signiﬁcantly diﬀerent (31.2 ± 8.3 vs. 32.7 ± 7.8). The comparative ﬁndings are remarkable given that the two samples diﬀered in ethnicity (white ethnicity reported for 14% in the previous sample and 88% in this sample) and education level (4-year college degree or higher for 18% vs. 65%; high school or less for 51% vs. <7%). Both samples were otherwise similar; for example, they were mostly female (89% vs. 86%), with a mean ecSI score >32 (33.6 ± 8.5 vs. 32.1 ± 8.0) and similar modeling of behaviors related to meals and FV (15.3 ± 4.8 vs. 15.2 ± 4.2). Author Contributions: Conceptualization, B.L. and L.C.-S.; methodology, B.L. and L.C.-S.; validation, B.L.; formal analysis, B.L. and M.P.P.; investigation, B.L., M.P.P., and L.C.-S.; resources, B.L. and L.C.-S.; data curation, B.L.; writing—original draft preparation, B.L.; writing—review and editing, B.L., M.P.P., and L.C.-S.; supervision, B.L. and L.C.-S; project administration, B.L. and L.C.-S.; funding acquisition, B.L. and L.C.-S. 4.3. Strengths and Limitations A strength of this study was that the sample was similar to the previously studied, mostly Hispanic group, facilitating generalization of ﬁndings from Hispanic to non-Hispanic white parents. Both samples were parents of 4th grade youth, similar in age; sex; frequency of eating dinner with their child; ecSI 2.0TM and subscale scores; and the primary outcomes of modeling, SE/OE, and FV availability in the home. As a result, this study was a replication across groups diﬀering in proportion of Hispanic ethnicity and related level of education; the mostly Hispanic sample included 49% with at least some college post-secondary education, compared with 94% for this mostly non-Hispanic white sample. Both studies utilized identical, validated instruments, although data collection was via paper survey in the Hispanic sample rather than the online survey platform used in the current study. In addition to the strength of sample similarity, the current study also included measures, for example, self-reported weight, that were noted as missing factors in the earlier study, enabling further exploration of relationships. For example, this study allowed for comparisons across clusters that controlled for BMI and income status, thus strengthening the earlier ﬁndings with Hispanic parents. Findings from this study increased the certainty that EC parents are better aligned with many child obesity prevention practices. However, limitations or threats to reproducibility of ﬁndings were still apparent. Although participants were from two school districts with children in eight elementary schools, generalization is attenuated because participants were from Fort Collins and Loveland, Colorado, which is a uniquely healthy state. It has the United States’ lowest rate of adult obesity, second highest level of physically active adults, and is eighth highest in the number of adults eating ﬁve or more daily FV servings. Coloradan children also stand out, being ranked nationally with the ﬁfth lowest rate of obesity and above the national average for physical activity, with 67% of school-age children participating in vigorous physical activity four or more days each week. In addition, at the time of this study, Fort Collins had just implemented a policy removing unhealthy beverages from vending machines in all city buildings and several hospitals across Colorado joined this action, revealing a strong health promotion culture [20]. Findings are also limited because height, weight, and income-based assistance program use was self-reported, possibly misrepresenting these sample characteristics and EC relationships for comparison with future studies. 4.2. Consequences of Reproducibility Replication has been construed as either “conceptual” or “direct”. Conceptual replication tries to validate and further deﬁne the underlying theory, whereas direct replication involves validating a speciﬁc fact or result. Direct replication studies retain all features of the original study, but conceptual replication intentionally varies one or more features to examine generalizability across conditions [30–32,45]. Earp and Traﬁmow [30] note that the distinction between the two types is not absolute; and so it is with this study. We sought to determine if the ﬁndings relating EC and parent modeling behaviors were reproduced, but intentionally evaluated for this in a sample that diﬀered in ethnicity from the original study, that is, conceptual replication. Usually, little support is given for this type of endeavor, especially for publication and promotion credit [31,45], and the consequences of failure to replicate challenge this undertaking. However, as noted in a seminal paper published in Science as an Open Science Collaboration [46], “Innovation points out paths that are possible; replication points out paths that are likely; progress relies on both. Replication can increase certainty when ﬁndings are reproduced and promote innovation when they are not.” Findings from this study point out a likely path that being EC is a desired state for 12 of 15 12 of 15 Nutrients 2019, 11, 1501 either white or Hispanic parents’ own health as well as their child’s. They also encourage ascribing the ﬁndings to other races, ethnicities, cultures, and countries, especially the non-English speaking cultures. However, the results also show that aﬃrming replication in said groups is feasible, reassuring, and will enhance the study of the EC construct, furthering the development of strategies, education, and policies that can promote healthful eating behaviors through the adoption of an EC approach. Funding: This research was funded by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award number 2012-68001-19603. (LCS PI; BL Co-PI). Clinical trials.gov registration number NCT02491294. Registered 7/7/15. 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Eating competence of Hispanic parents is associated with attitudes and behaviors that may mediate fruit and vegetable-related behaviors of 4th grade youth. J. Nutr. 2012, 142, 1903–1909. [CrossRef] [PubMed] 6. Lohse, B.; Psota, T.; Estruch, R.; Zazpe, I.; Sorli, J.V.; Salas-Salvado, J.; Serra, M.; Krall, J.S.; Marquez, F.; Ros, E. on behalf of the PREDIMED Study Investigators. Eating competence of elderly Spanish adults is associated with a healthy diet and a favorable cardiovascular disease risk proﬁle. J. Nutr. 2010, 140, 1322–1327. [CrossRef] 6. Lohse, B.; Psota, T.; Estruch, R.; Zazpe, I.; Sorli, J.V.; Salas-Salvado, J.; Serra, M.; Krall, J.S.; Marquez, F.; Ros, E. on behalf of the PREDIMED Study Investigators. 5. Conclusions Parental supports, for example, eating meals together, serving FV as snacks, encouraging breakfast, and facilitating an active lifestyle, impact child health-related behaviors and outcomes [47]. 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https://openalex.org/W2554537052	https://europepmc.org/articles/pmc5111343?pdf=render	English	null	Quality of life of caregivers of patients diagnosed with severe mental illness at the national referral hospitals in Uganda	BMC psychiatry	2,016	cc-by	7,262	© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: cndikuno@gmail.com 1Department of Nursing, College of Health Sciences, Makerere University, Kampala, Uganda 4Department of Nursing, College of Health Sciences, Makarere University, Mulago Hill road, P.O BOX 7072, Kampala, Uganda Full list of author information is available at the end of the article Abstract Background: Worldwide, 450 million people suffer from mental and behavioral disorders. In Uganda, it is estimated that 35% of the population that is 9,574,915 people suffer from some form of mental illness. Caregivers are increasingly bearing the responsibility of taking care of these patients, which can influence their QoL due to the social and economic costs they incur. The aim of the study was to assess the QoL of caregivers for patients diagnosed with severe mental illness attending the National Referral Hospitals in Uganda. Method: This was a cross sectional study. A pretested tool with two parts; a sociodemographic part and a validated WHOQOL-BREF, was used to collect data from 300 consecutive eligible participants. SPSS (Statistical Package for Social Sciences) Version 22 and Stata Version 14 were used in data entry and analysis. Results: Of the 300 participants, 57.3% of the caregivers had a poor QoL. The statistically significant factors associated with QoL were environment (Adjusted coefficient = 0.016, 95% CI = 0.009–0.023), caregiver satisfaction with their health (Adjusted coefficient = 0.405, 95% CI = 0.33–0.487), psychological wellbeing (Adjusted coefficient = 0.007, 95% CI = 0.0002–0.013), and education level (Adjusted coefficient = 0.148, 95% CI = 0.072–0.225). Conclusion: QoL of caregivers for patients diagnosed with mental illness is generally poor due to the added responsibilities and occupation of their time, energy and attention. This additional responsibility results in high levels of stress and caregivers may fail to have appropriate coping mechanisms. Interventions like support groups or counseling should be put in place to aid caregivers in their role and therefore improve QoL. This study adds to the international database of QoL literature and calls for more attention to be placed on caregivers in supporting their role and improving their QoL so as to lead to better patient outcomes among those diagnosed with mental illness. Keywords: Caregivers, Quality of life, Severe mental illness, WHOQOL-BREF, Uganda Cynthia Ndikuno1,4, Mariam Namutebi1, Job Kuteesa2, David Mukunya3 and Connie Olwit1 Cynthia Ndikuno1,4, Mariam Namutebi1, Job Kuteesa2, David Mukunya3 and Connie Olwit1 Ndikuno et al. BMC Psychiatry (2016) 16:400 DOI 10.1186/s12888-016-1084-2 Ndikuno et al. BMC Psychiatry (2016) 16:400 DOI 10.1186/s12888-016-1084-2 Background In an African perspective, the individuals diagnosed with mental illness are cared for in the community by caregivers who may be relatives or friends [3]. Caregivers offer care that may be physical, economical, psychological, and social which eventually changes their life since it is in- volving and demanding [4]. Globally, mental health disorders are known to represent two of the ten leading causes of disability and account for 35.6% of the total burden of disease according to the Global Burden of Disease (GBDCollaborators) Collabo- rator’s study [1]. In Uganda, it is estimated that 35% of the population that is 9,574,915 people suffer from some form of mental illness with atleast 15% requiring treatment [2]. Caregivers may experience reduced productivity at home and in the workplace thus losing wages [5]. This combined with the health care costs for the patients diagnosed with severe mental illness affect the caregivers’ financial situation thus creating or worsening poverty [5]. Caregivers are also prone to experiencing social conse- quences including; disrupted social networks, stigma and discrimination, which exposes them to high levels of Ndikuno et al. BMC Psychiatry (2016) 16:400 Page 2 of 9 Page 2 of 9 depression, stress and anxiety [5, 6]. These social, economic and psychological changes may greatly impact on the caregivers’ Quality of Life (QoL) [7]. hospital and the caregivers who were visiting the people they care for that were admitted on the inpatient wards in Butabika Hospital. QoL is an individual’s perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expecta- tions, standards and concerns. Study design This was a descriptive cross sectional study of caregivers for patients diagnosed with mental illness. Methods Study site A pre-tested data collection tool was used to collect the data from the caregivers. The tool had two parts with the first part seeking Sociodemographic and the second part being the WHOQOL-BREF. This study was conducted at the National Referral Hospitals of Uganda including Mulago Hospital and Butabika Hospital. These national referral hospitals are located in Kampala district in Uganda and they are both teaching hospitals. The sociodemographic section had questions on age, sex tribe, religion, occupation, monthly income, educa- tion level, marital status, period of time spent caring for the patient diagnosed with mental illness and the mental illness the patient is diagnosed with. Mulago hospital has an Outpatient mental health clinic known as S.B Bosa Mental Health Unit. On the other hand Butabika Hospital is a psychiatric hospital with inpatient wards and an outpatient clinic. The patients usually come with their caregivers. The WHOQOL-BREF is a 26 item, self-administered questionnaire (it can also be interviewer assisted or interviewer administered), which contains a total of 24 questions and two items that measure overall QoL and general health. Participants express how much they have experienced the items in the preceding 2 weeks on a 5-point Likert scale ranging from 1 (not at all) to 5 (completely) and the administration time is usually 10–15 min. Sample size As recommended by the WHOQOL-BREF (World Health Organisation Quality of Life-BREF) manual, the sample size used in this study was 300 caregivers [9]. As recommended by the WHOQOL-BREF (World Health Organisation Quality of Life-BREF) manual, the sample size used in this study was 300 caregivers [9]. The QoL of caregivers is important in reflecting the quality of care that is given to patients diagnosed with mental illness to aid recovery or stability and can also affect the progress and outcome of the patients [6]. This is considered an adequate sample size by the WHOQOL Group to give significant results that can be used in establishing the relationships between the inde- pendent and dependent variables. Caregivers are fundamental in giving care to patients diagnosed with severe mental illness however there is limited documentation on the QoL of the caregivers for these patients. Due to the limited documentation of QoL, there are few interventions for caregivers that are incorporated in the Uganda health policy to help them handle with their role [8]. The few interventions include the community mental health program run at Butabika Hospital that offers caregiver counseling to people living within the 10 km radius of the hospital and the Schizo- phrenia fellowship caregiver support group found in Jinja district. Therefore it is important to understand their QoL, which may give baseline information on their way of life and guide health workers and policy makers on the development of appropriate interventions to support the caregivers in their role. Sampling procedure All eligible caregivers who consented to participate were enrolled consecutively in the study from January 2015 to May 2015 until the required sample was obtained. The participants included in the study were caregivers for a patient diagnosed with severe mental illness including Schizophrenia, Bipolar Affective disorder and Major De- pressive disorder; caregivers who had taken care of a patient diagnosed with mental illness for atleast six months; caregivers who consented to participate in the study and caregivers who were above 18 years of age. The caregivers who were excluded had people diag- nosed with severe mental illness who were aggressive or had the potential to harm themselves or people around them and those who were unable to stay around the clinic long enough to fill in the tool. This study sought to determine the QoL and associ- ated factors of caregivers of mentally ill patients attend- ing the National Referral Hospitals in Uganda. Study population The WHOQOL-BREF contains 2 items from the overall QoL and general health facet and 1 item from each of the remaining 24 facets making a total of 26 question items. The 24 facets of QoL make up the four domain s namely: physical health that comprises 7 The study population comprised caregivers of patients diag- nosed with severe mental illness including Schizophrenia, Bipolar Affective Disorder and Major Depressive Disorder who were attending S.B Bosa Mental health outpatient clinic in Mulago Hospital, the Outpatient clinic in Butabika Ndikuno et al. BMC Psychiatry (2016) 16:400 Ndikuno et al. BMC Psychiatry (2016) 16:400 Page 3 of 9 Page 3 of 9 items, psychological wellbeing that comprises 6 items, social relationship that comprises 3 items, and environ- ment that comprises 8 items. Question 1 and 2 plus the domain scores were calculated and transformed using WHOQOL-BREF SPSS syntax file. The transformed scores were on a scale of 0–100. In this study, all items that were rated with a higher score indicated a higher or better QoL [9]. The internal consistency of the various multi-item domains used in this study was assessed in an international field trial involving 23 countries representing various regions and cultures [10]. The Internal consistency was found to high with an average Cronbach’s alpha of 0.81 for the physical health, psychological wellbeing and environmental domains but a marginal score of 0.68 for social rela- tionship domain. Univariate analysis was performed for caregiver factors of the study participants, which were categorized and presented as categorical variables. For categorical vari- ables proportions and percentages were reported and findings displayed in frequency distribution tables. For continuous variables such as age, mean and standard deviations were reported. The WHOQOL- BREF domain scores were reported in a table reflect- ing minimum and maximum scores, the mode, mean and median. The QoL was presented in a pie chart with the percentages for poor and good QoL. Bivariate and multivariate analysis was performed to assess the relationship of associated factors with QoL using the linear regression method to derive the coeffi- cients with associated confidence intervals. The associ- ated factors were in domains that were scored and the sociodemographic characteristics. The scalar or continu- ous variables were used in bivariate analysis and these include; age, physical health, psychological wellbeing, social relationship and environment. Results Sociodemographic characteristics of the participants A total of 300 participants were interviewed using interviewer-administered questionnaires. The response rate for this study was 0.94. 128 (42.7%) of the partici- pants were male and 172 (57.3%) were female and the age ranged from 18 to 75 years. The mean age was 36.59 (SD ± 0.73) and 111 (37.0%) participants were within the age group of < 30 years. Majority of the participants, 163 (54.3%), were Baganda and the most common religion was Catholic that constituted 143 (41.3%) (See Table 5 in Appendix section). Data was collected from the caregivers and their patients’ medical records using the interviewer-administered tool. The time required for the interview was approximately 15– 20 min. For those who did not understand English, a Luganda version of the tool was used. There was no imme- diate benefit or compensation for the caregivers’ time. The most common diagnosis of the patients was Bipo- lar Affective Disorder, which constituted 45%. Of the 300 caregivers; 123 (42.3%) had taken care of their rela- tives or friends diagnosed with mental illness for a period of time between 1 year to <5 years (see Table 6 in Appendix section). Study procedure A l Approvals were obtained to collect data in Mulago Hospital SB Bosa Mental health clinic and Butabika hospital from the heads of department and the nurses in charge of the wards. All participants were screened for eligibility and eligible caregivers were recruited consecutively until the desired sample size was ob- tained. The eligible caregivers were taken to a room within the facility to maintain privacy, the study was explained to them and informed consent obtained from them. This was done before the patients went to see the psychiatrist for review and treatment or before the caregivers commenced their visits with the patients who were admitted on the wards. This is because of the long time which the caregivers spent before they were seen by the psychiatrist. Study population Associations or relationships from bivariate analysis with p values less than 0.2 and variables that were associated with QoL from our literature search were considered for stepwise linear regression model using Stata. The stepwise regres- sion was used to identify confounders that were vari- ables that caused a difference of ≥10% between the unadjusted and adjusted coefficients. The confounders were dropped and variables that had a strong relation- ship with the outcome variable were maintained. The variables with p values less than 0.05 were considered statistically significant in association to QoL. The WHOQOL-BREF was translated to Luganda and field tested among a sample of people living with or without HIV in Uganda [11] as part of the validation process. The results showed that the Luganda version of the WHOQOL-BREF had acceptable validity and reli- ability of 0.915. Quality of life of caregivers The QoL was measured on a scale of 1–5 from very poor to very good where 1 indicated very poor and 5 indicated very good. The scores 1–5 were then trans- formed using the WHOQOL-BREF syntax file to a scale of 0–100. The QoL measure was further catego- rized based on the score onto two parameters: good and poor QoL. Scores > 50 represented a good QoL while scores ≤50 represented a poor QoL. Of the 300 caregivers, 172 (57.30%) had a poor QoL while 18 (42.70%) had good QoL. WHOQOL domain scores There are four WHOQOL domains that include physical health, psychological wellbeing, social relationships and environment. Each of these domains was scored and the mean scores for each domain were; physical health 52.20 (SD ± 15.90), psychological wellbeing 55.97 (SD ± 15.94), social relationships 51.64 (SD ± 21.08) and environment 50.9 (SD ± 17.35) (see Table 2 below). = 0.038, 95% CI = 0.033 – 0.043), and social relation- ships (Unadjusted Coefficient = 0.74, 95% CI = 0.527 – 0.957). All these factors were statistically significant in association with QoL with p values < 0.001. However the above factors plus the categorical variables including gender, monthly income, period of time spent caring for the patient, education level, marital status and caregiver satisfaction with health were adjusted for in multivariate stepwise linear regression model (Table 4 below). Out of the 11 inde- pendent variables entered in the model, 7 variables were dropped due to their confounding effect. The confounders included; age, gender, monthly income, period of time spent caring for the patient, marital status, and physical health. The statistically significant factors associated with QoL that were derived included; environment (Adjusted coefficient = 0.016, 95% CI = 0.009 – 0.023), caregiver satisfaction with their health (Adjusted coefficient = 0.405, 95% CI = 0.33 – 0.487), psychological wellbeing (Adjusted coef- ficient = 0.007, 95% CI = 0.0002 – 0.013), and educa- tion level (Adjusted coefficient = 0.148, 95% CI = 0.072 – 0.225). Caregivers’ satisfaction with health Table 2 The Caregivers’ WHOQOL domain scores Domain Minimum score Maximum score Mode Median Mean SD Physical health 10.71 100 64.29 57.14 55.20 15.90 Psychological 12.50 91.67 50.00 58.33 55.97 15.94 Social relationships 0.00 100 58.33 50.00 51.64 21.08 Environment 0.00 96.88 56.25 53.13 50.49 17.35 Table 2 The Caregivers’ WHOQOL domain scores Domain Minimum Maximum Mode Median Mean SD Table 2 The Caregivers’ WHOQOL domain scores g Concerning the caregivers’ satisfaction with health, 146 (48.67%) were very dissatisfied and 154 (51.33%) were satisfied with the state of their health (see Table 1 below). Data analysis plan Data was analyzed using SPSS Version 22 statistical software and Stata. The total QoL score was calculated and transformed using SPSS syntax file and the final scores were categorized as good QoL and Poor QoL. Caregivers with scores above 50 were categorized as having good QoL. A few caregivers had missing data for income, educa- tional level and marital status. This is because they did not feel comfortable giving the information and consid- ered it sensitive. Ndikuno et al. BMC Psychiatry (2016) 16:400 Page 4 of 9 Factors associated with the QoL of caregivers of patients diagnosed with mental illness Bivariate analysis and multivariate analysis of factors as- sociated with QoL was done using linear regression to determine the factors significantly affecting QoL. The variables in this study fulfilled the assumptions for linear regression since they had a linear relationship, and multivariate normality. The variables also had no auto- correlation and no multicolinearity between them the highest VIF being 2.191. The final model had n R2 (variance) of 0.6 and an F static value of 85.6 with p < 0.00. This showed that the linear regression model used had adequate explanatory power for the relationships observed between the inde- pendent and dependent variables in this study. According to Table 3 below, the QoL decreased with increasing age of the participants (Unadjusted Coeffi- cient = −0.018, 95% CI = −0.027 – -0.009). QoL in- creased with increasing scores of the WHOQOL-BREF domains including physical health (Unadjusted Coeffi- cient = 0.033, 95% CI = 0.027 – 0.039), psychological wellbeing (Unadjusted Coefficient = 0.036, 95% CI = 0.030 – 0.042), environment (Unadjusted Coefficient QoL of caregivers of patients diagnosed with mental illness In this study we found that of the 300 participants, 172 (57.30%) had a poor QoL. These results are consistent with the different studies done in Hong Kong and Europe that suggested the QoL of caregivers is poor in comparison with the general population [7, 12]. QoL research is rare in sub-Saharan Africa [13], which makes it difficult to find comparable studies among the African population. Table 1 Caregiver satisfaction with their health Level of satisfaction Frequency Percentage (%) Very dissatisfied 24 8.0 Dissatisfied 59 19.7 Neither satisfied nor dissatisfied 63 21.0 Satisfied 137 45.7 Very satisfied 17 5.7 p p This poor QoL of life is probably due to the task of caregiving, which results in additional responsibilities on the caregivers’ daily life, and occupies their time, Ndikuno et al. BMC Psychiatry (2016) 16:400 Page 5 of 9 Table 3 Bivariate analysis of individual factors associated with QoL of caregivers Variable Unadjusted coefficient 95% CI p value Age −0.018 −0.027 – -0.009 <0.001* Physical health 0.033 0.027 – 0.039 <0.001* Psychological wellbeing 0.036 0.030 – 0.042 <0.001* Environment 0.038 0.033 – 0.043 <0.001* Social relationship 0.74 0.527 – 0.957 <0.001* Statistically significant since p < 0.05 Table 3 Bivariate analysis of individual factors associated with QoL of caregivers energy, and attention. This additional responsibility results in high levels of stress that impact negatively on the QoL of the caregivers [14, 15]. Due to this additional responsibility and high levels of stress, care- givers may fail to have appropriate means of dealing with this added role [16]. This puts the caregivers at risk of developing psychosocial or physical illness therefore interventions should be put in place to aid them in dealing with their role and thus improve their QoL [14]. It was suggested that people with good health status may have a better QoL [19] since they are usually satis- fied with the state of their health. Those with a low health status are usually dissatisfied with their health since they have anxiety about their own health and the health of their patients. Professional health care providers can have a sig- nificant impact on the health and well-being of care- givers [6]. Therefore health workers should be vigilant in identifying healthcare problems early in this group so as to manage them adequately. QoL of caregivers of patients diagnosed with mental illness This can be done when the caregivers visit the hospital with their patient or while they are on the inpatient ward. Health care programs can be put in place to raise awareness of the important role of caregivers in management of patients diagnosed with mental illness among health care providers so that they are not ignored during the routine clinic or hospital visits. Statistically significant since p < 0.05 Factors associated with QoL of caregivers of patients diagnosed with severe mental illness Caregiver satisfaction with health, education level, psychological wellbeing and environment were the four main factors, which were significantly associated with QoL of caregivers of patients diagnosed with severe mental illness. The education level of the caregiver was positively correlated with QoL showing that the higher the education level attained by the caregiver, the better their chances of having a good QoL. The caregivers who were dissatisfied with their health were more likely to have a poor QoL. This is consistent with the results of a study done in China which reflected that caregivers sacrifice themselves to care for their patients, resulting in strain on their physical and mental health [17]. This dissatisfaction in health is influenced by the inadequate time the care- giver spends on their health concerns since most of their time is invested in their role. As caregivers adjust their social lives to the needs of patients diagnosed with mental illness, they usually have concerns about their own health but neglect them therefore their health deteriorates which eventually impacts on their perception of their overall QoL [18]. These findings are consistent with the findings of studies carried out in Hong Kong and Turkey [6, 7]. As caregivers attain higher levels of education, they be- come more knowledgeable and develop more effective skills in managing the demand that comes with their role thus improving their QoL. For example they can be able to read and understand basic instructions re- quired to take care of patients diagnosed with severe mental illness, which makes their role easier. Highly educated caregivers tend to have better paying jobs or Table 4 Multivariate analysis of factors associated with poor QoL of caregivers of patients diagnosed with mental illness Variable Adjusted coefficient 95% CI p value Environment 0.016 0.009 – 0.023 <0.001* Caregiver satisfaction with their health 0.405 0.33 – 0.487 <0.001* Psychological wellbeing 0.0065 0.0002 – 0.013 0.044* Education level 0.148 0.072 – 0.225 <0.001* Social relationship 0.003 −0.001 – 0.007 0.180 Constant 0.231 −0.084 – 0.547 0.150 *Statistically significant since p < 0.05 Ndikuno et al. BMC Psychiatry (2016) 16:400 Page 6 of 9 Page 6 of 9 Page 6 of 9 sources of income and are able to adequately use their financial and social resources available in their communities to deal with the caregiving burden resulting in better QoL. transport. Factors associated with QoL of caregivers of patients diagnosed with severe mental illness These components may not significantly influ- ence the QoL on their own but have an effect in com- bination. The significance of the environment’s influence on QoL of caregivers is consistent with a study done in China [19]. The Ugandan education system is comprised of three major levels including primary, secondary and tertiary [20]. The primary level includes students from 6 to14 years, secondary includes 14 to 19 years and tertiary level includes students from 19 to 24 years. Since basic education is a fundamental human right and a component of wellbeing, the government of Uganda has put in place certain measures to avail education to all citizens. This has been done through introduction of Universal Primary Education (UPE) and Universal Secondary Education. The schools under these programs offer free education for those who may not be able to afford paying for it. This free education leads to increase in number of liter- ate caregivers therefore leading to improvement in QoL. In Uganda, certain sociocultural aspects dictate the caregiving role. The responsibilities of a caregiver are carried out both in the hospital setting and in the community [8]. While in the hospital after admission of the patient diagnosed with mental illness, the caregiver ensures that the patient’s basic needs are met from feeding to washing clothes, bathing and hygiene. Doctors and nurses utilise the caregivers in administering medica- tion and monitoring the patients. Occasionally when drugs are unavailable in the hospital, the caregiver is incharge of purchasing them from pharmacies or drug shops within the community. While in the com- munity after discharge, caregivers ensure physical wellbeing of the patient by providing the basic needs, protecting them from harm and watching them so that they do not wander and get lost or go missing. Caregivers also ensure that the patients adhere to drugs and go for their follow-up appointments or visits in the mental health clinics. Caregivers also offer emotional and social support to the patients diagnosed with mental illness. Concerning psychological wellbeing, the results showed that the lower the score, the lower the QoL of the caregiver. Low scores reflected a poor psycho- logical wellbeing. The caregiving role usually adds responsibilities on the caregivers’ daily life, and occupies their time, energy, and attention [21]. Limitations This study had a number of limitations including cul- tural bias as some participants feared to answer ques- tions about their sex life and intimate or personal relationships. The results of this study cannot be gener- alized to the entire population of caregivers worldwide due to differences in culture and environment in differ- ent areas. Environment domain score had a positive correlation with QoL of caregivers. The environmental domain was assessed using a number of components which included: financial resources, freedom, physical safety and security, health and social care, home environment, opportunities for acquiring new information and skills, participation in recreation/leisure activities, physical environment and Factors associated with QoL of caregivers of patients diagnosed with severe mental illness This results in care- giver stress and strain and predisposes them to develop- ment of mental or psychological illness especially if they have genetic predisposition [14, 16]. Opportunities for acquiring new information concern- ing the illness of the patient and how to care for the pa- tient in day to day life is important and it reduces the caregivers sense of strain or burden in their role [19]. As ones financial resources increase, their ability to afford care and hospitalization plus medication for their pa- tients increases while strain and stress in caring role is reduced. Drug banks have been created in caregiver sup- port groups where money is pooled and drugs are bought in bulk cheaply thus aiding the caregiver role. Therefore caregivers require information, support, good communication with mental health services and ad- equate financial resources inorder to effectively handle caring for the patients diagnosed with mental illness [12]. The most common psychological illnesses observed among caregivers include depression and anxiety dis- orders [6, 22]. Inorder to improve their psychological wellbeing and avoid development of any mental disor- ders, caregivers require social support or assistance in caring for their relatives or friends that are diagnosed with severe mental illness. This assistance ensures that caregivers are left with enough time and re- sources to address their own psychological needs [19]. Psychological wellbeing can be improved through support groups where caregivers are able to share the stressors related to their role so that they can adequately handle their role [8]. Support groups usually offer instrumental and emotional support. Im- proved psychological wellbeing results in better QoL of caregivers and they are able to carry out their role so that the patients diagnosed with mental illness can have good health outcomes. a10 participants did not answer this question Others bstands for other tribes including Itesot, Banyoro, Madi, Dinka, Batoro, Bagisu, Lugbara, Banyala, Japhadola, Alur, Acholi, Langi, Banyarwanda, Bakonjo, Bafumbira, Bakiga and Basamya. Others cfor other religions including SDA, Orthodox, Moon-Worshipper and Baptist. Others dstands for other occupations including student, construction workers and pastor Conclusion The QoL of the majority of caregivers of patients diag- nosed with mental illness attending the National Referral Ndikuno et al. BMC Psychiatry (2016) 16:400 Page 7 of 9 Appendix Table 5 Socio-demographic characteristics of the study participants Variable Frequency (N = 300) Percentage (%) Age ≤30 122 40.7 31–40 77 25.7 41–50 56 18.7 51–60 24 8.0 ≥60 21 7.0 Gender Male 128 42.7 Female 172 57.3 Tribe Baganda 163 54.3 Banyankole 20 6.7 Basoga 25 8.8 Othersb 92 30.7 Religion Catholic 124 41.3 Moslem 42 14.0 Pentecostal 49 16.3 Protestant 71 23.7 Othersc 14 4.7 Marital status (N = 290)a Single 100 34.5 Married 111 38.3 Living as married 44 15.2 Separated 15 5.2 Divorced 12 4.1 Widowed 8 2.8 Occupation Housewife 37 12.3 Subsistence Farmer 27 9.0 Self-employed 95 31.7 Professional 51 17.0 Unemployed 63 21.0 Othersd 27 9.0 a10 participants did not answer this question Others bstands for other tribes including Itesot, Banyoro, Madi, Dinka, Batoro, Bagisu, Lugbara, Banyala, Japhadola, Alur, Acholi, Langi, Banyarwanda, Bakonjo, Bafumbira, Bakiga and Basamya. Others cfor other religions including SDA, Orthodox, Moon-Worshipper and Baptist. Others dstands for other occupations Hospitals in Uganda is poor. From this study the main factors associated with the QoL are caregiver satisfaction with health, environment, education level and psycho- logical wellbeing. Attention to these factors should therefore be considered central to any intervention for caregivers in this population. Nurses and other health care professionals therefore need to be aware of the poor QoL of caregivers so as to put in place interventions like support groups or passing on information about the mental illness and how to care for the patients diagnosed with mental illness in order to contribute to improving the QoL of caregivers. This research done in a low-income country setting adds to the body of evidence that has accrued from high-income country settings to call for increased atten- tion to caregivers for patients diagnosed with mental illness. Internationally, interventions need to be put in place to aid caregivers in their role based on the socio- cultural setting in the different countries. a10 participants did not answer this question b Funding h d 7. Wong DF, Lam AY, Chan SK, Chan SF. Quality of life of caregivers with relatives suffering from mental illness in Hong Kong: roles of caregiver characteristics, caregiving burdens, and satisfaction with psychiatric services. Health Qual Life Outcomes. 2012;10:15. doi:10.1186/1477-7525-10-15. 7. Wong DF, Lam AY, Chan SK, Chan SF. Quality of life of caregivers with relatives suffering from mental illness in Hong Kong: roles of caregiver characteristics, caregiving burdens, and satisfaction with psychiatric services. Health Qual Life Outcomes. 2012;10:15. doi:10.1186/1477-7525-10-15. The study was self-funded from the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. References 1. GBDCollaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015;386(9995):743–800. doi:10.1016/s0140- 6736(15)60692-4. a12 participants did not answer the question. b9 participants did not answer this question 2. Sanyu R. Mental illness & exclusion: putting mental health on the development agenda in Uganda. 2007. 3. Schulz R, Beach SR. Caregiving as a risk factor for mortality: the Caregiver Health Effects Study. JAMA. 1999;282(23):2215–9. 4. Seloilwe ES. Experiences and demands of families with mentally ill people at home in Botswana. J Nurs Scholarsh. 2006;38(3):262–8. 5. Dalui A, Guha P, De A, Chakraborty S, Chakraborty I. Assessment of stress & related albuminuria in caregivers of severe mentally ill persons. Indian J M d R 2014 139(1) 174 7 Authors’ contributions CN, MN, JK, DM, CO participated in the conception, study design, data analysis and manuscript preparation. CN participated in the data collection. All authors read and approved the final manuscript. 11. Martin F, Russell S, Seeley J. The WHOQOL BREF questionnaire in Luganda: validation with a sample including people living with HIV in Uganda DEV reports and policy paper series. University of East Anglia: The School of International Development; 2013. Ethics approval and consent to participate Ethical approval to carry out the study was sought and obtained from Makerere University School of Health Sciences IRB, Mulago Hospital Research and Ethics Committee and Butabika Research and Ethics Committee. Ethical approval to carry out the study was sought and obtained from Makerere University School of Health Sciences IRB, Mulago Hospital Research and Ethics Committee and Butabika Research and Ethics Committee. Informed consent was obtained from the study participants. A translated consent form was availed to Luganda speaking participants. For illiterate caregivers, an impartial witness was present for informed consent. Privacy was ensured during data collection by carrying out interviews from the Nurse’s office in the mental health Outpatient clinics. Confidentiality was observed during and after data collection by not including names of the participants and access to the questionnaire was limited to the principle investigator and the supervisors. Availability of data and materials 8. Carroll D. ‘The Core Resource’ the role of informal caretakers of the mentally Ill as seen in Uganda. 2008. 8. Carroll D. ‘The Core Resource’ the role of informal caretakers of the mentally Ill as seen in Uganda. 2008. The dataset generated and analyzed during the study is not publicly available due to the fact that it has a number of indirect identifiers. This could lead to breach of confidentiality. However the dataset can be obtained from the corresponding author on reasonable request. 9. WHOQOLGroup. WHOQOL-BREF: introduction, administration, scoring and generic version of the assessment: field trial version, December 1996. In: W. H. O. Geneva: Programme on Mental Health (Ed.). 1996. 9. WHOQOLGroup. WHOQOL-BREF: introduction, administration, scoring and generic version of the assessment: field trial version, December 1996. In: W. H. O. Geneva: Programme on Mental Health (Ed.). 1996. 10. Skevington SM, Lotfy M, O’Connell KA, Group W. The World Health Organization’s WHOQOL-BREF quality of life assessment: psychometric properties and results of the international field trial. A report from the WHOQOL group. Qual Life Resour. 2004;13(2):299–310. Acknowledgements g We want to acknowledge Nandawula Valeria and Ddembe Emma who helped in data collection and Mr. Tom Ngabirano for help in data management. The nurses of Mulago hospital Bbosa Mental Health Unit and Butabika Hospital for all their invaluable help and support during the study. We would like to thank the caregivers who participated in the study by giving information that was used to answer the different questions in the study. 6. Yikilkan H, Aypak C, Gorpelioglu S. Depression, anxiety and quality of life in caregivers of long-term home care patients. Arch Psychiatr Nurs. 2014;28(3): 193–6. doi:10.1016/j.apnu.2014.01.001. 6. Yikilkan H, Aypak C, Gorpelioglu S. Depression, anxiety and quality of life in caregivers of long-term home care patients. Arch Psychiatr Nurs. 2014;28(3): 193–6. doi:10.1016/j.apnu.2014.01.001. Implications for practice However the dataset can be obtained from the corresponding author on reasonable request. Authors’ contributions CN, MN, JK, DM, CO participated in the conception, study design, data analysis and manuscript preparation. CN participated in the data collection. All authors read and approved the final manuscript. Authors’ information CN Nurse BSN (MakCHS). Table 6 Other Sociodemogragraphic characteristics of caregivers Variable Frequency (N = 300) Percentage Education level (n = 288)a None at all 29 10.1 Primary 61 21.2 Secondary 95 33.0 Tertiary 103 35.8 Income (n = 291)b Shs. 0–50,000 154 54.2 Shs. 50,001–100,000 41 14.4 Shs. 100,001–500,000 63 22.2 Shs. 500,001 and above 26 9.2 Diagnosis of the patient Schizophrenia 105 35 Bipolar Affective Disorder 135 45 Major Depressive Disorder 60 20 Period of time spent in taking care of the patient < 1 year 63 21.6 1 year to < 5 years 123 42.3 5 years to < 10 years 49 16.8 10 years to < 20 years 41 14.1 20 years and above 15 5.0 a12 participants did not answer the question. b9 participants did not answer this question Table 6 Other Sociodemogragraphic characteristics of caregivers Variable Frequency (N = 300) Percentage Education level (n = 288)a None at all 29 10.1 Primary 61 21.2 Secondary 95 33.0 Tertiary 103 35.8 Income (n = 291)b Shs. 0–50,000 154 54.2 Shs. 50,001–100,000 41 14.4 Shs. 100,001–500,000 63 22.2 Shs. 500,001 and above 26 9.2 Diagnosis of the patient Schizophrenia 105 35 Bipolar Affective Disorder 135 45 Major Depressive Disorder 60 20 Period of time spent in taking care of the patient < 1 year 63 21.6 1 year to < 5 years 123 42.3 5 years to < 10 years 49 16.8 10 years to < 20 years 41 14.1 20 years and above 15 5.0 a12 participants did not answer the question. b9 participants did not answer this question Received: 6 June 2016 Accepted: 21 October 2016 Received: 6 June 2016 Accepted: 21 October 2016 Author details 1D f 1Department of Nursing, College of Health Sciences, Makerere University, Kampala, Uganda. 2Department of Surgery, College of Health sciences, Makerere University, Kampala, Uganda. 3Department of Pediatrics, College of Health Sciences, Makerere University, Kampala, Uganda. 4Department of Nursing, College of Health Sciences, Makarere University, Mulago Hill road, P.O BOX 7072, Kampala, Uganda. Received: 6 June 2016 Accepted: 21 October 2016 Implications for practice Health workers should be aware of the poor QoL among caregivers so as to routinely measure this QoL and intervene where there is impairment. Support groups should also be put in place for the caregivers where they can share and assist each other to reduce the burden of care thus improving their QoL. Within these support groups they can so being drug banks or collection of funds that can be used to aid caregivers that may be lacking resources to use in their role. In addition, counseling sessions should be put in place for the caregivers at the different health facilities or within the community. The policy makers at different government levels should put in place policies that ensure integration of caregiver interventions within the mental health system. Community mental health programs should also be integrated in the different decentralized health facilities where awareness about mental illness can be increased among the community members and thus reduce stigma against patients and their caregivers. The increased awareness can so lead to provision of social support to caregivers by the community members thus reducing social isolation among the caregivers and eventually leading to better QoL. a10 participants did not answer this question b Further research should be carried out to assess the relationship between caregiving situation (objective and subjective burden) and QoL of caregivers. Further research should also be carried out involving a case control study to compare QoL of caregivers of patients diagnosed with severe mental illness with the QoL of the general population. Ndikuno et al. BMC Psychiatry (2016) 16:400 Page 8 of 9 Acknowledgements We want to acknowledge Nandawula Valeria and Ddembe Emma who helped in data collection and Mr. Tom Ngabirano for help in data management. The nurses of Mulago hospital Bbosa Mental Health Unit and Butabika Hospital for all their invaluable help and support during the study. We would like to thank the caregivers who participated in the study by giving information that was used to answer the different questions in the study. Funding The study was self-funded from the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Availability of data and materials The dataset generated and analyzed during the study is not publicly available due to the fact that it has a number of indirect identifiers. This could lead to breach of confidentiality. Authors’ information CN Nurse BSN (MakCHS). MN Midwife, lecturer BSN (MakCHS), MSN (Midwifery and Women’s health) (MakCHS). l h k MN Midwife, lecturer BSN (MakCHS), MSN (Midwifery and Women’s health) (MakCHS). 12. Margetic BA, Jakovljevic M, Furjan Z, Margetic B, Marsanic VB. Quality of life of key caregivers of schizophrenia patients and association with kinship. Cent Eur J Public Health. 2013;21(4):220–3. JK Registrar surgery (Surgical trainee), MBChB (MakCHS), MCS (ECSA). DM Medical Officer, MBChB (MakCHS), PhD student (UIB, Norway). CO Psychiatrist Nurse, lecturer BSN (MakCHS), MSN (Psychiatric Nursing) (MUHAS). JK Registrar surgery (Surgical trainee), MBChB (MakCHS), MCS (ECSA). DM Medical Officer, MBChB (MakCHS), PhD student (UIB, Norway). CO Psychiatrist Nurse, lecturer BSN (MakCHS), MSN (Psychiatric Nursing) (MUHAS). 13. Muhwezi WW, Okello ES, Turiho AK. Gender-based profiling of Quality of Life (QOL) of primary health care (PHC) attendees in central Uganda: a cross sectional analysis. Afr Health Sci. 2010;10(4):374–85. Page 9 of 9 Ndikuno et al. BMC Psychiatry (2016) 16:400 14. Beinart N, Weinman J, Wade D, Brady R. Caregiver burden and psychoeducational interventions in Alzheimer’s disease: a review. Dement Geriatr Cogn Disord Extra. 2012;2:638–48. 15. Kasuya RT, Polgar-Bailey P, Takeuchi R. Caregiver burden and burnout. A guide for primary care physicians. Postgrad Med. 2000;108:119–23. 16. Olwit C, Musisi S, Leshabari S, Sanyu I. Chronic sorrow: Lived experiences of caregivers of patients diagnosed with Schizophrenia in Butabika Mental Hospital, Kampala, Uganda. Arch Psychiatr Nurs. 2015;29(1):43–8. http://dx. doi.org/10/1016/j.apnu.2014.09.007. 17. Yang X, Hao Y, George SM, Wang L. Factors associated with healthrelated quality of life among Chinese caregivers of the older adults living in the community: a cross-sectional study. Health Qual Life Outcomes. 2012;10:143. 18. Mavundla TR, Toth F, Mphelane ML. Caregiver experience in mental illness: a perspective from a rural community in South Africa. Int J Mental Health Nurs. 2009;18(5):357–67. doi:10.1111/j.1447-0349.2009.00624.x. 19. Li J, Lambert CE, Lambert VA. Predictors of family caregivers’ burden and quality of life when providing care for a family member with schizophrenia in the People’s Republic of China. Nurs Health Sci. 2007;9(3):192–8. doi:10. 1111/j.1442-2018.2007.00327.x. BOS, U. B. o. S. (2010). Uganda National Household Survey 2009/10: U 21. Leow MQH, Chan SWC. Factors affecting caregiver burden of terminally ill adults in the home setting‐A systematic review. JBI Database of Systematic Reviews and Implementation Reports. 2011;9(45):1883–916. 22. Idstad M, Ask H, Tambs K. Mental disorder and caregiver burden in spouses: the Nord- Trondelag health study. BMC Public Health. 2010;10:516. doi:10. 1186/1471-2458-10-516. 22. Idstad M, Ask H, Tambs K. Mental disorder and caregiver burden in spouses: the Nord- Trondelag health study. BMC Public Health. 2010;10:516. doi:10. 1186/1471-2458-10-516. 21. Leow MQH, Chan SWC. Factors affecting caregiver burden of terminally ill adults in the home setting‐A systematic review. JBI Database of Systematic Reviews and Implementation Reports. 2011;9(45):1883–916. 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https://openalex.org/W1990631683	https://europepmc.org/articles/pmc3636091?pdf=render	English	null	LABCG2, a New ABC Transporter Implicated in Phosphatidylserine Exposure, Is Involved in the Infectivity and Pathogenicity of Leishmania	PLoS neglected tropical diseases	2,013	cc-by	14,808	Abstract Leishmaniasis is a neglected disease produced by the intracellular protozoan parasite Leishmania. In the present study, we show that LABCG2, a new ATP-binding cassette half-transporter (ABCG subfamily) from Leishmania, is involved in parasite virulence. Down-regulation of LABCG2 function upon expression of an inactive mutant version of this half-transporter (LABCG2K/M) is shown to reduce the translocation of short-chain analogues of phosphatidylserine (PS). This dominant- negative phenotype is specific for the headgroup of the phospholipid, as the movement of phospholipid analogues of phosphatidylcholine, phosphatidylethanolamine or sphingomyelin is not affected. In addition, promastigotes expressing LABCG2K/M expose less endogenous PS in the stationary phase than control parasites. Transient exposure of PS at the outer leaflet of the plasma membrane is known to be one of the mechanisms used by Leishmania to infect macrophages and to silence their immune response. Stationary phase/metacyclic promastigotes expressing LABCG2K/M are less infective for macrophages and show decreased pathogenesis in a mouse model of cutaneous leishmaniasis. Thus, mice infected with parasites expressing LABCG2K/M did not develop any lesion and showed significantly lower inflammation and parasite burden than mice infected with control parasites. Our results indicate that LABCG2 function is required for the externalization of PS in Leishmania promastigotes, a process that is involved in the virulence of the parasite. Citation: Campos-Salinas J, Leo´n-Guerrero D, Gonza´lez-Rey E, Delgado M, Castanys S, et al. (2013) LABCG2, a New ABC Transporter Implicated in Phosphatidylserine Exposure, Is Involved in the Infectivity and Pathogenicity of Leishmania. PLoS Negl Trop Dis 7(4): e2179. doi:10.1371/journal.pntd.0002179 Editor: Genevieve Milon Institut Pasteur France Citation: Campos-Salinas J, Leo´n-Guerrero D, Gonza´lez-Rey E, Delgado M, Castanys S, et al. (2013) LABCG2, a New ABC Transporter Implicated in Phosphatidylserine Exposure, Is Involved in the Infectivity and Pathogenicity of Leishmania. PLoS Negl Trop Dis 7(4): e2179. doi:10.1371/journal.pntd.0002179 Editor: Genevieve Milon, Institut Pasteur, France Editor: Genevieve Milon, Institut Pasteur, France Received September 25, 2012; Accepted March 15, 2013; Published April 25, 2013 Copyright: 2013 Campos-Salinas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Spanish Grants SAF2009-07440 and SAF2012-34267 (to F.G.), SAF2011-28215 (to J.M.P.V.) and SAF2011-28102 (to S.C.), the EU Marie Curie Research Training Network Grant MRTN-CT-2004-005330 (to F.G.), the Plan Andaluz de Investigacio´n (Cod. Jenny Campos-Salinas., David Leo´ n-Guerrero., Elena Gonza´lez-Rey, Mario Delgado, Santiago Castanys, Jose´ M. Pe´rez-Victoria, Francisco Gamarro* Instituto de Parasitologı´a y Biomedicina ‘‘Lo´pez-Neyra’’, CSIC, (IPBLN-CSIC), Parque Tecnolo´gico de Ciencias de la Salud, Armilla, Granada, Spain Biomedicina ‘‘Lo´pez-Neyra’’, CSIC, (IPBLN-CSIC), Parque Tecnolo´gico de Ciencias de la Salud, Armilla, Granada, Spain nstituto de Parasitologı´a y Biomedicina ‘‘Lo´pez-Neyra’’, CSIC, (IPBLN-CSIC), Parque Tecnolo´gico de Ciencias de la Salud, Armilla, Granad Abstract BIO130) and by FEDER funds from the EU to F.G., S.C. and J.M.P.V. and funds from Junta de Andalucia (Spain) P09-CTS-4705 (E. G-R). 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. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gamarro@ipb.csic.es . These authors contributed equally to this work. Citation: Campos-Salinas J, Leo´n-Guerrero D, Gonza´lez-Rey E, Delgado M, Castanys S, et al. (2013) LABCG2, a New ABC Transporter Implicated in Phosphatidylserine Exposure, Is Involved in the Infectivity and Pathogenicity of Leishmania. PLoS Negl Trop Dis 7(4): e2179. doi:10.1371/journal.pntd.0002179 PLOS Neglected Tropical Diseases \| www.plosntds.org LABCG2, a New ABC Transporter Implicated in Phosphatidylserine Exposure, Is Involved in the Infectivity and Pathogenicity of Leishmania Jenny Campos-Salinas., David Leo´ n-Guerrero., Elena Gonza´lez-Rey, Mario Delgado, Santiago Castanys, Jose´ M. Pe´rez-Victoria, Francisco Gamarro* Author Summary Author Summary Leishmania is a protozoan parasite that infects human macrophages, producing the neglected tropical disease known as leishmaniasis. As is the case for apoptotic cells, transient exposure of phosphatidylserine (PS) on the surface of the parasite is required for macrophage engulfment and infection. Although the mechanism involved in this lipid translocation remains unknown, inhibition of PS exposure could therefore prove to be a novel way to combat this parasitic disease. Here, we have identified a new ABC transporter from Leishmania, namely LABCG2, as a protein involved in this process. The dominant-negative inhibition of LABCG2 showed that this transporter is required for the normal exposure of PS on the outer leaflet of the plasma membrane. This altered phenotype was subsequently found to be correlated with a deficient ability to infect mouse peritoneal macrophages. In addition, studies in a mouse model of cutaneous leishmaniasis showed that animals infected with parasites with down-regulated LABCG2 activity did not develop any lesions. Taken together, these results suggest a role for the Leishmania LABCG2 transporter in PS exposure, determin- ing the virulence of the parasite. The aim of our work was to study the functionality of the transporter LABCG2 from Leishmania, specifically its involvement in PS translocation and its implication in parasite virulence. The results show that down-regulation of LABCG2 produces a defect in the exposure of endogenous PS at the external surface of the parasite, and that this defect correlates with a significant decrease in the ability of these parasites to infect mouse peritoneal macrophages and to produce pathology in a mouse model of cutaneous leishmaniasis. of L. amazonensis is associated with a modified host inflammatory response, correlating with parasite infectivity and with clinical parameters of diffuse cutaneous leishmaniasis [17]. Thus, Leish- mania parasites able to expose higher amounts of PS, induce a more severe and persistent human disease [17]. The plasma membrane PL asymmetry in eukaryotic cells is maintained due to the bidirectional transport of PL (flip-flop), which involves three protein-mediated activities [18]: i) flippases, which promote active inward-directed PL migration, mediated by aminophospholipid translocases (APT); ii) floppases, which are responsible for the active outward transport of PL from the cytoplasmic to the exoplasmic leaflet of the membrane, mediated by various ATP-binding cassette (ABC) transporters; and iii) scramblases, which are translocases that not require ATP to equilibrate the PL between the two membrane bilayers. Introduction outer leaflet of the plasma membrane of apoptotic mammalian cells [10] constitutes the most central ‘‘eat-me’’ signal known for macrophages, which also ‘‘silent’’ its activity to avoid an inflammatory reaction [11]. In a process known as apoptotic mimicry, surface exposure of PS in Leishmania promastigotes and amastigotes is required for the infection of new mammalian cells [6,7] and for down-regulation of the microbicidal activity of macrophages [8,9,12] by inhibiting their nitric oxide production and increasing IL-10 synthesis and TGFb1 secretion [8,13]. In addition, the well-characterized higher infectivity of the stationary phase promastigotes (metacyclic), as compared to the log phase promastigotes, is also due to the specific exposure of PS on their surface [14], among others factors including the lipophosphogly- can (LPG) or the phosphatidylinositol-anchored surface molecule gp63 [15]. Interestingly, it has been suggested that these PS- exposing promastigotes could be genuine apoptotic cells destined for death [12,16] instead of apoptotosis-mimicking parasites. Indeed, their presence in the virulent inoculum, in an altruistic behaviour, provides survival advantages for the viable parasites and is necessary for progress of the disease [16]. Recently, it has been demonstrated that PS exposure by intracellular amastigotes Leishmaniasis is a neglected disease that is caused by different species of the protozoan parasite Leishmania [1]. This parasite has a digenetic life cycle in which it alternates between promastigote and amastigote stages. Inside the insect (sandfly) vector, non-infective promastigotes are transformed into infective parasites during metacyclogenesis. After the host is bitten by the sandfly, an intense neutrophilic infiltrate into the skin bite sites occurs accompanied by a significant recruitment of macrophages. Afterwards, Leish- mania metacyclic promastigotes attach to neutrophils as the initial host cell, and are taken up by phagocytosis [2]. The uptake of infected neutrophils by macrophages is a mechanism for ‘‘silent’’ entry of parasites into macrophages, where they differentiate into the replicative amastigote forms that are responsible for mainte- nance and propagation of the infection in the phagolysosomal compartment of the mammal host [3,4]. Phosphatidylserine (PS), a phospholipid (PL) normally asym- metrically confined on the inner leaflet of the plasma membrane of eukaryotic cells [5], seems to play a critical role in the infection of macrophages by Leishmania [6–9]. Indeed, PS exposure on the April 2013 \| Volume 7 \| Issue 4 \| e2179 1 PLOS Neglected Tropical Diseases \| www.plosntds.org Leishmania Infectivity Involves an ABC Transporter homo-/heterodimerization to reconstitute the ATP sites. Author Summary PS externalization in apoptotic cells has been suggested to be due to i) a scramblases activity, enhanced by loss of the APT function [19]; and ii) to a higher activity of ABC efflux pumps such as ABCA1 [20]. Additionally, it has been suggested that PS is also delivered to the surface of lysosomes that fuse with the plasma membrane during apoptosis [21]. In the case of Leishmania, a decrease in the active out-to-in PS translocation, thus allowing ATP-independent PS movement, has also been suggested to be responsible for the loss of PL asymmetry [14]. However, disruption of the plasma membrane APT of Leishmania (LdMT) does not lead to an increased infectivity [22,23]. In addition, although a scramblase activity has been described in Leishmania, its role in parasite infectivity remains to be elucidated [24]. The molecular basis of PS exposure in Leishmania therefore remains unsolved. Introduction Members of the ABCA, ABCB and ABCG human subfamilies have been implicated in PL translocation [18,27]. For example, human ABCG2 (BCRP/MXR/ABCP), a protein involved in multidrug resistance in cancer cells [28,29], is responsible for enhanced exposure of PS at the plasma membrane of ABCG2 overexpress- ing cells due to increased outward PS transport [30]. Members of the ABCG subfamily of half-transporters have been identified in Leishmania [31], and three of these have already been functionally characterized. Thus, LABCG4 is localized at the plasma membrane of the parasite and is involved in the translocation of phosphatidylcholine (PC) analogues; it also confers resistance to alkyl phospholipids [32]. LABCG6 is also localized at the plasma membrane and is probably involved in PL trafficking as it reduces the accumulation of PL analogues of PC, phosphatidylethanol- amine (PE) and PS [33], and confers resistance to camptothecin [34], miltefosine and sitamaquine [33]. LABCG5, in contrast, is not involved in the translocation of PL at the plasma membrane or drug resistance but participates in salvage of the heme released after the breakdown of internalized haemoglobin [35]. Addition- ally, it has been reported that other Leishmania ABC transporters such as LABCB4 [36], LABCA1 [37] and LABCA2 [38] are involved in PL translocation. PLOS Neglected Tropical Diseases \| www.plosntds.org Leishmania strains and cell cultures Promastigote forms of Leishmania major clone V1 (MHOM/IL/ 80/Friedlin), Leishmania infantum (MHOM/ES/1993/BCN-99) and Leishmania donovani (MHOM/ET/67/HU3) were maintained in vitro at 28uC in modified RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 20% heat-inactivated foetal bovine serum (hiFBS, Invitrogen), as described previously [37,38]. To determine parasite sensitivity to the toxic peptides papuamide B and Ro-peptide, and to amphotericin B, 106/mL parasites were incubated in RPMI-1640 containing different concentrations of peptides and the parasite viability determined by MTT analysis after 72 h, as described previously [39]. Measurement of NBD-PS outward transport in L. major lines To measure the NBD-PS outward transport from the cytoplas- mic to the exoplasmic leaflet, Leishmania stationary-phase promas- tigotes (107/ml) were labeled with 30 mM (control parasites) or 15 mM (LABCG2K/M) NBD-PS for 30 min at 28uC in HPMI buffer (20 mM HEPES, 132 mM NaCl, 3.5 mM KCl, 0.5 mM MgCl2, 5 mM glucose, 1 mM CaCl2, pH 7.4) supplemented with 0.1% (w/v) BSA to allow that inward movement of the NBD analogue was equally, as previously described [30]. Afterwards, NBD-PS remaining on the cell surface was extracted twice by incubation with 2% (w/v) BSA in HPMI (supplemented with 5 mM glucose and 500 mM PMSF) for 5 min on ice. Before starting the outward transport assay, the medium was removed and parasites were washed with ice-cold PBS. For t = 0 min, the cells were resuspended in HPMI (supplemented with 2% BSA, 5 mM glucose and 500 mM PMSF). Time dependent outward transport was monitored at 28uC at different time points (5, 15, 30, 60 min) in the supernatants and the samples were analyzed by SLM-Aminco 8000C spectrofluorimeter. Leishmania Infectivity Involves an ABC Transporter Leishmania Infectivity Involves an ABC Transporter pXG-GFP+29 and pXG-’GFP, which can be used to express GFP fusion proteins in Leishmania with GFP at either the N- or the C- terminus, respectively, were kindly provided by Dr. Stephen M. Beverley. 28uC or 4uC. For mitochondrial labelling, 107 stationary-phase promastigotes were stained with 50 nM MitoTracker Deep Red 633 for 30 min at 28uC and then washed in ice-cold phosphate- buffered saline (PBS; 1.2 mM KH2PO4, 8.1 mM Na2HPO4, 130 mM NaCl and 2.6 mM KCl adjusted to pH 7). Parasites were fixed for 30 min at 4uC with 2% paraformaldehyde and then observed under a microscope. Images (one stack) were acquired using an Olympus IX81 microscope and deconvolved using Huygens Professional. Gene expression analysis Total RNA was prepared from control (empty vector) and LABCG2K/M expressing promastigotes using the total RNA isolation kit (Roche Biochemicals). cDNA was synthesized from 60 ng of total RNA using Superscript II TM RNaseH Reverse Transcriptase (Invitrogen) and oligo (dT)12–18 primers (Invitro- gen) following the manufacturer’s instructions. Semi-quantitative PCR was performed with 50 mL aliquots using 50 pmol each of sense and antisense primers corresponding to LABCG2 and LmGAPDH using the following profile: initial denaturation at 95uC for 5 min followed by 25 cycles with denaturation at 95uC for 1 min, annealing at 54uC for 30 s and extension at 68uC for 35 s, with a final extension of 5 min. DNA constructs and cell transfection DNA constructs and cell transfection LABCG2 (GeneDB-L. major, Accession Code LmjF06.0090) was isolated from the genomic DNA of L. major by PCR using sense (59- ATATCGCTGTCTCTGCGTCG) and antisense (59- GGCA- AACACACAGAGCGATG) primers. The nucleotide sequences were determined automatically as previously described [40]. To obtain parasites expressing non-functional LABCG2, a mutation was introduced in the Walker A motif of the ATP binding domain, replacing lysine 108 with a methionine (K108M) using the QuikChange XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). The resulting mutated gene LABCG2K108M (LABCG2K/M) was cloned into the Leishmania expression vector pUCNeoPlus [37]. The vector pXG-GFP+29 was used to create GFP- LABCG2 and -LABCG2K/M versions with GFP fusions at N-terminus [41]. The LABCG2 and LABCG2K/M open reading frame for these N-terminus tagged versions was amplified by PCR using sense (59-GCGGCCGCATGCCCCCTCCCGCAGCAA- CACGTGC) and antisense (59-GCGGCCGCTCATGCC- GTTCTCGCACAGCTCGCCA) primers. For the C-terminus tagged versions, LABCG2 and LABCG2K/M were cloned in a pXG-’GFP+ using sense (59-ACCGGTATGCCCCCTCCCG- CAGCAACACGTGC) and antisense (59- GATATCTGCCGTT- CTCGCACAGCTCGCCACGG) primers. Promastigotes of L. major were transfected with the different constructs and selected for G-418 resistance as described previously [42]. Materials 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), PMSF (phenylmethylsulfonyl fluoride), DFP (diisopropyl- fluorophosphate), monoclonal anti a-tubulin, and amphotericin B were obtained from Sigma Chemical Company (St. Louis, USA). Anti-histone H2A was courtesy of Dr. Stephen M. Beverley (Washington University, School of Medicine, St. Louis, Missouri, USA). Polyclonal anti-GFP antibody was from Rockland Compa- ny. Mouse monoclonal anti-gp63 was from Life Span BioSciences. Polyclonal antisera against metacyclic marker protein HASPB was a kind gift from Dr. Deborah F. Smith (University of York, UK). The fluorescent analogues 1-palmitoyl-2-[6-(7-nitrobenz-2- oxa-1,3-diazol-4-yl)amino]hexanoyl-sn-glycero-3-phosphocholine (NBD-PC), -phosphoethanolamine (NBD-PE), -phosphoserine (NBD-PS) and 6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-hexa- noyl-sphingosine-1 phosphocholine (NBD-sphingomyelin; NBD- SM) were purchased from Avanti Polar Lipids (Birmingham, AL, USA). Annexin V-Alexa 488, FM4-64, concanavalin A-Alexa Red, MitoTracker Deep Red 633, Cell Tracker TM Green and DAPI were from Molecular Probes (Invitrogen, Carlsbad, CA). Ro-peptide (Ro09-0198), a tetracyclic peptide antibiotic, was kindly provided by Dr. Masato Umeda (The Tokyo Metropolitan Institute of Medical Science, Japan). Papuamide B (Flintbox, LynseyHuxham), a novel depsipeptide obtained from extracts of marine sponges, was kindly provided by Dr. Thomas Gunther Pomorski and Dr. Rosa Lo´pez (Department of Plan Biology and Biotechnology, University of Copenhagen, Denmark). Peanut agglutinin (PNA) and fluorescein-conjugated ricin agglutinin was purchased from Vector (Burlingame, CA). The plasmids Functional ABC transporters consists of two homologous halves, each of which is composed of a transmembrane domain (TMD), which is involved in substrate binding and a cytosolic nucleotide binding domain (NBD), which hydrolyses ATP to provide the energy required for the transport [25]. The ATP sites are reconstituted upon dimerization of both NBDs, which pack together in a head-to-tail configuration to generate two ATP binding and hydrolysis sites between the conserved Walker A and B motifs of one NBD and the signature motif of the other [26]. ABC half-transporters with a single NBD therefore require PLOS Neglected Tropical Diseases \| www.plosntds.org April 2013 \| Volume 7 \| Issue 4 \| e2179 2 Annexin V- binding assay g y Leishmania promastigotes were harvested in RPMI-1640 and centrifuged at 25006g for 10 min at 4uC. The cells were washed with Annexin V-binding buffer (20 mM HEPES, 132 mM NaCl, 3.5 mM KCl, 5 mM CaCl2 and 0.5 mM MgCl2, pH 7.4 and 10 mM glucose), then resuspended in the same buffer and incubated with Annexin V–Alexa 488 (1/20 dilution; at the concentration indicated by the manufacturer) at 4uC for 15 min. The parasites were subsequently labelled with propidium iodide (0.4 mg/ml) and the mixture incubated for 5 min at 4uC. The cells were washed for 1 min at 25006g and 4uC, and resuspended to a cell density of 46106 cells/mL. Controls measurements in the absence of calcium were included using Annexin V–Alexa 488 plus 8 mM EGTA. Cellular fluorescence was quantified by scanning the emission in a FACSCalibur and analysed using the Cell Quest Pro software application. A total of 10,000 events were harvested from each sample. The control cells were incubated in Analysis of fluorescent PL uptake The NBD-phospholipid accumulation was determined by flow cytometry as described previously [43]. Briefly, stationary-phase promastigotes (107/mL) were incubated in HPMI buffer (20 mM HEPES, 132 mM NaCl, 3.5 mM KCl, 0.5 mM MgCl2, 5 mM glucose, 1 mM CaCl2, pH 7.4) supplemented with 0.3% (w/v) BSA for 30 min at 28uC, then labelled with 10 mM NBD-PC, 10 mM NBD-PE, 10 mM NBD-SM or 30 mM NBD-PS for 30 min at 28uC. HPMI was supplemented with either 500 mM PMSF or 5 mM DFP to block the catabolism of NBD-lipids [43]. Parasites were washed twice with ice-cold PBS, supplemented with 0.3% BSA and resuspended in PBS for flow cytometry analysis, using a Beckton Dickinson FACScan (San Jose´, CA) equipped with an argon laser operating at 488 nm. Fluorescence microscopy of Leishmania promastigotes 7 For endosome/lysosomal labelling, 107 stationary-phase pro- mastigotes obtained after 4 day culture were incubated in 1 mL of RPMI 1640 medium containing 50 mg/mL of concanavalin A- Alexa Red for 2 h at 28uC or with 1 mM FM4-64 for 30 min at PLOS Neglected Tropical Diseases \| www.plosntds.org April 2013 \| Volume 7 \| Issue 4 \| e2179 3 Leishmania Infectivity Involves an ABC Transporter Annexin V-binding buffer alone, without Annexin V–Alexa 488, under identical conditions. Annexin V-binding buffer alone, without Annexin V–Alexa 488, under identical conditions. lin G (Sigma). These cells were washed three times with PBS-0.1% BSA and the parasite-associated fluorescence was analyzed by flow cytometry using a FACSCalibur. Metacyclic purification assay p p g Peritoneal macrophages from BALB/c mice (Charles River Ltd.) were harvested by lavage with ice-cold RPMI 1640 medium, plated at a density of 56105 macrophages/well in RPMI-1640 medium plus 10% hiFBS in 24-well plates provided with glass coverslips (22 mm2) and allowed to adhere for 4 h at 37uC under 5% CO2, as described previously [7,8]. The adherent macrophages were infected at 35uC with stationary-phase promastigotes of control and LABCG2K/M- expressing L. major parasites with or without Annexin V (0.05 mg/ ml6107promastigotes), at a parasite-to-cell ratio of 5:1 in RPMI-1640 medium supplemented with 5% hiFBS. After 4 h of infection, unphagocytosed parasites were removed by washing with serum-free medium. The infected macrophages were further incubated in RPMI 1640 medium supplemented with 10% hiFBS for 24 h at 37uC in a 5% CO2 atmosphere. Following incubation, the cultures were fixed with 2% paraformaldehyde/glucose, stained with DAPI and the rate of infected macrophage analyzed using images acquired with an Olympus IX81 microscope as described previously [44]. Parasites were quantified using a cell counter provide with the Image J software (http://rsb.info.nih.gov/ij/). The images were deconvolved using Huygens Professional from Scientific Volume Imaging (http://www. svi.nl). The percentage of macrophage infection was calculated by dividing the number of infected macrophages by the number of counted macrophages. The mean number of amastigotes per infected macrophage was determined by dividing the total number of amastigotes counted by the number of infected macrophages. Three independent experiments were performed with duplicates. Metacyclic promastigotes were isolated from stationary cultures of L. major promastigotes by negative selection using a previously described peanut agglutinin (PNA) methodology [46]. Briefly, stationary-phase promastigotes were collected after culture for 4 days, washed with PBS and then incubated with 100 mg/mL of PNA. After incubation for 10 min at room temperature, cells were separated by centrifugation at 5006 g for 10 min. The non-agglutinated promastigotes (metacyclic) collected in the supernatant were washed twice with PBS and resuspended in PBS for further experiments. Ethics statement All experiments were performed according to the National/EU Guidelines for the Care and Use of Laboratory Animals in Research and the approval of the Ethics Committee of the Spanish National Research Council (CSIC, file CEA-213-1-11). Analysis of in vivo infection Six-week-old female BALB/c mice were purchased from Charles River Breeding Laboratories and maintained in the Animal Facility Service of our Institute under pathogen-free conditions. Animals (10 mice/group) were injected subcutaneously in their left hind footpads with 104 L. major purified metacyclic promastigotes resuspended in PBS, as described above. Disease progression was monitored by measuring the inflammation edema and the area of the cutaneous lesion of the infected footpad using a digital caliper (Mitutoyo, Japan), in comparison with the values obtained in the uninfected contralateral footpad. Parasite burdens in target tissues were determined from the presence of amastigotes isolated from footpad, spleen and lymph nodes at week five post-infection, after tissue homogeneization and culture in promastigote culture medium, using a limiting dilution assay, as described previously [48]. LPG and gp63 surface expression analysis Statistical comparisons between groups were performed using Student’s t test. Differences were considered significant at a level p,0.05. Expression analysis of the surface molecule LPG was performed as described previously [45]. Thus, stationary-phase promastigotes (107/mL) were washed twice with PBS and incubated with 5 mg/ mL fluorescein-conjugated ricin agglutinin in PBS for 10 min at 28uC, then washed with PBS and analyzed by flow cytometry using a FACSCalibur (Beckton Dickinson). For quantification of cell surface gp63, parasites were incubated with a 1:500 dilution of a mouse monoclonal anti-gp63 on ice. The cells were subsequently washed with PBS supplemented with 0.1% BSA and fixed at 4uC in 2% paraformaldehyde for 20 min, then washed again and incubated at room temperature with a 1:500 dilution of FITC fluorescein isothiocyanate-labeled goat anti-mouse immunoglobu- Leishmania binding assays The interactions between L. major stationary-phase promasti- gotes and mouse peritoneal macrophages were measured as previously described with some modifications [22]. Mouse peritoneal macrophages (56105/well), maintained at 37uC with 5% CO2 in RPMI 1640 medium supplemented with 10% of hiFBS, were labeled with 5 mM FM4-64 for 30 min at RT in RPMI 1640 medium. LABCG2K/M and control L. major promastigotes (107/ml) in stationary phase were labeled with 1 mM Cell Tracker TM Green for 40 min at 28uC in RPMI 1640 medium. Then, parasites and peritoneal macrophages were washed four times in RPMI 1640 medium and finally resuspended in RPMI 1640 medium supplemented with 5% of hiFBS. Binding assays were performed using a parasite:macrophage ratio of 5:1. Promastigote forms of L. major lines were added to the monolayer cells. After 4 h incubation at 37uC, unbound promastigotes were removed by thorough washing. The monolayers and bound promastigotes were analysed by a Confocal Leyca SP5 microsco- py. All the experiments were done in triplicate. Western blot analysis Proteins from whole stationary-phase promastigotes (107/well) were resolved in 10% SDS-PAGE and electroblotted onto PVDF membranes. Western blot analysis was performed as described previously [47], using a polyclonal antibody against metacyclic promastigote protein HASPB (1:2000) or GFP (1:5000; Invitro- gen), followed by detection with a horseradish peroxidase- conjugated secondary goat anti-rabbit IgG (1:5000; Dako Den- mark) antibody. Monoclonal antibodies against H2A histone or a- tubulin (1:5000 or 1:10000; Sigma-Aldrich) were used to confirm equivalent protein loading. Detection was carried out by enhanced chemiluminescence reaction using the ECL kit (Amersham). PLOS Neglected Tropical Diseases \| www.plosntds.org LABCG2 is involved in the exposure of PS on the outer surface of Leishmania To study the possible role of LABCG2 in PL transport, we first investigated the accumulation level of fluorescent short-chain PL analogues by flow cytometry. Thus, stationary-phase L. major promastigotes transfected with the empty vector (control) or the vector containing LABCG2K/M (LABCG2K/M parasites) were incubated with NBD-PE, -PC, -PS and -SM, and the cell- associated fluorescence analyzed by flow cytometry after back- exchange with BSA to extract the NBD-PL located in the outer plasma membrane leaflet. Under these conditions, accumulation of NBD-PS by the LABCG2K/M parasites was significantly higher than that observed for control cells (4.2 fold, n = 12, p,0.05; Fig. 2A). In contrast, no significant differences were observed for NBD-PC, NBD-PE and NBD-SM accumulation between control and LABCG2K/M parasites (Fig. 2B–D). The change in NBD-PS accumulation was not due to differences in endocytosis, as the internalization of NBD-SM, which is taken up by this process, was not affected by the functionality of LABCG2 (Fig. 2D). The above results were validated in a second transfection event with LABCG2K/M (Fig. 2E). To verify that the higher NBD-PS accumulation observed was due to the dominant-negative inhibition of LABCG2 activity, LABCG2K/M parasites were cured for the plasmid pUCNeoplusLABCG2K/M (reverted line) by culturing the parasites in the absence of plasmid drug selection pressure for three months. This reverted line showed a similar NBD-PS accumulation to the control line (Fig. 2F). We subsequently tested whether LABCG2 was involved in NBD-PS internalization in two other Leishmania species (L. infantum and L. donovani). Down-regulation of LABCG2 function was also assayed by expressing LABCG2K/M and a similar phenotype was observed in these Leishmania species (Appendix in Supporting information, Fig. S4A and B). To confirm that the higher accumulation of NBD-PS in the LABCG2K/M parasites was due to a reduced floppase activity, we measured the outward translocation of NBD- PS from the cytoplasmic to the exoplasmic leaflet of the plasma membrane in both Leishmania lines. Thus, control and LABCG2K/M L. major promastigotes were loaded under conditions that yielded similar amounts of intracellular NBD-PS after the back-exchange step. Then, parasites were maintained in probe- free culture medium containing BSA and the amount of NBD-PS extracted from the external side of the plasma membrane was measured at different time points. The results showed that the outward translocation of NBD-PS was higher in control versus LABCG2K/M promastigotes (Fig. 2G) suggesting a PS floppase activity of LABCG2. Leishmania Infectivity Involves an ABC Transporter Leishmania Infectivity Involves an ABC Transporter Leishmania Infectivity Involves an ABC Transporter transfected with GFP-LABCG2K/M were incubated with 1 mM FM4-64 for 30 min at 4uC. The parasites were fixed for 10 min in 2% with paraformaldehyde at 4uC. (a) Nomarski images of b, c, d and e. Representative co-localization sites (f) are indicated by yellow arrows in the merged images. Scale bar: 5 mm. N (nucleus) and K (kinetoplast) are stained with DAPI. FP: flagellar pocket; AP: aflagellar pole. The figure illustrates a representative parasite of a total population of parasites with a similar fluorescence pattern. doi:10.1371/journal.pntd.0002179.g001 tagged LABCG2 protein at the COOH-terminal showed a similar pattern of localization (Appendix in Supporting information, Fig. S3B), but its expression was unstable after few culture passages, thus suggesting that the C-terminal TMD region is critical for maintaining LABCG2 stability. Overall, these studies suggest that LABCG2 localizes to intracellular vesicles of the endosomal pathway, at the flagellar pocket and at the aflagellar pole of Leishmania. LABCG3, accession code LmjF06.0100) [31]. LABCG1, LABCG2 and LABCG3 are ‘‘half-transporters’’ with a single NBD and a single TMD localized at their C-terminus (Appendix in Supporting information, Fig. S1A). LABCG1 and LABCG2 codes for a 657 and 663 amino acid protein, with a predicted molecular weight of approximately 73.4 and 74.0 kDa, respectively. LABCG1 and LABCG2 are almost identical (95.7% of identity). LABCG3 protein is truncated, with Walker A and several transmembrane segments being absent (Appendix in Supporting information, Fig. S1B). LABCG2 shares 19.5% amino acid identity with human ABCG1, 24.6% with human ABCG2 and 27.3% with the White protein from Drosophila [35]. LABCG2 is involved in the exposure of PS on the outer surface of Leishmania The dimerization requirement for ABC half-transporters (such as LABCG2) to become functional led us to test a dominant- negative approach to down-regulate LABCG2 function, as recently described for Leishmania LABCG5 [35]. To this end, we first mutated in LABCG2 the lysine residue inside the Walker A motif (108 position), which is known to be critical for ATP hydrolysis in ABC transporters [35,49], to a methionine (K108M). The expression of other ABCG half-transporters with a similar K/ M substitution produces a dominant-negative inhibition in the wild-type transporters [35,50]. The resulting construct was transfected into L. major and the expression of LABCG2K108M (LABCG2K/M) was verified by RT-PCR (Appendix in Supporting information, Fig. S2A). In contrast to the phenotype observed after LABCG5K/M expression [35], parasites transfected with LABCG2K/M grew at normal rates (Appendix in Supporting information, Fig. S2B). Sequence features of LABCG2 and generation of a Leishmania line expressing an inactive version of the protein LABCG2 (GeneDB-L. major, accession code LmjF06.0090) has two additional tandem imperfect repeats in chromosome 6 of Leishmania (LABCG1, accession code LmjF06.0080, and April 2013 \| Volume 7 \| Issue 4 \| e2179 April 2013 \| Volume 7 \| Issue 4 \| e2179 April 2013 \| Volume 7 \| Issue 4 \| e2179 PLOS Neglected Tropical Diseases \| www.plosntds.org 4 Leishmania Infectivity Involves an ABC Transporter Figure 1. LABCG2 localizes to the intracellular vesicles of Leishmania parasites. L. major stationary promastigotes transfected with GFP- LABCG2 (A and C) and GFP-LABCG2K/M (B and D) were incubated at 28uC with 1 mM FM4-64 (A and B) and 50 mg/mL concanavalin A-Alexa Red (C and D) for 30 min and 120 min, respectively. (E) LABCG2K/M localizes into the flagellar pocket of Leishmania parasites. L. major stationary promastigotes Figure 1. LABCG2 localizes to the intracellular vesicles of Leishmania parasites. L. major stationary promastigotes transfected with GFP- LABCG2 (A and C) and GFP-LABCG2K/M (B and D) were incubated at 28uC with 1 mM FM4-64 (A and B) and 50 mg/mL concanavalin A-Alexa Red (C and D) for 30 min and 120 min, respectively. (E) LABCG2K/M localizes into the flagellar pocket of Leishmania parasites. L. major stationary promastigotes PLOS Neglected Tropical Diseases \| www.plosntds.org April 2013 \| Volume 7 \| Issue 4 \| e2179 April 2013 \| Volume 7 \| Issue 4 \| e2179 5 Subcellular localization of LABCG2 To study the localization of LABCG2, we fused LABCG2 and LABCG2K/M with an N-terminal GFP-tag. These constructs were transfected into L. major promastigotes and expression of these proteins determined by Western-blot analysis of whole parasite lysates. As expected, a band corresponding to GFP-LABCG2 was observed at around 100 kDa (Appendix in Supporting informa- tion, Fig. S3A). Additional higher molecular weight signals could correspond to dimeric forms of the protein, as described for LABCG5 [35], whereas the lower bands probably correspond to degraded proteins. g p Fluorescence microscopy studies showed that GFP-LABCG2 partially overlap with the endosomal markers FM4-64 [51] and concanavalin A [52] in the stationary growth phase promastigotes, which is depicted in representative micrographs in Fig. 1A and 1C. In contrast, the stained vesicular structures do not co-localize with the mitochondrial marker MitoTracker (data not shown). The localization pattern of the protein does not change when GFP- LABCG2K/M was expressed in Leishmania parasites (Fig. 1B and 1D). To evaluate whether GFP-LABCG2 was also localized in the flagellar pocket, the sole site for endo-/exocytosis in Leishmania, we subsequently performed the co-localization experiments with FM4-64 at 4uC to block its vesicular trafficking. The results showed that GFP-LABCG2K/M co-localizes in the flagellar pocket of the stationary-phase promastigotes (Fig. 1E, yellow arrows). Another part of the GFP-LABCG2K/M pool was detected in intracellular vesicles localized in the apical part of the cell, at the tip of the aflagellar pole of the parasite (Fig. 1E), a site that is known to be involved in the interaction with host cells [53]. GFP- Next, we studied the translocation of endogenous, long-chain PS labelling control and LABCG2K/M stationary growth phase promastigotes with Annexin V-Alexa Fluor 488. This probe binds PLOS Neglected Tropical Diseases \| www.plosntds.org April 2013 \| Volume 7 \| Issue 4 \| e2179 6 Leishmania Infectivity Involves an ABC Transporter Figure 2. Fluorescent PL accumulation in Leishmania parasites. Stationary promastigotes of Leishmania were incubated with the fluorescent PL analogues NBD-PS (A), NBD-PC (B), NBD-PE (C) or NBD-SM (D) for 30 min at 28uC. After washing and back-exchange with BSA, cell-associated fluorescence was measured by flow cytometry analysis. The grey histogram represents control cells transfected with the empty vector, the uncoloured histogram represents parasites expressing LABCG2K/M and the dotted histogram represents non-labelled cells. (E) NBD-PS accumulation in Leishmania lines after a second transfection event. Subcellular localization of LABCG2 The reverted line (F), which was maintained for 3 months without drug pressure, was also included. The histograms correspond to a representative experiment from three independent experiments. (G) Outward transport of NBD-PS in Leishmania parasites. Stationary promastigotes of control (black circles) and LABCG2K/M (black squares) Leishmania lines were incubated with the fluorescent analogue NBD-PS for 30 min at 28uC. After washing and back-exchange with BSA, cells were incubated for different time points in a free NBD-PS medium containing BSA and the fluorescence of the supernatant was measured by spectrofluorimetry. Results represent the means 6 SD of four independent duplicated experiments. * P,0.05 vs. control parasites. doi:10.1371/journal.pntd.0002179.g002 Figure 2. Fluorescent PL accumulation in Leishmania parasites. Stationary promastigotes of Leishmania were incubated with the fluorescent PL analogues NBD-PS (A), NBD-PC (B), NBD-PE (C) or NBD-SM (D) for 30 min at 28uC. After washing and back-exchange with BSA, cell-associated fluorescence was measured by flow cytometry analysis. The grey histogram represents control cells transfected with the empty vector, the uncoloured histogram represents parasites expressing LABCG2K/M and the dotted histogram represents non-labelled cells. (E) NBD-PS accumulation in Leishmania lines after a second transfection event. The reverted line (F), which was maintained for 3 months without drug pressure, was also included. The histograms correspond to a representative experiment from three independent experiments. (G) Outward transport of NBD-PS in Leishmania parasites. Stationary promastigotes of control (black circles) and LABCG2K/M (black squares) Leishmania lines were incubated with the fluorescent analogue NBD-PS for 30 min at 28uC. After washing and back-exchange with BSA, cells were incubated for different time points in a free NBD-PS medium containing BSA and the fluorescence of the supernatant was measured by spectrofluorimetry. Results represent the means 6 SD of four independent duplicated experiments. * P,0.05 vs. control parasites. doi:10.1371/journal.pntd.0002179.g002 higher expression of LABCG2 in stationary growth phase/ metacyclic promastigotes versus log phase parasites (Fig. 3C and 3D). To validate that the differences in Annexin V-Alexa Fluor 488 labelling were due to a defect in PS exposure, we tested the sensitivity of control and LABCG2K/M parasites to papuamide B, a pore-forming cytolytic peptide that specifically binds to PS at the external surface of the plasma membrane [54]. The results showed that LABCG2K/M parasites were less sensitive to papuamide B than controls (EC50 = 3.2660.67 mM vs. EC50 = 2.1260.33 mM, p,0.05, respectively) (Fig. 4A). Subcellular localization of LABCG2 Thus, differences in papuamide B sensitivity might be indirectly caused by a general change in membrane lipid organization in LABCG2K/M parasites. We study this possibility by analyzing both sensitivity of control and LABCG2K/M parasites to amphotericin B; the results shown that there were no significant differences in sensitivity of both lines to amphotericin B (Fig. 4C), suggesting that LABCG2 does not contribute greatly in the distribution of sterols into the plasma membrane. Finally, we confirmed that the parasites expressing GFP-LABCG2K/M used for the subcellular localization analysis also maintained their papuamide B resistance phenotype (data not shown). evaluated whether down-regulation of LABCG2 function correlated with a decreased infectivity of the parasites. Thus, we measured the ability of control and LABCG2K/M stationary-phase promastigotes to infect mouse peritoneal macrophages. The results showed that whereas 80% of macrophages were infected by control parasites after 24 h post-infection, only 20% of cells were infected by LABCG2K/M parasites (Fig. 5A). In contrast, the number of parasites per infected macrophage was similar in both cases (Fig. 5B). We have shown that the different infectivity values observed in LABCG2K/M parasites are not due to differences in the interaction parasite-macrophages as determined after 4 hours post-infection binding assays (Appendix in Supporting information, Fig. S6A and B. The results showed there were not differences in the percentage of interaction in control cells (80.5065.77) compared with LABCG2K/M parasites (84.3367.35). Additionally, we have determined that the overexpression of LABCG2 in Leishmania parasites did not show differences in the PS exposition nor in the % infectivity of mouse peritoneal macrophages (data not shown). Down-regulation of LABCG2 function decreases in vitro parasite infectivity As PS externalization by the parasite is a key mediator for infection of the macrophages [12] and polymorphonuclear cells [16], we Figure 3. The externalization of endogenous PS by stationary Leishmania promastigotes depends on LABCG2 function. (A) PS exposure at the outer leaflet of the parasite plasma membrane was analyzed by flow cytometry using Annexin V–Alexa 488 as described in Materials and Methods. The lower right quadrant in the density plots represents the percentage of Annexin V positive/Propidium iodide negative in control or LABCG2K/M parasites. Markers were placed using non-stained parasites. (B) Density of PS molecules (GeoMean) on the cell surface. The grey histogram represents control cells transfected with the empty vector, the uncoloured histogram represents parasites expressing LABCG2K/M and the dotted histogram represents non-labelled cells. The results shown are representative of three independent duplicated experiments. (C and D) Gene expression analysis of LABCG2 from L. major control determined by RT-PCR analysis through the different growth phases of Leishmania parasites: early log (day 2), late log (day 3), stationary (day 4) and late stationary phase (day 5). RT-PCR was carried out for 35 cycles using RNA isolated from the above parasites and the products were run in 2% agarose gel as described in Materials and Methods. Lower imagine in C shows the expression of GADPH used as internal loading control. The arrows indicate amplified 280 bp LABCG2 fragment and 227 bp GADPH fragment. Lower imagine in D shows the mRNA level of LABCG2 normalized with GADPH in different points of the growth curve. The results shown are the means of three independent experiments 6 SD. doi:10.1371/journal.pntd.0002179.g003 Figure 3. The externalization of endogenous PS by stationary Leishmania promastigotes depends on LABCG2 function. (A) PS exposure at the outer leaflet of the parasite plasma membrane was analyzed by flow cytometry using Annexin V–Alexa 488 as described in Materials and Methods. The lower right quadrant in the density plots represents the percentage of Annexin V positive/Propidium iodide negative in control or LABCG2K/M parasites. Markers were placed using non-stained parasites. (B) Density of PS molecules (GeoMean) on the cell surface. The grey histogram represents control cells transfected with the empty vector, the uncoloured histogram represents parasites expressing LABCG2K/M and the dotted histogram represents non-labelled cells. The results shown are representative of three independent duplicated experiments. (C and D) Gene expression analysis of LABCG2 from L. Subcellular localization of LABCG2 As a control experiment, we tested the sensitivity of both lines to Ro-peptide, which strictly recognizes PE residues at the external surface of biological membranes [55]. The results of this study indicated that there were no differences in sensitivity between LABCG2K/M and control parasites (EC50 = 1.5860.09 mM vs. EC50 = 1.6460.12 mM, respectively) (Fig. 4B). These results are in agreement with the absence of PS exposed in mammalian apoptotic cells [8] and Leishmania promastigotes in a calcium dependent manner [16] (Appendix in Supporting information, Fig. S5). Quantitative analysis by flow cytometer (Fig. 3A) showed that LABCG2K/M parasites presented a significantly reduced exposure of PS in the outer leaflet of the plasma membrane compared with control cells (20.1% vs. 52.7% of Annexin V positive/propidium iodide negative, respectively, p,0.05). Additionally, we determined that the density of PS molecules on the cell surface is significantly higher in the control (53.3566.12) than in the LABCG2K/M parasites (38.1063.49), as measured by the mean fluorescence intensity (Fig. 3B); however, control and LABCG2K/M log phase parasites showed a low and similar Annexin V-binding (10.97% vs. 11.06% of Annexin V positive/propidium iodide negative, respectively, p,0.05; data not shown). These results were supported by RT-PCR analysis of expression of LABCG2 through the life cycle of L major that shows PS exposed in mammalian apoptotic cells [8] and Leishmania promastigotes in a calcium dependent manner [16] (Appendix in Supporting information, Fig. S5). Quantitative analysis by flow cytometer (Fig. 3A) showed that LABCG2K/M parasites presented a significantly reduced exposure of PS in the outer leaflet of the plasma membrane compared with control cells (20.1% vs. 52.7% of Annexin V positive/propidium iodide negative, respectively, p,0.05). Additionally, we determined that the density of PS molecules on the cell surface is significantly higher in the control (53.3566.12) than in the LABCG2K/M parasites (38.1063.49), as measured by the mean fluorescence intensity (Fig. 3B); however, control and LABCG2K/M log phase parasites showed a low and similar Annexin V-binding (10.97% vs. 11.06% of Annexin V positive/propidium iodide negative, respectively, p,0.05; data not shown). These results were supported by RT-PCR analysis of expression of LABCG2 through the life cycle of L major that shows PLOS Neglected Tropical Diseases \| www.plosntds.org April 2013 \| Volume 7 \| Issue 4 \| e2179 7 Leishmania Infectivity Involves an ABC Transporter alterations in NBD-PE translocation in LABCG2K/M parasites. Several ABCG transporters have been implicated in sterol transport [56]. PLOS Neglected Tropical Diseases \| www.plosntds.org Down-regulation of LABCG2 function decreases in vitro parasite infectivity major control determined by RT-PCR analysis through the different growth phases of Leishmania parasites: early log (day 2), late log (day 3), stationary (day 4) and late stationary phase (day 5). RT-PCR was carried out for 35 cycles using RNA isolated from the above parasites and the products were run in 2% agarose gel as described in Materials and Methods. Lower imagine in C shows the expression of GADPH used as internal loading control. The arrows indicate amplified 280 bp LABCG2 fragment and 227 bp GADPH fragment. Lower imagine in D shows the mRNA level of LABCG2 normalized with GADPH in different points of the growth curve. The results shown are the means of three independent experiments 6 SD. doi:10.1371/journal.pntd.0002179.g003 April 2013 \| Volume 7 \| Issue 4 \| e2179 PLOS Neglected Tropical Diseases \| www.plosntds.org 8 Leishmania Infectivity Involves an ABC Transporter Figure 4. Leishmania LABCG2K/M parasites present resistance to papuamide B. Sensitivity of control and LABCG2K/M parasites to the PS- binding peptide papuamide B (A), PE-binding peptide Ro09-0198 (B) and amphotericin B (C). Logarithmic-phase promastigotes were diluted to 106/ mL in RPMI 10% hiFBS containing different concentrations of these peptides; after 72 h, cell viability was analysed by a MTT analysis as described in Materials and Methods. Results represent the means 6 SD of four independent duplicated experiments. * P,0.05 vs. control parasites. doi:10.1371/journal.pntd.0002179.g004 Figure 4. Leishmania LABCG2K/M parasites present resistance to papuamide B. Sensitivity of control and LABCG2K/M parasites to the PS- binding peptide papuamide B (A), PE-binding peptide Ro09-0198 (B) and amphotericin B (C). Logarithmic-phase promastigotes were diluted to 106/ mL in RPMI 10% hiFBS containing different concentrations of these peptides; after 72 h, cell viability was analysed by a MTT analysis as described in Materials and Methods. Results represent the means 6 SD of four independent duplicated experiments. * P,0.05 vs. control parasites. doi:10.1371/journal.pntd.0002179.g004 We have repeated the infection experiments masking most of the PS in the metacyclic forms by incubating stationary-phase control and LABCG2K/M parasites with Annexin V. As expected, PS masking reduced the macrophage infection percentage of control parasites by approximately 82%. Annexin V-mediated masking of PS in LABCG2K/M parasites did not significantly altered their lower ability to infect peritoneal macrophages, reaching similar values to those obtained with Annexin V treated control parasites (Fig. 5C). Down-regulation of LABCG2 function decreases in vitro parasite infectivity Furthermore, we assessed whether other molecules, such as lipophosphoglycan (LPG) or the phosphatidylinositol-anchored surface molecule gp63 [15], both of which are implicated in Leishmania infectivity, could be altered in LABCG2K/M parasites. Flow cytometry analysis of stationary- phase promastigotes marked with fluorescein-conjugated ricin agglutinin, which specifically label LPG [45], showed no differences between control and LABCG2K/M parasites (Appendix in Supporting information, Fig. S6C). Additionally, flow cytometry analysis using a specific monoclonal antibody for Leishmania gp63 showed no significant differences between expression of this surface molecule in the control and LABCG2K/M stationary-phase promastigotes (Appendix in Supporting information, Fig. S6D). Finally, we evaluated whether the infectivity differences observed may be due to an alteration in the metacyclogenesis of the parasites produced by down-regulation of LABCG2 function. Thus, we purified infective metacyclic forms from stationary-phase promastigotes of control and LABCG2K/M parasites by binding to the lectin peanut agglutinin (PNA) [57], and found that the percentage of metacyclic parasites (PNA2) obtained was similar in both cell lines (Appendix in Supporting information, Fig. S7A). Furthermore, both parasite lines were morphologically elongated, highly mobile, there were no rounded shapes and differences in size (FSC-H) between control (427.26613.82) and LABCG2K/M (413.61614.53) lines, respectively (Appendix in Supporting information, Fig. S7B). We also analysed expression of the metacyclic marker protein HASPB (hydrophilic acylated surface protein B) [58], which is implicated in host-cell infection. Western blot analysis indicated that there were no differences in expression of this 32 kDa protein between control and LABCG2K/M parasites (Appendix in Supporting information, Fig. S7C). LABCG2 is required for disease development in a mouse model of cutaneous leishmaniasis LABCG2 is required for disease development in a mouse model of cutaneous leishmaniasis Infection of mouse peritoneal macrophages with stationary Leishmania promastigotes from control and LABCG2K/M parasites was performed as described in Materials and Methods. The percentage of infected macrophages (A) and the mean number of parasites per macrophage (B) were determined 24 h post-infection. The results represent the means 6 SD of three independent experiments. P,0.05 vs. infection level of control parasites. Additionally, the effect of Annexin V-binding on macrophage infectivity was determined (C) using control and LABCG2K/M stationary parasites incubated in the presence (+) or absence (2) of Annexin V (0.05 mg/ml6107 stationary promastigotes) for 4 h. The results shown are the means of three independent experiments 6 SD. P,0.05 untreated control vs.: Annexin V-treated control, untreated LABCG2K/M parasites and Annexin V-treated LABCG2K/M parasites. doi:10.1371/journal.pntd.0002179.g005 Leishmania Infectivity Involves an ABC Transporter Leishmania Infectivity Involves an ABC Transporter Figure 5. LABCG2K/M parasites are less infective to mouse peritoneal macrophages. Infection of mouse peritoneal macrophages with stationary Leishmania promastigotes from control and LABCG2K/M parasites was performed as described in Materials and Methods. The percentage of infected macrophages (A) and the mean number of parasites per macrophage (B) were determined 24 h post-infection. The results represent the means 6 SD of three independent experiments. P,0.05 vs. infection level of control parasites. Additionally, the effect of Annexin V-binding on macrophage infectivity was determined (C) using control and LABCG2K/M stationary parasites incubated in the presence (+) or absence (2) of Annexin V (0.05 mg/ml6107 stationary promastigotes) for 4 h. The results shown are the means of three independent experiments 6 SD. P,0.05 untreated control vs.: Annexin V-treated control, untreated LABCG2K/M parasites and Annexin V-treated LABCG2K/M parasites. doi:10.1371/journal.pntd.0002179.g005 the footpad of mice infected with the LABCG2K/M parasites (Fig. 7A), and that its phenotype of increased NBD-PS accumula- tion remained unaltered (Fig. 7B). To confirm that the loss of virulence exhibited by the mutant line was due to the expression of LABCG2K/M, these experiments were repeated with a second line of LABCG2K/M parasites generated from an independent trans- fection event. The results of this study were similar to those described above (Appendix in Supporting information, Fig. S8 ), thus indicating that the dramatic differences in the virulence of LABCG2K/M parasites were due to down-regulation of LABCG2 function and suggesting that the Leishmania LABCG2 gene is crucial for disease development. LABCG2 is required for disease development in a mouse model of cutaneous leishmaniasis As down-regulation of LABCG2 function decreased the in vitro macrophage infectivity of metacyclic parasites, we analysed whether this defect was correlated with a lower in vivo virulence of the parasites using a mouse model of cutaneous leishmaniasis. Thus, susceptible female BALB/c mice were infected with 104 metacyclics purified from control and LABCG2K/M parasites by footpad inoculation. As we had previously observed during the assays to cure LABCG2K/M parasites for the plasmid pUCNeo- plusLABCG2K/M (reverted line) that the defect on the external- ization of NBD-PS remained unaltered for at least five weeks in the absence of antibiotic pressure, we were able to use transfected parasites for this model. After infection, the measure of inflam- mation and the development of skin lesions in the footpad with time were monitored weekly up to a maximum of five weeks. At this time, control animals had to be sacrificed due to the severity of the lesions. Mice infected with control parasites showed progres- sive inflammation and lesion pathology after the first two weeks (Fig. 6A–C), whereas mice infected with LABCG2K/M parasites showed no detectable lesions pathology at any time, and presented significantly lower footpad inflammation (Fig. 6A–C). As observed in Fig. 6B, the curve for footpad lesion size is the same for non- infected control animals and for the animals infected with LABCG2K/M L. major metacyclic parasites, which shows no lesions during the time of the infection assay. Additionally, we decided to determine whether these infected mice presented parasites in different target tissues and whether these parasites maintained the expression of LABCG2K/M and increased NBD-PS accumulation. Thus, at the indicated time, mice were euthanized and their footpad, spleen and lymph nodes collected for parasite isolation following the limiting dilution assay. As can be seen from Fig. 6D–E, a lower parasite burden was recovered from the footpad of mice infected with the LABCG2K/M parasites compared to the control mice (Fig. 6D), and no parasites were isolated from the spleen or lymph nodes of mice infected with the LABCG2K/M parasites (Fig. 6E) after one week of in vitro culture. Moreover, we confirmed by RT-PCR that the mutant LABCG2K/M gene was still expressed in parasites isolated from April 2013 \| Volume 7 \| Issue 4 \| e2179 PLOS Neglected Tropical Diseases \| www.plosntds.org 9 Figure 5. LABCG2K/M parasites are less infective to mouse peritoneal macrophages. Discussion The exposure of PS on their cell surface is one of the mechanisms known to be used by Leishmania amastigotes and metacyclic promastigotes to infect host macrophages and to April 2013 \| Volume 7 \| Issue 4 \| e2179 PLOS Neglected Tropical Diseases \| www.plosntds.org April 2013 \| Volume 7 \| Issue 4 \| e2179 PLOS Neglected Tropical Diseases \| www.plosntds.org 10 inhibit their microbicidal activity [7–9,11,14,16]. PS is usually asymmetrically distributed in the cell membrane of eukaryotic cells and is present only in the cytoplasmic leaflet of the plasma been a matter of intense debate [59,60], in which t state of Leishmania parasites could be the possible d factor it could be concluded that parasites in late l Figure 6. LABCG2K/M parasites are less infective in a cutaneous leishmaniasis mouse model. Susceptible BALB/c mice were in 104 control and LABCG2K/M L. major metacyclic parasites as described in Materials and Methods. Disease development was monitored measuring the inflammation (A) and lesion size (B). The pictures in C show the lesion at week 5 post-infection. Parasite burden was de footpad (D) and tissues (E): lymph nodes (LN) and spleen (SP). The results represent the means 6 SD of three independent experiments, w per group. Mice were euthanized when the lesion size in controls reached a value of 50–70 mm2. * P,0.05 vs. control parasites; n.d. sta detected. doi:10.1371/journal.pntd.0002179.g006 Leishmania Infectivity Involves an ABC Leishmania Infectivity Involves an ABC Transporter Figure 6. LABCG2K/M parasites are less infective in a cutaneous leishmaniasis mouse model. Susceptible BALB/c mice were infected with 104 control and LABCG2K/M L. major metacyclic parasites as described in Materials and Methods. Disease development was monitored weekly by measuring the inflammation (A) and lesion size (B). The pictures in C show the lesion at week 5 post-infection. Parasite burden was determined in footpad (D) and tissues (E): lymph nodes (LN) and spleen (SP). The results represent the means 6 SD of three independent experiments, with 10 mice per group. Mice were euthanized when the lesion size in controls reached a value of 50–70 mm2. * P,0.05 vs. control parasites; n.d. stands for not detected. doi:10.1371/journal.pntd.0002179.g006 doi:10.1371/journal.pntd.0002179.g006 been a matter of intense debate [59,60], in which the growth state of Leishmania parasites could be the possible discrepancy factor, it could be concluded that parasites in late logarithmic phase contain PS. inhibit their microbicidal activity [7–9,11,14,16]. PLOS Neglected Tropical Diseases \| www.plosntds.org April 2013 \| Volume 7 \| Issue 4 \| e2179 Discussion This finding is in agreement with previous reports for L. donovani and L. tropica [14] and suggests that LABCG2-mediated PS exposure could be induced during metacyclogenesis of the parasite and is probably maintained in the intracellular amastigote stage. The constitutive expression of LABCG2 in both life-cycle stages of L. donovani has been described recently [34]. Although not discussed in detail in that study, LABCG2 expression seems to be higher in the amastigote forms than in the log-phase promastigote forms [34], as would be expected for a protein involved in PS externalization. In our case, the results have shown that the expression of LABCG2 increases in the early and late stationary phase compare with the log phase and this increase would be involved in the redistribution of PS to the outer leaflet of the plasma membrane in the metacyclic forms of the parasite. On the other hand, the localization of LABCG2 at both the flagellar pocket and the aflagellar pole as well as in intracellular vesicles, suggests a possible mechanism for PS exposure in Leishmania, similar to that described for human ABCG2, namely as an intracellular sterol transporter [64]. It remains possible that LABCG2 could transfer PS and other factors (as virulent factors) to the inner leaflet of these vesicles before their fusion with the plasma membrane at the flagellar pocket, followed by a redistribution of PS/other factors to the outer leaflet of the plasma membrane of the parasite (Fig. 8). In the case of LABCG2 K/M, the content of PS and other factors could be reduced in the intracellular vesicles and in the outer leaflet of plasma membrane, consequently influencing in the infectivity and virulence. A similar situation has been described for the LtrABCA2 transporter of Leishmania, involved in phospholipid trafficking and localized at the flagellar pocket and internal vesicles [43]. Additionally, LABCG2 could function as a floppase of PS/other factors due to their localization in the flagellar pocket of the parasite (Fig. 8). Thus, down-regulation of LABCG2 function in L. major upon expression of an inactive version of the transporter (LABCG2K/M) diminishes the outward translocation of fluorescent short-chain analogues of PS (NBD-PS). This dominant-negative phenotype seems to be specific for the head of PL, as the movement of fluorescent analogues of PC, PE or SM was not affected. Discussion PS is usually asymmetrically distributed in the cell membrane of eukaryotic cells and is present only in the cytoplasmic leaflet of the plasma membrane [18]. Although the PS synthesis in Leishmania have April 2013 \| Volume 7 \| Issue 4 \| e2179 PLOS Neglected Tropical Diseases \| www.plosntds.org PLOS Neglected Tropical Diseases \| www.plosntds.org 11 Leishmania Infectivity Involves an ABC Transporter Figure 7. LABCG2K/M parasites isolated from footpad lesions of infected mice retained the increased NBD-PS accumulation phenotype. (A) RT-PCR gene-expression analysis of wild-type and mutated LABCG2 in control and LABCG2K/M Leishmania parasites. The lower image shows the expression of GADPH used as internal loading control. RT-PCR was carried out for 35 cycles using RNA isolated from the above parasites and the products were run in 2% agarose gel. The positions of molecular markers (bp) are indicated on the left. (B) Accumulation of NBD-PS in control (grey histogram) and LABCG2K/M (uncoloured histogram) parasites. Parasites recovered from the footpad lesions of infected mice were maintained in culture medium and the stationary promastigotes incubated with the fluorescent phospholipid analogue NBD-PS for 30 min at 28uC. After washing and back-exchange with BSA, cell-associated fluorescence was measured by flow cytometry analysis as described in Materials and Methods. The dotted line represents non-labelled parasites. doi:10.1371/journal.pntd.0002179.g007 Figure 7. LABCG2K/M parasites isolated from footpad lesions of infected mice retained the increased NBD-PS accumulation phenotype. (A) RT-PCR gene-expression analysis of wild-type and mutated LABCG2 in control and LABCG2K/M Leishmania parasites. The lower image shows the expression of GADPH used as internal loading control. RT-PCR was carried out for 35 cycles using RNA isolated from the above parasites and the products were run in 2% agarose gel. The positions of molecular markers (bp) are indicated on the left. (B) Accumulation of NBD-PS in control (grey histogram) and LABCG2K/M (uncoloured histogram) parasites. Parasites recovered from the footpad lesions of infected mice were maintained in culture medium and the stationary promastigotes incubated with the fluorescent phospholipid analogue NBD-PS for 30 min at 28uC. After washing and back-exchange with BSA, cell-associated fluorescence was measured by flow cytometry analysis as described in Materials and Methods. The dotted line represents non-labelled parasites. doi:10.1371/journal.pntd.0002179.g007 Although the molecular basis for this outward PS translocation in Leishmania remains unknown, herein we provide experimental evidence supporting the involvement of a new ABCG half- transporter (LABCG2) from Leishmania in this process. very low. Discussion It remains possible that LABCG2 could transfer PS and other factors (as virulent factors) to the inner leaflet of intracellular vesicles before their fusion with the plasma membrane at the flagellar pocket, followed by a redistribution of PS/other factors to the outer leaflet of the plasma membrane of the parasite. doi:10.1371/journal.pntd.0002179.g008 doi:10.1371/journal.pntd.0002179.g008 infective parasites invade the host cell, in a truly cooperative system [16,66]. Should be noted considered that these experiments were made using late stationary phase Leishmania parasites, where they begin to appreciate the apoptotic round shapes described above. In our case, the experiments were performed using early stationary phase Leishmania parasites, where rounded apoptotic forms were not detected. In any case, PS exposure is the relevant phenotype for macrophages infection and subsequent inactivation of microbicidal activity in both these scenarios, thereby allowing parasite persistence in the host [65]. macrophages by LABCG2K/M parasites. This finding agrees with previous reports [7,14], which showed that PS exposure was important for macrophage engulfment of the promastigote and amastigote forms of Leishmania. As suggested [12], we believe that the only process probably affected would be the phagocytosis and consequently the variation of PS exposure could not be important for the interaction; this consideration would explain the observed differences in % of infected macrophages. Additionally, the reduced infectivity was not due to changes in the metacyclogenesis process considering that LABCG2 function does not affect either expression of the metacyclic marker protein HASPB or the number of metacyclic promastigotes produced. The need for ABC half-transporters such as ABCG proteins to dimerize in order to reconstitute the ATP-binding sites and become functional has allowed us to test a dominant-negative approach to down-regulate LABCG2 function. Dominant-nega- tive inhibition of mammalian ABCG2 [50,67] and Leishmania LABCG5 [35] has also been described upon expression of different mutants of these transporters. Although the inhibition of homodimeric LABCG2 function is the most likely explanation for expression of the inactive version of LABCG2, we cannot rule out the inhibition of other putative partners. Indeed, some ABCG proteins, such as human ABCG5/8 [68] and Drosophila White, Brown and Scarlet, work as heterodimers [69]; the latter can even change the substrate transported as a function of the partner of the White protein. Discussion This substrate specificity contrasts with the broader spectrum of PL translocated by other lipid floppases [33,36] and flippases [42] characterized in Leishmania. Similar results were obtained after expression of LABCG2K/M in L. donovani and L. infantum, thereby suggesting that the functionality of LABCG2 could be conserved in Leishmania spp. It should be noted, however, that this phenotype was not due to an LABCG2-mediated alteration of PL endocytosis as the internalization of NBD-SM (which is taken up by lipid- phase endocytosis) was not altered. In addition, Annexin V-Alexa 488 binding assays showed that LABCG2K/M promastigotes in the stationary phase exposed less endogenous PS than control parasites. As Annexin V is not entirely specific for PS, despite its widespread use in labelling it, and could bind to other anionic PL [60–62], we performed an additional control using papuamide B, a cytolytic peptide that specifically recognizes PS residues in biological membranes [54]. The reduced exposure of PS on the outer face of the plasma membrane in LABCG2K/M parasites was correlated with an increased resistance to papuamide B. Although these differences in resistance to papuamide B were not so higher, such differences were statistically significant and reproducible even in GFP-LABCG2K/M parasites, considering that extreme changes in the phospholipid asymmetry of the eukaryotic membranes lead to cell death [5,22,63]. The differences in Annexin V labelling between control and LABCG2K/M parasites were not observed during the log growth-phase of parasites, where the labelling was In addition, the defect in PS externalization produced by down- regulation of LABCG2 function was found to be correlated with a significant reduction of the infection of mouse peritoneal April 2013 \| Volume 7 \| Issue 4 \| e2179 PLOS Neglected Tropical Diseases \| www.plosntds.org 12 Figure 8. Schematic diagram of the localization of LABCG2 in intracellular vesicles and flagellar pocket of Leishmania parasites. It remains possible that LABCG2 could transfer PS and other factors (as virulent factors) to the inner leaflet of intracellular vesicles before their fusion with the plasma membrane at the flagellar pocket, followed by a redistribution of PS/other factors to the outer leaflet of the plasma membrane of the parasite. doi:10.1371/journal.pntd.0002179.g008 Leishmania Infectivity Involves an ABC Transporter Leishmania Infectivity Involves an ABC Transporter Figure 8. Schematic diagram of the localization of LABCG2 in intracellular vesicles and flagellar pocket of Leishmania parasites. PLOS Neglected Tropical Diseases \| www.plosntds.org Supporting Information Figure S1 (A) Membrane topology model of the Leish- mania half-transporters LABCG1, LABCG2 and LABCG3. The nucleotide binding domain (NBD) is located N- terminal with respect to the transmembrane domain (TMD). The putative membrane-spanning helices of the TMD are shown as cylinders passing through the lipid bilayer. The ATPase catalytic Walker A, Walker B, and the signature motif C localized in the nucleotide binding domain (NBD) are shown (boxes A, B and C, respectively). The arrow indicates the catalytic site mutation (K108M) engineered into the Walker A motif. The topology model was predicted using TMHMM (http://www.cbs.dtu.dk/services/ TMHMM-2.0/) and TMRPres2D ((http://biophysics.biol.uoa. gr/TMRPres2D/) softwares. (B) Amino acid sequences and alignment (Clustal W) of L. major LABCG1, LABCG2 and LABCG3. Putative transmembrane segments predicted by TMHMM and TMRPres2D are underlined. The Walker A/ Walker B motifs, and the ABC family signature motif C are boxed. Identical amino acids present in the three sequences are indicated by , the amino acid similarity is indicated by : and weak similar amino acid are indicated by . . Gaps introduced for the sequence alignment are indicated by -. (TIF) Figure S5 The externalization of endogenous PS in Leishmania parasites. PS exposure at the outer leaflet of the parasite plasma membrane was analyzed by flow cytometry using Annexin V–Alexa 488 in control parasites as described in Materials and Methods. Controls measurements in the absence of calcium were included using Annexin V–Alexa 488 plus 8 mM EGTA. The results shown are representative of three independent duplicated experiments 6 SD. P,0.05 vs. control parasites. (TIF) Figure S6 LABCG2K/M parasites has not affected its capacity of binding to macrophages. (A) Binding of control and LABCG2K/M metacyclic promastigotes to mouse peritoneal macrophages. Percentage of positive interaction of promastigotes to macrophages after 4 h interaction was determined by a fluorescence microscopy analysis counting 100 macrophages/well. The results represent the means 6 SD of three independent experiments. (B) Micrograph of double-fluorescence labeling of the binding of Leishmania control and LABCG2K/M metacyclic parasites to mouse peritoneal macrophages. Cell Tracker TM Green-labeled parasites were added (5:1) to mouse peritoneal macrophages relabeled with FM4-64 (red). a and c, Nomarski images of b and d, respectively. b and d shows the binding and intracellular localization of control and LABCG2K/M parasites. Scale bar: 10 mm. Discussion Nonetheless, new approaches will be needed to fully understand the molecular mechanism by which LABCG2 affects the infectivity and virulence of Leishmania. Additionally, these findings indicate that LABCG2 could be considered as a promising drug target for leishmaniasis. Further- more, as mutant parasites could be isolated from infected mice despite the absence of lesions, null mutants for LABCG2 could potentiality be used for live vaccination studies and to understand the role of LABCG2 in Leishmania pathogenesis. Finally, LABCG2 belongs to the same subfamily as mammalian ABCG2, a well-characterized PS transporter [30] that also pumps drugs conferring a MDR phenotype in cancer cells [70,71]. Others LABCG proteins, such as LABCG4 and LABCG6, also confer resistance to leishmanicidal agents [32,33]. Future work in our group will examine the implication of LABCG2 in drug resistance. In conclusion, we have provided evidence that strongly suggests the involvement of Leishmania LABCG2 function in the PS externalization required for the infection of host macrophages and development of the disease. Nonetheless, new approaches will be needed to fully understand the molecular mechanism by which LABCG2 affects the infectivity and virulence of Leishmania. Additionally, these findings indicate that LABCG2 could be considered as a promising drug target for leishmaniasis. Further- more, as mutant parasites could be isolated from infected mice despite the absence of lesions, null mutants for LABCG2 could potentiality be used for live vaccination studies and to understand the role of LABCG2 in Leishmania pathogenesis. Figure S4 Fluorescent PS accumulation in Leishmania parasites. Stationary promastigotes of L. infantum (A) or L. donovani (B) were incubated with the fluorescent PL analogue NBD-PS for 30 min at 28uC. After washing and back-exchange with BSA, cell-associated fluorescence was measured by flow cytometry analysis. The grey histogram represents control parasites transfected with the empty vector, the uncoloured histogram represents parasites expressing LABCG2K/M and the dotted histogram represents non-labelled cells. The histograms correspond to a representative experiment from three independent experiments. (TIF); Supporting Information The expression levels of two surface molecules, LPG (C) and gp63 (D), were determined in control and LABCG2K/M parasites marked with fluorescein-conjugated ricin agglutinin that specifically labels LPG (C) and a specific monoclonal antibody for Leishmania gp63 (D). The fluorescence intensity was determined by flow cytometry analysis, as described in Materials and Methods. The data are means of the geometrical mean channel fluorescence values (g.m.) 6 SD of three independent experiments versus controls. Figure S2 Gene-expression analysis of LABCG2 in Leishmania lines. (A) Upper panel: gene expression of LABCG2 by RT-PCR as indicated by the amplified 280 bp LABCG2 fragment. Lower panel: gene expression of GADPH as internal loading control. The arrow indicates amplified 227 bp GADPH fragment. Lane 1: DNA marker phi 174 HaeIII; lane 2: control parasites; lane 3: LABCG2K/M parasites. RT-PCR was carried out for 35 cycles using RNA isolated from the above- mentioned parasites and the products run in 2% agarose gel. (B) Growth curve of control (black circles) and LABCG2K/M (black squares) parasites at different time points (24, 48, 72, 96 and 120 h). The results represent the means 6 SD of three independent experiments. (TIF) p (TIF) Discussion However, as expression of inactive versions of LABCG4 [32] and LABCG5 does not affect the translocation of PS [35] or the infectivity of the parasites, it is more likely that LABCG2 does not heterodimerize with these other Leishmania ABCG proteins; it could however dimerize with LABCG1, which is almost identical in its TMD. Furthermore, the virulent factors LPG and gp63 are probably not affected by the function of LABCG2, as deduced by the absence of variations in the expression levels of these surface molecules, both of which have been suggested that play important roles in establishment of the infection. However, we cannot discard the possibility that other unknown factors involved in the virulence of Leishmania could be transported by LABCG2, and their expression could be affected in the LABCG2K/M parasites. The decrease of the in vitro ability to infect macrophages observed in mutant parasites with a defect in PS exposure was found to be correlated with lower infectivity in an in vivo mouse model of cutaneous leishmaniasis, thereby supporting the proposal that LABCG2 function is required in order to develop the lesion. The role of the apoptosis-like PS exposure in the establishment of a Leishmania infection has been widely discussed. One possibility in this respect is that PS-positive cells do not necessarily die but use PS exposure, in an apoptosis-mimicking fashion, to infect macrophages and inhibit their microbicidal activity [8,11,16,65]. The second possibility suggests that PS-positive forms are indeed ‘‘altruistic’’ apoptotic parasites that have been sentenced to death but are nevertheless required in order that the PS-negative A further attractive alternative could be that truncated LABCG3, which only includes the conserved Walker B, the signature motifs and two transmembrane segments and is expressed at least at mRNA levels [31,34], could dimerize with PLOS Neglected Tropical Diseases \| www.plosntds.org April 2013 \| Volume 7 \| Issue 4 \| e2179 13 Leishmania Infectivity Involves an ABC Transporter LABCG2 to produce an inhibitory effect on LABCG2 function. This could be a novel way to regulate protein function in this parasite, although further experiments will be required to confirm this hypothesis. GFP (A) or H2A histone (lower inset) in L. major lines expressing control GFP (lane 1), GFP-LABCG2 (lane 2) and GFP- LABCG2K/M (lane 3). Western blot analysis of total proteins from parasites incubated with antibodies against GFP or H2A histone, as loading control, at a 1:5000 dilution. Discussion The molecular mass standards (kDa) from Bio-Rad are indicated on the left. (B) L. major stationary promastigotes transfected with pXG-GFP+ and LABCG2K/M-GFP were fixed for 10 min in 2% paraformalde- hyde at 4uC. a and c, Nomarski images of b and d, respectively. b shows the cytoplasmic localization of the protein GFP and d corresponds to localization sites of LABCG2K/M-GFP, indicated by white arrows in the merged images. Scale bar: 5 mm. FP: flagellar pocket; AP: aflagellar pole. The figure illustrates a representative parasite of a total population of parasites with a similar fluorescence pattern. (TIF); LABCG2 to produce an inhibitory effect on LABCG2 function. This could be a novel way to regulate protein function in this parasite, although further experiments will be required to confirm this hypothesis. GFP (A) or H2A histone (lower inset) in L. major lines expressing control GFP (lane 1), GFP-LABCG2 (lane 2) and GFP- LABCG2K/M (lane 3). Western blot analysis of total proteins from parasites incubated with antibodies against GFP or H2A histone, as loading control, at a 1:5000 dilution. The molecular mass standards (kDa) from Bio-Rad are indicated on the left. (B) L. major stationary promastigotes transfected with pXG-GFP+ and LABCG2K/M-GFP were fixed for 10 min in 2% paraformalde- hyde at 4uC. a and c, Nomarski images of b and d, respectively. b shows the cytoplasmic localization of the protein GFP and d corresponds to localization sites of LABCG2K/M-GFP, indicated by white arrows in the merged images. Scale bar: 5 mm. FP: flagellar pocket; AP: aflagellar pole. The figure illustrates a representative parasite of a total population of parasites with a similar fluorescence pattern. (TIF); LABCG2 to produce an inhibitory effect on LABCG2 function. This could be a novel way to regulate protein function in this parasite, although further experiments will be required to confirm this hypothesis. p , g p q this hypothesis. Finally, LABCG2 belongs to the same subfamily as mammalian ABCG2, a well-characterized PS transporter [30] that also pumps drugs conferring a MDR phenotype in cancer cells [70,71]. Others LABCG proteins, such as LABCG4 and LABCG6, also confer resistance to leishmanicidal agents [32,33]. Future work in our group will examine the implication of LABCG2 in drug resistance. In conclusion, we have provided evidence that strongly suggests the involvement of Leishmania LABCG2 function in the PS externalization required for the infection of host macrophages and development of the disease. References Wanderley JL, Benjamin A, Real F, Bonomo A, Moreira ME, et al. (2005) Apoptotic mimicry: an altruistic behavior in host/Leishmania interplay. Braz J Med Biol Res 38: 807–812. 25. Higgins CF (2001) ABC transporters: physiology, structure and mechanism–an overview. Res Microbiol 152: 205–210. 10. Schlegel RA, Williamson P (2001) Phosphatidylserine, a death knell. Cell Death Differ 8: 551–563. 26. Locher KP (2009) Review. Structure and mechanism of ATP-binding cassette transporters. Philos Trans R Soc Lond B Biol Sci 364: 239–245. transporters. Philos Trans R Soc Lond B Biol Sci 364: 239–24 11. van Zandbergen G, Solbach W, Laskay T (2007) Apoptosis driven infection. Autoimmunity 40: 349–352. 27. Nagao K, Kimura Y, Mastuo M, Ueda K (2010) Lipid outward translocation by ABC proteins. FEBS Lett 584: 2717–2723. 12. Wanderley JL, Pinto da Silva LH, Deolindo P, Soong L, Borges VM, et al. (2009) Cooperation between apoptotic and viable metacyclics enhances the pathogenesis of Leishmaniasis. PLoS One 4: e5733. p 28. Woodward OM, Kottgen A, Kottgen M (2011) ABCG transporters and disease. FEBS J 278: 3215–3225. J 29. 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Immunodetection of (TIF) April 2013 \| Volume 7 \| Issue 4 \| e2179 PLOS Neglected Tropical Diseases \| www.plosntds.org 14 Leishmania Infectivity Involves an ABC Transporter Mice were euthanized when the lesion size in controls reached a value of 50–70 mm2. *P,0.05 vs. control parasites; n.d. stands for not detected. (TIF) Figure S7 LABCG2K/M Leishmania parasites do not show defective metacyclogenesis. (A) Control and LABCG2K/M metacyclic parasites were purified from stationary promastigotes using negative selection with the lectin PNA, as described in Materials and Methods. The results represent the means 6 SD of four independent experiments. (B) Analysis by flow cytometry of the FSC-H of control and LABCG2K/M metacyclic parasites; right panel shows a Nomarsky micrography of the same samples. Scale bar: 5 mm. (C) Total cell lysates from stationary promastigotes were analyzed by Western blotting with an antibody to the metacyclic protein HASPB. Anti-alpha tubulin antibody was used as loading control. The positions of molecular marker (kDa) are indicated on the left. Figure S7 LABCG2K/M Leishmania parasites do not show defective metacyclogenesis. (A) Control and LABCG2K/M metacyclic parasites were purified from stationary promastigotes using negative selection with the lectin PNA, as described in Materials and Methods. The results represent the means 6 SD of four independent experiments. (B) Analysis by flow cytometry of the FSC-H of control and LABCG2K/M metacyclic parasites; right panel shows a Nomarsky micrography of the same samples. Scale bar: 5 mm. (C) Total cell lysates from stationary promastigotes were analyzed by Western blotting with an antibody to the metacyclic protein HASPB. Anti-alpha tubulin antibody was used as loading control. The positions of molecular marker (kDa) are indicated on the left. Acknowledgments We thank Stephen M. Beverley (Washington University, School of Medicine, USA) for providing the vectors pXG-GFP+29 and pXG- ’GFP+, Barbara A. Smith (University of York, UK) for providing the polyclonal antisera against metacyclic marker protein HASPB, and Thomas Gunther Pomorski and Rosa Lo´pez (Department of Plan Biology and Biotechnology, University of Copenhagen, Denmark), for providing the papuamide B peptide. Author Contributions Figure S8 LABCG2K/M parasites are less infective in a mouse model of cutaneous leishmaniasis. A second independent transfection event using control and LABCG2K/M parasites was inoculated in mice, as described in Materials and Methods. The inflammation (A), lesion size (B) and parasite burden in footpad (D) and tissues (E) such as lymph nodes (LN) and spleen (SP) were determined weekly. The pictures in C show the lesion at week 5 post-infection. The results represent the means 6 SD of three independent experiments, with 10 mice per group. Conceived and designed the experiments: FG JMPV SC. Performed the experiments: JCS DLG EGR MD. Analyzed the data: FG JMPV SC JCS EGR MD. Contributed reagents/materials/analysis tools: FG. Wrote the paper: FG JMPV SC JCS. Obtained permission for use of the vectors pXG-GFP+29 and pXG-’GFP+, for use of the polyclonal antisera against metacyclic marker protein HASPB, and for use of the papuamide B peptide: FG. Leishmania Infectivity Involves an ABC Transporter Leishmania Infectivity Involves an ABC Transporter 35. Campos-Salinas J, Cabello-Donayre M, Garcia-Hernandez R, Perez-Victoria I, Castanys S, et al. (2011) A new ATP-binding cassette protein is involved in intracellular haem trafficking in Leishmania. Mol Microbiol 79: 1430–1444. 52. Denny PW, Lewis S, Tempero JE, Goulding D, Ivens AC, et al. (2002) Leishmania RAB7: characterisation of terminal endocytic stages in an intracellular parasite. Mol Biochem Parasitol 123: 105–113. p 53. 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https://openalex.org/W4223967328	https://www.mdpi.com/2072-666X/13/4/610/pdf?version=1650245648	English	null	Pedestrian Dead Reckoning with Low-Cost Foot-Mounted IMU Sensor	Micromachines	2,022	cc-by	5,349	Article Article Shunsei Yamagishi and Lei Jing * Graduate School of Computer and Information Systems, The University of Aizu, Aizuwakamatsu 965-0006, Japan; shunsei_research@icloud.com * Correspondence: leijing@u-aizu.ac.jp Graduate School of Computer and Information Systems, The University of Aizu, Aizuwakamatsu 965-0006, Japan; shunsei_research@icloud.com * Correspondence: leijing@u-aizu.ac.jp Abstract: In this paper, we researched Pedestrian Dead Reckoning (PDR) with one foot-mounted IMU sensor. The issues of PDR are magnetism noise and accumulated error due to the noise included in acceleration and gyro data. Two methods are proposed in this paper. First is the gait-phase-estimation method with pitch angle for the Zero Velocity Update algorithm. Second is a method for avoiding accumulated errors by updating the roll and pitch angles with acceleration. The two experiments were conducted to examine the error of gait-phase estimation and distance estimations. The relative error of distance was about 7.40% in the case of walking straight and about 12.27% in the case of a shifting travel direction. Keywords: PDR; zero velocity update; gait phase estimation; kalman ﬁlter micromachines micromachines micromachines micromachines 1. Introduction PDR is the one of the technologies to estimate pedestrian position. PDR contributes to navigation systems. Navigation systems should use GPS or PDR technology depending on the situation. For example, a navigation system navigates a person using GPS when they are in the outdoors and navigates a person using PDR when they are in an underground pathway. There are technologies for navigation systems, such as GPS; however, GPS is unsuitable for an indoor navigation system. Navigation systems with IMU sensors are suitable for indoor navigation systems. • The roll/pitch update method that, during the mid-stance phase, updates the roll and pitch angles using the acceleration measured in each mid-stance phase. This paper is organized in ﬁve sections, with the Introduction in Section 1, Related Works in Section 2, the System and Method in Section 3, Experiments in Section 4 and our Conclusions and Future Work in Section 5. This paper is organized in ﬁve sections, with the Introduction in Section 1, Related Works in Section 2, the System and Method in Section 3, Experiments in Section 4 and our Conclusions and Future Work in Section 5. Citation: Yamagishi, S.; Jing, L. Pedestrian Dead Reckoning with Low-Cost Foot-Mounted IMU Sensor. Micromachines 2022, 13, 610. https:// doi.org/10.3390/mi13040610 IMU-based PDR technologies have been explored in detail by the other papers, and there are advantages, such as no effects from circumjacent objects except for elec- tronic goods in IMU-based PDR. There are technologies to estimate a pedestrian’s position, such as methods using ultrasound waves or beacons. Pedestrian position estimation by these methods are affected by circumjacent objects. Academic Editor: Burak Eminoglu However, there are two main disadvantages in IMU-based PDR methods. First is the accumulated error in estimating velocity. Second is the accumulated error in estimating attitude angles. Generally, the ﬁrst issue is handled by a Zero Velocity Update algorithm, and the second issue is handled by sensor-fusion methods, such as a Kalman ﬁlter and Complementary ﬁlter. The development of high accuracy mid-stance detection algorithms for Zero Velocity Updates is needed. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Therefore, in this paper, we proposed new methods to solve these two issues. First is the gait-phase-estimation method for the Zero Velocity Update algorithm. This detects the mid-stance phase. The pitch angle is the focus in this method. When a foot is in mid-stance phase, the pitch angle and the gyro of the foot-mounted IMU sensor will be nearly 0. Secondly, the roll/pitch update method handles the accumulated error in attitude angles. The roll and pitch angles are calculated by integrating the gyro during gait phases, except for mid-stance, and they update the roll and pitch angles using the acceleration measured in each mid-stance phase. The main contributions of this paper are two proposed methods: 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/micromachines Micromachines 2022, 13, 610. https://doi.org/10.3390/mi13040610 2 of 14 Micromachines 2022, 13, 610 • The gait-phase estimation method for Zero Velocity Updates. • The gait-phase estimation method for Zero Velocity Updates. g p y p • The roll/pitch update method that, during the mid-stance phase, updates the roll and pitch angles using the acceleration measured in each mid-stance phase. 2.3. Related Works for the First Issue and Second Issue In the paper [6] (H. Fourati), a method using the Complementary ﬁlter instead of Extended Kalman ﬁlter was proposed. The paper [6] presented the Quaternion-based complementary ﬁlter. MTI-IMU produced by Xsens was used for the experiments of [6]. The measurement error in positioning was about 0.4%. In the paper [7] (Z. Xiao-dong et al.), a new Zero Velocity Update algorithm using Kalman ﬁlter and Particle ﬁlter was proposed. An IMU sensor produced by Xsens was used in the experiments of [7]. The measurement error in positioning was less than 0.5%. In the paper [8] (L.-F.Shi et al.), a novel orientation estimation and gait-phase detection algorithm were proposed. The paper [8] proposed a Zero Velocity Update and orientation and velocity calibration method. The experiments of [8] were conducted indoors and outdoors, attaching an MPU9250 IMMU to the foot. The average distance error was less than 1.2%. 2.2. IMU Sensors and Deployment Positions for PDR 2.2. IMU Sensors and Deployment Positions for PDR 2.2. IMU Sensors and Deployment Positions for PDR In this research, a low-cost IMU sensor was used. There are tactical-grade IMU sensors, such as STIM202 [1], STIM300 [2]. Tactical-grade IMU sensors have much better performance compared with low-cost IMU sensors. However, tactical-grade IMU sensors are not suitable for navigation system in daily life because the price is expensive. In this paper, a low-cost IMU sensor is mounted on a shoe because it is easy to detect the mid- stance phase for Zero Velocity Updates. There are papers on PDR using smartphones, such as [3–5]. A paper [4] (A. Poulose et al.) described that the advantage of using smartphone- based position-estimation systems is that this requires no additional peripherals devices except for the smartphone itself. However, according to the paper [3] (S. Park et al.), the zero velocity phase rarely occurs in the case of Zero Velocity Updates (ZUPT) in the smartphone. 2.3. Related Works for the First Issue and Second Issue 2.1. Application Model 2.1. Application Model 2.1. Application Model pp A pedestrian wears an IMU sensor on their left shoe. Figure 1 shows application model. A pedestrian wears an IMU sensor on their left shoe. Figure 1 shows application model. Figure 1. Application model. Figure 1. Application model. Figure 1. Application model. 3.3. Low-Pass Filter (Step 1, 2, 3) Acceleration, gyro, and magnetism data are ﬁltered with a low-pass ﬁlter and simple moving average. The deﬁnition of the simple moving average is shown in Equation (1). a f iltered,n and araw,n denote ﬁltered data and raw data, respectively. a f iltered,n = 1 2w + 1 n+w ∑ k=n−w araw,k (1) (1) In this paper, n denotes the index. 2w + 1 is the window size. The window size is set to 21 (w = 10). 3.2. Data Preprocessing (Step 0) Before the data are processed, the gyro and magnetism data are calibrated. In the gyro calibration, gyro offsets are eliminated by subtracting the gyro data for calibration from the raw gyro data. In the magnetism calibration, the gap of the ellipse center is measured, and the raw magnetism data are modiﬁed with the measured gap. 3.3. Low-Pass Filter (Step 1, 2, 3) 3.1. System Architecture Figure 2 shows the system architecture. There are 11 steps. The gyro and magnetism data are calibrated in step 0. After that, the acceleration, gyro and magnetism data are input. In step 1 to step 3, all input data are ﬁltered by the simple moving average. In step 4, gait-phase estimation is conducted to detect mid-stance phase. In step 5, the gyro data of the pitch angle and roll angles are integrated, and the roll and pitch angles are updated mid-stance using acceleration. In step 6, the gyro and magnetism data are fused to calculate the yaw angle. In step 7, the acceleration is converted from local coordinates to global coordinates. In step 8, the acceleration is integrated to calculate the velocity, and the Zero Velocity Update is conducted. In step 9, the velocity is integrated to calculate the position vector. In step 10, the pedestrian position is estimated using the yaw angle and position vector. Figure 2. System architecture. Figure 2. System architecture. Figure 2. System architecture. 2.4. Summary In previous studies, the orientation and velocity calibration method [8] and methods using sensor-fusion methods, such as Quaternion-based Complementary ﬁlter [6], Kalman ﬁlter and Particle ﬁlter [7] were proposed to handle accumulated error issues. In addi- tion, many gait-phase detection algorithms have been proposed to improve Zero Velocity Updates as well. Micromachines 2022, 13, 610 3 of 14 3 of 14 Therefore, in this paper, the roll/pitch update method and gait-phase-estimation method are proposed with the aims of handling accumulated errors in attitude angles and improving the Zero Velocity Update method. 3.4. Gait Phase Estimation (Step 4) We propose a method to estimate the gait phase with pitch angle. According to the papers [6,9], there are two phases, the stance phase and the swing phase in the gait phase. Furthermore, according to [9], there are the pre-stance, mid-stance and terminal stance Micromachines 2022, 13, 610 4 of 14 in the stance phase, and there are the pre-swing, mid-swing and terminal swing in the swing phase. Figure 3 shows six gait phases. According to the paper [10], the pitch angle is obtained with Equation (2). in the stance phase, and there are the pre-swing, mid-swing and terminal swing in the swing phase. Figure 3 shows six gait phases. According to the paper [10], the pitch angle is obtained with Equation (2). θa = tan−1 −ax q a2y + a2z (2) (2) Equation (2) is used for our method. Let θa,n = tan−1 −ax,n q a2y,n+a2z,n . θa,n denotes θa q y, , measured in n-th time. The algorithm to estimate the gait phases is as follows. Step 1: Conduct the low-pass ﬁlter to θa,n2 and ωy,n2. q y measured in n-th time. The algorithm to estimate the gait phases is as follows. S 1 C d h l ﬁl θ 2 d 2 measured in n-th time. The algorithm to estimate the gait phases is as follows. Step 1: Conduct the low-pass ﬁlter to θa,n2 and ωy,n2. Step 1: Conduct the low-pass ﬁlter to θa,n2 and ωy,n2. θ′ a,n 2 =      θa,n2 if n ≤w ∨N −w ≤n 1 2w+1 n+w ∑ i=n−w θa,n2 else (3) (3) ω′ y,n 2 =      ωy,n2 if n ≤w ∨N −w ≤n 1 2w+1 n+w ∑ i=n−w ωy,n2 else (4) if n ≤w ∨N −w ≤n (4) N: Length of the data g Step 2: Estimate the gait phase into Label Ln g Step 2: Estimate the gait phase into Label Ln Ln = ( 1 if θ1 ≤θa,n2 ∨θ2 ≤ωy,n2 0 else (5) (5) where θ1, θ2 denote thresholds. θ1 is set to 0.1, and θ2 is set to 0.2. If Ln = 0, it is estimated as mid-stance. Otherwise, if Ln = 1, it is not estimated as mid-stance. Figure 3. Gait phases (there are six gait phases). 3.5. 3.4. Gait Phase Estimation (Step 4) Integration and Roll and Pitch Update (Step 5) The method for avoiding accumulated errors in attitude angles is proposed for use in a ramp. In the paper [8] (L.-F.Shi et al.), they make an assumption that the roll and pitch angles are assumed to be the same as the initial value during each stance phase. In the . Figure 3. Gait phases (there are six gait phases). 3.5. Integration and Roll and Pitch Update (Step 5) The method for avoiding accumulated errors in attitude angles is proposed for use in a ramp. In the paper [8] (L.-F.Shi et al.), they make an assumption that the roll and pitch angles are assumed to be the same as the initial value during each stance phase. In the Micromachines 2022, 13, 610 5 of 14 paper [8], a method based on this assumption is proposed. However, in the case of a ramp, their assumption does not hold. paper [8], a method based on this assumption is proposed. However, in the case of a ramp, their assumption does not hold. In the method in this paper, during each mid-stance phase, the roll and pitch angles are updated to φa and θa as measured in each mid-stance phase. On the other hand, the gyro is integrated during the other gait phases. According to the paper [10], Equation (6) can be used to obtain the pitch angle. a φa = tan−1 ay az (6) (6) Equations (2) and (6) are used for the roll/pitch update method. The trapezoidal rule is used for integration. The trapezoidal rule is shown in the following equation. Z b a f (t)dt ≈ N−1 ∑ n=0 ∆t 2 ( fn + fn+1) (7) (7) ∆t is 1/Sampling rate, f0 = f (a), fN = f (b) and fn+1 = f (t + ∆t) when fn = f (t). The integration and roll and pitch update method is implemented with the recurrence Formula (8).  1 a  ∆t is 1/Sampling rate, f0 = f (a), fN = f (b) and fn+1 = f (t + ∆t) when fn = f (t). The integration and roll and pitch update method is implemented with the recurrence Formula (8). 3.4. Gait Phase Estimation (Step 4) θn φn =                tan−1 −ax,n q a2y,n+a2z,n tan−1 ay,n az,n   if Ln = 0 " θn−1 φn−1 # + ∆t 2 ( " ωy,n ωx,n # + " ωy,n+1 ωx,n+1 # ) if Ln = 1 (8) (8) Figure 4 shows the comparison between roll and pitch angles obtained by only inte- gration and roll and pitch angles obtained by (8). It is conﬁrmed that the orange lines show less drift than the blue lines. Figure 4 shows the comparison between roll and pitch angles obtained by only inte- gration and roll and pitch angles obtained by (8). It is conﬁrmed that the orange lines show less drift than the blue lines. Figure 4. Comparison between attitude angles by only integration and attitude angles by integration with the roll/pitch update (the horizontal axes and vertical axes denote the index and roll or pitch angle [rad], respectively). Figure 4. Comparison between attitude angles by only integration and attitude angles by integration with the roll/pitch update (the horizontal axes and vertical axes denote the index and roll or pitch angle [rad], respectively). 3.7. Coordinate Conversion (Step 7) The acceleration is converted from local coordinates to global coordinates with Quater- nion. Quaternion q is represented as q = q0 + q1i + q2j + q3k. Let u = u0 + u1i + u2j + u3k, al = axi + ayj + azk and ag = aXi + aYj + aZk. u, al and ag denote Quaternion, which represents rotation, acceleration in the local coordinate and acceleration in the global coordinate, respectively.   u0 u1 u2 u3  =   cos ψ 2 cos θ 2 cos φ 2 + sin ψ 2 sin θ 2 sin φ 2 cos ψ 2 cos θ 2 sin φ 2 −sin ψ 2 sin θ 2 cos φ 2 cos ψ 2 sin θ 2 cos φ 2 + sin ψ 2 cos θ 2 sin φ 2 −cos ψ 2 sin θ 2 sin φ 2 + sin ψ 2 cos θ 2 cos φ 2   (16) (16) ψ, θ and φ denote the yaw angle, pitch angle and roll angle, respectively. The local coordi- nate is converted to a global coordinate with the following equation. ag = ual ¯u (17) (17) ag = ual ¯u (17) ¯u denotes the conjugate Quaternion of u. Figure 5 shows the comparison between the accelerations in the local coordinate and in the global coordinate. ¯u denotes the conjugate Quaternion of u. Figure 5 shows the comparison between the accelerations in the local coordinate and in the global coordinate. 3.8. Integration and Zero Velocity Update (Steps 8 and 9) 3.8. Integration and Zero Velocity Update (Steps 8 and 9) Zero Velocity Update is used for reducing the drift. The foot velocity is almost 0 when the foot state is in the mid-stance. From the trapezoidal rule and Zero Velocity Update, the foot velocity vn and position vector rn are calculated with the following recurrence equation. 3.6. Kalman Filter (Step 6) ( p ) The Kalman ﬁlter is implemented with the following status equation and observa- tion equation The Kalman ﬁlter is implemented with the following status equation and observa- tion equation. (9) ψk = ψk−1 + ∆tωz,k + wk (9) Micromachines 2022, 13, 610 6 of 14 zk = ψk + vk (10) (10) zk = ψk + vk zk = ψk + vk wk, vk and ψk denote the system noise, the observation noise and the yaw angle in the k −th time, respectively. The observation vector zk is −atan2(mx,k, my,k) −ψ′ 0. ψ′ 0 is the average of −atan2(mx,k, my,k) from index k = 0 to k = 399. Let ψk be status vector ˆxk, A = 1, B = ∆t, uk = ωz,k and H = 1. k z,k According to [11], the prediction equations are deﬁned from (11) to (12). ˆx− k = A ˆxk−1 + Buk−1 (11) T ˆx− k = A ˆxk−1 + Buk−1 (11) P− k = APk−1AT + Q (12) ˆx− k = A ˆxk−1 + Buk−1 (11) (11) (11) P− k = APk−1AT + Q (12) (12) According to [11], the update equations are deﬁned from (13) to (15). According to [11], the update equations are deﬁned from (13) to (15). Kk = P− k HT(HP− k HT + R)−1 (13) ˆxk−1 = ˆx− k + Kk(zk −H ˆx− k ) (14) Pk = (I −KkH)P− k (15) (13) (14) (15) I denotes the unit matrix. The ﬁlter deviation matrix P0 is set to 100, and Q and R are set to 1.0 × 10−5, 1.0 × 102. 3.7. Coordinate Conversion (Step 7) vn =      0 ∈R2 if Ln = 0 vn−1 + ∆t 2 ( " aX,n aY,n # + " aX,n+1 aY,n+1 # ) if Ln = 1 (18) rn = rn−1 + ∆t 2 ( vX,n vY,n + vX,n+1 vY,n+1 ) (19) (v0 = 0 ∈R2, r0 = 0 ∈R2) vn =      0 ∈R2 if Ln = 0 vn−1 + ∆t 2 ( " aX,n aY,n # + " aX,n+1 aY,n+1 # ) if Ln = 1 (18) (18) rn = rn−1 + ∆t 2 ( vX,n vY,n + vX,n+1 vY,n+1 ) (19) (v0 = 0 ∈R2, r0 = 0 ∈R2) (19) 7 of 14 Micromachines 2022, 13, 610 7 of 14 Figure 6 shows the comparison between the velocity obtained by only integration and the velocity obtained by the Zero Velocity Update and integration. Figure 6 shows the comparison between the velocity obtained by only integration and the velocity obtained by the Zero Velocity Update and integration. Figure 5. Comparison between local coordinates and global coordinates (The horizontal axes and Figure 5. Comparison between local coordinates and global coordinates (The horizontal axes and vertical axes denote the index and acceleration [m/s2], respectively). Figure 5. Comparison between local coordinates and global coordinates (The horizontal axes and vertical axes denote the index and acceleration [m/s2], respectively). Figure 6. Comparison between the velocity obtained by only integration and the velocity obtained by the Zero Velocity Update and integration (the horizontal axes and vertical axes denote the index and velocity [m/s], respectively). Figure 6. Comparison between the velocity obtained by only integration and the velocity obtained by the Zero Velocity Update and integration (the horizontal axes and vertical axes denote the index and velocity [m/s], respectively). 3.9. Pedestrian Position Estimation (Step 10) The pedestrian position is estimated on the complex plane using the following equations (Figure 7). p′ n = pn−1 + \|\|rn −rn−1\|\|i (20) pn = (p′ n −pn−1eiψn) + pn−1 (21) (20) (21) 8 of 14 8 of 14 Micromachines 2022, 13, 610 (p0 = 0) Figure 7. The pedestrian position in the global coordinates (pn and pn−1 denote the current pedestrian position and previous pedestrian position, respectively. Coordinate (pX, pY) is the global coordinate of the ﬂoor). 4. Experiments The two experiments were conducted for accuracy examination of the system. The ex- periments were conducted indoors. Figure 8 shows the implementation of the system, and Figure 9 shows the location of the IMU sensor. The IMU sensor (Wonder-Sense, developed in our laboratory) was used. Figure 8. Implementation of the experiments. Figure 8. Implementation of the experiments. 4.1. Exp. 1: Gait Phase Estimation Test 4.1. Exp. 1: Gait Phase Estimation Test This experiment’s objective is accuracy examination for gait-phase estimation. We de- ﬁned the True label by synchronizing the collected data with videos taken during collecting the data. If the True label is 0, this indicates mid-stance. On the other hand, if the True label is 1, this indicates the other gait phases. The estimation error (ER) is calculated with the below equation. ER = (1 − True Positive True Positive + False Negative) × 100 [%] (22) (22) Table 1 shows the estimation error of mid-stance by the gait-phase-estimation algo- rithm shown in Section 3.4. Three sets of straight walking data of one person were collected. Figures 10–12 show the wave-form of data collected in ﬁrst time, second time and third time. The wave-form in the ﬁrst line shows the acceleration data in the x axis (acceleration data of the traveling direction) in local coordinates. The wave-form in the second line Micromachines 2022, 13, 610 9 of 14 shows the squared pitch angle θ′ a,n 2. The wave-form in the third line shows the squared pitch angle’s gyro ω′ y,n 2. The wave-form in the fourth line shows a comparison between the true label and estimated label. Figure 9. The placement location of the IMU sensor on a shoe. Figure 9. The placement location of the IMU sensor on a shoe. Figure 9. The placement location of the IMU sensor on a shoe. The gait-phase-estimation algorithm should be improved from the data in Table 1. Estimation error of mid-stance was about 7.34% in average. However, each estimation error is scattered. A reliable gait-phase-estimation algorithm should have a lower error and low variance. Table 1. The results of Experiment 1. p Estimation Error of Mid-Stance [%] Measurement Time [s] ﬁrst time 7.38 16.2 second time 4.66 15.3 third time 9.99 15.6 Figure 10. The wave-form of the data from the ﬁrst data collection. Figure 10. The wave-form of the data from the ﬁrst data collection. 10 of 14 Micromachines 2022, 13, 610 Figure 11. The wave-form of the data from the second data collection. Figure 11. The wave-form of the data from the second data collection. Figure 12. The wave-form of the data from the third data collection. 4.2. Exp. 2: Test for Estimation of Walking Trajectory Figure 11. The wave-form of the data from the second data collection. Figure 12. The wave-form of the data from the third data collection. 4.1. Exp. 1: Gait Phase Estimation Test Figure 12. The wave-form of the data from the third data collection. Figure 12. The wave-form of the data from the third data collection. 4.2. Exp. 2: Test for Estimation of Walking Trajectory This experiment’s objective was the examination of the accuracy of the trajectory estimation. The relative error (RE) shown in the following equation calculates the accuracy of the distance estimation. RE= Estimated distance −Walked distance Walked distance × 100 [%] (23) (23) Figure 13 shows the walking routes of this experiment. Figures 14–16 show the esti- mated walking trajectory. Tables 2–4 show the results of the estimated distance. The walking data of one person were collected. This experiment was conducted in the laboratory. Micromachines 2022, 13, 610 11 of 14 11 of 14 The relative error of distance is much larger than in related studies, and the yaw angle estimation is not good as shown Figures 15 and 16. One of the possible reasons for the bad yaw angle estimation is the effect of magnetism from the other electronic devices. It is possible that the Kalman ﬁlter could not accurately estimate the yaw angle due to the large magnetism noise. Therefore, the yaw angle estimation in this paper should be improved referring to papers on the heading estimation method, such as [12,13]. Figure 13. The walking routes of Experiment 2. Figure 14. The results of route 1. Table 2. The results of route 1. [m] Estimated Distance [m] Relative Error [%] Measurement Time [s] 0.88 −11.964 11.9 1.893 −5.355 13.3 2.887 −3.752 14.3 3.658 −8.545 12.5 Figure 13. The walking routes of Experiment 2. Figure 13. The walking routes of Experiment 2. Figure 13. The walking routes of Experiment 2. Figure 13. The walking routes of Experiment 2. Figure 13. The walking routes of Experiment 2. Figure 14. The results of route 1. Table 2. The results of route 1. Walked Distance [m] Estimated Distance [m] Relative Error [%] Measurement Time [s] 1 (d = 1) 0.88 −11.964 11.9 2 (d = 2) 1.893 −5.355 13.3 3 (d = 3) 2.887 −3.752 14.3 4 (d = 4) 3.658 −8.545 12.5 Figure 14. The results of route 1. 12 of 14 12 of 14 Micromachines 2022, 13, 610 Table 3. The results of route 2. Estimated Distance [m] Relative Error [%] Measurement Time [s] 4.911 4.491 17.0 7.846 17.103 17.6 9.061 4.147 18.3 Figure 15. 4.1. Exp. 1: Gait Phase Estimation Test The results of route 2. Figure 16. The results of route 3. Figure 15. The results of route 2. Figure 15. The results of route 2. Figure 16. The results of route 3. 13 of 14 13 of 14 Micromachines 2022, 13, 610 Table 4. The results of route 3. Walked Distance [m] Estimated Distance [m] Relative Error [%] Measurement Time [s] 8 7.883 −1.466 20.8 16 (1 round-trip) 19.688 23.049 28.9 32 (2 round-trip) 37.357 16.739 47.5 48 (3 round-trip) 54.02 12.541 67.8 64 (4 round-trip) 71.809 12.201 85.8 80 (5 round-trip) 91.037 13.797 101.4 96 (6 round-trip) 110.206 14.798 114.1 112 (7 round-trip) 128.723 14.931 129.9 Table 4. The results of route 3. 5. Conclusions and Future Work 5. Conclusions and Future Work The norm of the relative errors of distance was about 7.40% in the case of straight walking and a short distance. On the other hand, the relative error of distance was about 12.27% in the case of a shifting travel direction. In comparison with with related research, the relative errors of the system in this paper were larger. In particular, the relative error of distance was much larger in the shifting travel direction. Future work must verify the accuracy of the system for the case of walking on a ramp. We could not verify this due to time constraints in our research. In addition, the roll/pitch update method in Section 3.5 was proposed in the case of a ramp, and the gait-phase-estimation method in Section 3.4 was proposed in the case of no ramp. Therefore, the gait-phase-estimation method should be modiﬁed when the roll/pitch update is veriﬁed in the case of walking on a ramp. Author Contributions: Conceptualization, L.J.; Data curation, S.Y.; Formal analysis, L.J.; Funding acquisition, L.J.; Investigation, L.J.; Methodology, S.Y.; Project administration, L.J.; Resources, L.J.; Software, S.Y.; Supervision, L.J.; Writing—original draft, S.Y.; and Writing—review and editing, L.J. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by JSPS KAKENHI Grant Number 18K11400, and NEDO Younger Research Support Project Grant Number 21502121. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. 7. Zhang, X.; Ren, M.; Wang, P.; Pan, K. A new zero velocity update algorithm for the shoe- mounted personal navigation system based on IMU. In Proceedings of the 2015 34th Chinese Control Conference (CCC), Hangzhou, China, 28–30 July 2015; pp. 5297–5302. 6. Fourati, H. Heterogeneous Data Fusion Algorithm for Pedestrian Navigation via Foot- Mounted Inertial Measurement Unit and Complementary Filter. IEEE Trans. Instrum. Meas. 2015, 64, 221–229. [CrossRef] 8. Shi, L.; Zhao, Y.; Liu, G.; Chen, S.; Wang, Y.; Shi, Y. A Robust Pedestrian Dead Reckoning System Using Low-Cost Magnetic and Inertial Sensors. IEEE Trans. Instrum. Meas. 2019, 68, 2996–3003. [CrossRef] 9. Peng, F.; Peng, W.; Zhang, C.; Zhong, D. IoT Assisted Kernel Linear Discriminant Analysis Based Gait Phase Detection Algorithm for Walking With Cognitive Tasks. IEEE Access 2019, 7, 68240–68249. [CrossRef] 11. Li, Q.; Li, R.; Ji, K.; Dai, W. Kalman Filter and Its Application. In Proceedings of the 2015 Eighth International Conference on Intelligent Networks and Intelligent Systems (ICINIS), Tianjin, China, 1–3 November 2015; pp. 74–77. g g 10. Hirose, K.; Doki, H.; Kondo, A. Studies on orientation measurement in sports using inertial and magnet Sport. Ind. 2012, 22, 255–262. [CrossRef] 13. Lee, J.-S.; Huang, S.-M. An Experimental Heuristic Approach to Multi-Pose Pedestrian Dead Reckonin tometers for Indoor Localization. IEEE Sens. J. 2019, 19, 9532–9542. [CrossRef] 12. Tian, Y.; Peng, A.; Xu, X.; Zhang, W. A Heading Estimation Algorithm for Wrist Device Assisted by Sequential Geomagnetic Observations. IEEE Sens. J. 2022, 22, 5309–5317. [CrossRef] Kondo, A. Studies on orientation measurement in sports using inertial and magnetic ﬁeld sensors. J. Jpn. Soc 255–262. [CrossRef] g g y j pp 12. Tian, Y.; Peng, A.; Xu, X.; Zhang, W. A Heading Estimation Algorithm for Wrist Device Assisted Observations. IEEE Sens. J. 2022, 22, 5309–5317. [CrossRef] 13. Lee, J.-S.; Huang, S.-M. An Experimental Heuristic Approach to Multi-Pose Pedestrian Dead Reckoning without Using Magne- tometers for Indoor Localization. IEEE Sens. J. 2019, 19, 9532–9542. [CrossRef] o a k g t Cog t e asks 0 9, , 68 0 68 9 [C oss e ] 10. Hirose, K.; Doki, H.; Kondo, A. Studies on orientation measurement in sports using inertial and magnetic ﬁeld sensors. J. Jpn. Soc. Sport. Ind. 2012, 22, 255–262. [CrossRef] g g g g y q g Observations. IEEE Sens. J. 2022, 22, 5309–5317. [CrossRef] 13. Lee, J.-S.; Huang, S.-M. An Experimental Heuristic Approach to Multi-Pose Pedestrian Dead Reckoning without Using Magne- References 1. Sensonor, STIM202 Inertial Measurement Unit. Available online: https://www.sensonor.com/products/gyro-modules/stim202/ (accessed on 17 March 2022). 2. Sensonor, STIM300 Inertial Measurement Unit. Available online: https://www.sensonor.com/products/inertial-measurement- units/stim300/ (accessed on 19 March 2022). 3. Park, S.; Lee, J.H.; Park, C.G. Robust Pedestrian Dead Reckoning for Multiple Poses in Smartphones. IEEE Access 2021, 9, 54498–54508. [CrossRef] O.S.; Han, D.S. An Indoor Position-Estimation Algorithm Using Smartphone IMU Sensor Data. IEEE Access 7. [CrossRef] 4. Poulose, A.; Eyobu, O.S.; Han, D.S. An Indoor Position-Estimation Algorithm Using Smartphone IMU 2019, 7, 11165–11177. [CrossRef] 5. Deng, Z.-A.; Hu, Y.; Yu, J.; Na, Z. Extended Kalman Filter for Real Time Indoor Localization by Fusing WiFi and Smartphone Inertial Sensors. Micromachines 2015, 6, 523–543. [CrossRef] 6. Fourati, H. Heterogeneous Data Fusion Algorithm for Pedestrian Navigation via Foot- Mounted Inertial Measurement Unit and Complementary Filter. 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https://openalex.org/W3117470329	https://www.researchsquare.com/article/rs-135792/latest.pdf	English	null	Relationship Between the Accuracy of Acetabular Cup Angle and Body Mass Index in Posterolateral Total Hip Arthroplasty With CT-Based Navigation: A Retrospective Case-Control Study	Research Square (Research Square)	2,020	cc-by	3,681	Relationship Between the Accuracy of Acetabular Cup Angle and Body Mass Index in Posterolateral Total Hip Arthroplasty With CT-Based Navigation: A Retrospective Case-Control Study Hisatoshi Ishikura (  hishikura817@gmail.com ) Masaki Nakamura Toranomon Hospital Hanae Nishino Teikyo University: Teikyo Daigaku Shigeru Nakamura Nishitokyo Chuo General Hospital Takeyuki Tanaka University of Tokyo: Tokyo Daigaku Hirotaka Kawano Teikyo University: Teikyo Daigaku Sakae Tanaka University of Tokyo: Tokyo Daigaku Abstract Background: Precise acetabular cup placement is essential for successful total hip arthroplasty (THA). In obese patients, its accuracy is often difficult to achieve because of the thickness of the soft tissues. This study aimed to determine the relationship between the accuracy of acetabular cup angle and body mass index (BMI) in posterolateral THA using the computed tomography-based navigation (CT-navi) system. Methods: We retrospectively reviewed 145 consecutive primary THAs using the CT-navi system between January 2015 and January 2018. All surgeries were performed using cementless cups employing the posterolateral approach with the patient in the decubitus position. We compared the radiographic inclination and anteversion obtained from the angle displayed on the CT-navi screen with those measured by the postoperative CT using the three-dimensional templating software. We evaluated the relationship between the extent of errors and correlation with BMI. Statistical analyses were performed using the Student’s t-test and Spearman’s rank coefficient test. Results: In non-overweight patients (BMI < 25, 88 hips), the mean navigation errors for inclination were 2.8 ± 2.2° and for anteversion were 2.6 ± 2.3°. Meanwhile, in overweight patients (BMI ≥ 25, 57 hips), the mean navigation errors were 2.6 ± 2.4° for inclination and 2.4 ± 2.4° for anteversion. We found no significant difference between overweight and non-overweight patients in both inclination and anteversion. The Spearman’s rank correlation coefficients were -0.04 for inclination and -0.11 for anteversion, showing no correlation between the extent of errors and BMI. Conclusions: In posterolateral THA, CT-navi can aid the precise placement of the acetabular cup irrespective of a patient’s BMI. Trial registration: This trial was retrospectively registered and approved by the institutional ethics committee of Teikyo University. The registration number is 17-190, and the date of approval was March 1, 2018. URL of trial registry is: https://www.teikyo- u.ac.jp/application/files/7015/8432/1341/2016_all_syounin_1.pdf Research article Keywords: total hip arthroplasty, navigation, computed tomography, body mass index, obesity Posted Date: December 29th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-135792/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: December 29th, 2020 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/11 Participants We retrospectively reviewed 153 consecutive hips in which primary THA was performed using CT-navi from January 2015 to January 2018. We excluded patients (8 hips) who experienced intraoperative issues such as apex pin loosening, mechanical seizure, and incongruity of verification. As a result, 145 hips were reviewed in this study. A senior hip surgeon (M.N.) was responsible for all the surgeries. CT-navi (Stryker CT-based Hip Navigation System, Stryker-Leibinger, Freiberg, Germany) was used in all surgeries. Patients with a BMI of ≥ 25 were classified as overweight and those with a BMI < 25 were considered non- overweight, according to the criteria of the World Health Organization. The retrieved patient data were anonymized and de-identified before the analyses. Background Total hip arthroplasty (THA) has been around for over 50 years, is widely performed in patients with hip diseases, and has become one of the most common and successful surgeries. To guarantee a successful surgical outcome, the accurate positioning of the implant is vital. Acetabular component malposition is associated with instability, impingement, and accelerated wear, and may sometimes necessitate early revision [1–4]. Lewinnek suggested that the radiographic cup inclination should ideally be 40° ± 10° and anteversion should be 15° ± 10°, based on the rate of postoperative dislocation [1]. However, several reports have questioned the reliability of those results because dislocation occurs very often, even in the region of the Lewinnek’s safe zone [5]. Page 2/11 Page 2/11 Widmer et al. proposed combined anteversion (CA) to achieve a sufficient range of motion without impingement [6]. They proposed that the ideal CA, when the radiographic inclination (RI) of the cup lies between 40° and 45°, may be determined as follows: cup radiographic anteversion + 0.7 × stem antetorsion (SA) = 37.3°. Yoshimine et al. developed a model adding the cup inclination, which was given as the cup anatomic anteversion + cup RI + 0.8 × SA = 90.8° [7]. Past studies demonstrated that the accuracy of cup placement in THA without navigation is reduced in obese patients [8]. Moreover, the accuracy of cup placement also decreased in imageless navigation [9]. On the other hand, for the anterolateral THA, the accuracy of the acetabular cup angle using the computed tomography-based navigation system (CT-navi) was reported to be maintained irrespective of patients’ BMI [10]. However, to the best of our knowledge, there are no reports on the relationship between the accuracy of cup angle and obesity in posterolateral THA using CT-navi. The accuracy of cup placement decreases in the absence of CT-navi when the procedure is performed in the lateral position compared to the supine position in THA because it is more difficult to maintain its orientation in the lateral position [11]. In addition to posture, it is apparent that in posterolateral THA for an obese patient, the surgical view is deeper and narrower because of the thicker fat and gluteus maximus muscle. Our study aimed to ascertain the relationship between the precision of th patients’ body mass index (BMI) in posterolateral THA with CT-navi. Background Our study aimed to ascertain the relationship between the precision of the acetabular cup angle and the patients’ body mass index (BMI) in posterolateral THA with CT-navi. Preoperative planning CT examinations were performed on three 64-detector row scanners (Light Speed VCT; GE Healthcare, Milwaukee, USA/ Aquilion; Toshiba Medical Systems, Tochigi, Japan / SOMATOM Definition Flash; Siemens AG, Forchheim, Germany) with a slice thickness of 0.5 mm. CT images in DICOM format were transferred to both the three-dimensional planning software (Zed Hip, Lexi Co., Ltd., Tokyo, Japan) and the navigation planning station. Initially, using the three-dimensional planning software, the SA was determined to match the shape of the femoral medullary canal and to restore the hip centre. If the native SA was too large (> 40°) or too small (< 10°), the modular stem was used to revise the SA. The cup RI was Page 3/11 Page 3/11 Page 3/11 planned at 43° for all hips. Cup anteversion was obtained from those values and by calculation using the formula of Yoshimine [7]. Finally, we fine-adjusted the implant position, the length of the neck, and offset to strike a bilateral balance. Furthermore, in the navigation planning station, we set several bony landmarks including the bilateral anterior superior iliac spine (ASIS), pubic tubercles and ischia, the pubic symphysis, and the sacral midplane to coordinate the pelvic position. Cup size and position were determined according to the plan using the three-dimensional planning software. Surgical procedure with CT-navi The posterolateral approach was used in all surgeries, with patients in the lateral decubitus position. In all the procedures, we used CT-navi without intraoperative fluoroscopy. Two apex pins were inserted in the ipsilateral ilium through small stab incisions. A pelvic tracker was affixed to the apex pins with an external fixation device (Hoffman II, Stryker-Leibinger) to enable detection by the infrared sensor camera. After exposing the acetabulum, initial paired-point matching registration was performed by digitizing the seven or eight landmarks including ASIS and greater sciatic notch. Surface matching of the pelvis was then performed by digitizing more than 30 points around the acetabulum and the ilium. The reliability was verified by touching the bone surfaces, including both the inside and outside of the acetabulum, ASIS, and apex pin insertion site. During all the surgeries, we adhered to the preoperative plan as closely as possible. The cup angles on the display were recorded after achieving press-fit fixation of the cup. The femoral stem was positioned without the navigation system. Postoperative evaluation Postoperative CTs were obtained a week following the surgeries using a minimal radiation dose protocol [12]. The CT data were imported into the Zed Hip software for three-dimensional analyses. After matching the postoperative pelvis position with the preoperative one, the cup angles were measured. In this study, “navigation error” was defined as the difference between the calculated value, value shown on the navigation display during the surgeries, and the one measured by the Zed Hip software. Data analysis Statistical analysis was performed using IBM SPSS Statistics for Windows, version 24.0 (IBM Corp., Armonk, New York, USA). The student’s t-test was used to compare the mean navigation errors between obese and non-obese patients. The Spearman’s rank correlation coefficient was used to ascertain the correlation between the extent of navigation errors and BMI. The strength of the correlations, indicated by Spearman’s rank correlation coefficient, were as follows: <0.3, negligible; 0.3–0.5, low positive; 0.5–0.7: moderate positive; 0.7–0.9: high positive; 0.9–1.0: very high positive [13, 14]. Discussion This study indicated that a remarkable accuracy in cup placement could be achieved in THA using CT- navi (2.7° for inclination and 2.5° for anteversion). Also, CT-navi could aid in precise placement of the acetabular cup irrespective of a patient’s BMI. Many previous studies described the effectiveness of CT-navi. A retrospective study demonstrated that fewer cups were placed outside of the safe zone and fewer postoperative dislocations occurred in the CT- navi group compared to the control group [15]. According to previous studies, the mean error of the cup angle with CT-navi was 1.5–3.0° for inclination and 1.2–3.3° for anteversion [16–18]. The result of our study is in agreement with those results. Obesity makes the THA procedure more challenging. Previous studies revealed that obesity was associated with increased complication rates and poor outcomes following primary THA, including infection, dislocation, and early aseptic loosening [19, 20]. Previous studies compared the incidence of these complications among several approaches. In THA in obese patients, there was no difference in the incidence of wound complications and deep infections when direct anterior and posterior approaches were compared [21]. Another study suggested that the increase in complication rates with THA for obese patients in comparison to non-obese patients was similar among several approaches [22]. Therefore, in THA for obese patients, approach strategy is often based on the condition of each patient or the surgeon’s preference. In THA without navigation, the accuracy of acetabular cup angle can decrease in obese patients because of the depth of the surgical field and the narrow surgical view [8]. In THA with imageless navigation, the thick subcutaneous tissue makes the registration inaccurate, leading to the increased error of the cup angle [9]. On the contrary, in THA with CT-navi, the registration was performed by touching the bony surface directly. This procedure could help minimize the effect of being overweight in the posterior approach. The result of this study confirmed the usefulness of CT-navi, particularly in overweight or obese patients. Several limitations must be considered when interpreting the findings of this study. First, the BMI of Japanese persons tends to be lower than those of persons from other countries [23]. Therefore, the number of obese patients (BMI ≥ 30) was relatively small (n = 14). Moreover, there were no morbidly obese patients (BMI ≥ 40) were included in this study. Results Page 4/11 Thirty-six patients were male, and 109 were female (145 hips). The mean age was 64.0 ± 10.1. The demographics of the two groups are shown in Table 1. The patients in the overweight group were significantly younger than those in the non-overweight group. Overall, the mean navigation errors in patients were 2.7 ± 2.3 for inclination and 2.5 ± 2.3 for anteversion. Table 1 Patients' demographic characteristics Non-overweight Overweight P-value Number of hips 88 57 Age (years) 66.3 ± 11.3 60.5 ± 10.5 0.002* Female sex 70 (80%) 39 (68%) 0.13 Diagnosis OA 68 (77%) 45 (79%) 0.81 ON 17 (19%) 10 (18%) 0.91** FNF 2 1 RA 1 1 OA: osteoarthritis; ON: osteonecrosis; FNF: femoral neck fracture; RA: rheumatoid arthritis * Student’s t-test ** Chi-square test In non-overweight patients (BMI < 25, 88 hips), the mean navigation errors were 2.8 ± 2.2° for inclination and 2.6 ± 2.3° for anteversion. Meanwhile, in overweight patients (BMI ≥ 25, 57 hips), the mean navigation errors were 2.6 ± 2.4° for inclination and 2.4 ± 2.4° for anteversion (Table 2). There were no significant differences between overweight and non-overweight patients in both inclination and anteversion. The Spearman’s rank correlation coefficients were − 0.04 for inclination and − 0.11 for anteversion, indicating that there was no correlation between the extent of navigation errors and BMI (Fig. 1, 2). Table 2 Navigation errors of the acetabular cup angle Non-overweight Overweight P-value RI (°) 2.8 ± 2.2 2.6 ± 2.4 0.67* RA (°) 2.6 ± 2.3 2.4 ± 2.4 0.58* RI: radiographic inclination; RA: radiographic anteversion * Student’s t-test Acknowledgements Not applicable. Consent for publication Consent for publication was obtained from all the patients. Funding The authors did not receive any funding for this study. Authors’ contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Hisatoshi Ishikura, Masaki Nakamura, Hanae Nishino, and Shigeru Nakamura. The first draft of the manuscript was written by Hisatoshi Ishikura and all authors reviewed and edited on previous versions of the manuscript. All authors read and approved the final version of the manuscript. Ethics approval and consent to participate The study was approved by the institutional ethics committee of Teikyo University (no. 17-190) and all the procedures were conducted in accordance with the Declaration of Helsinki and its later amendments. All participants enrolled in this study gave their written informed consent. Availability of data and materials The datasets generated during the current study are not publicly available due to patients’ privacy but are available from the corresponding author upon reasonable request. Discussion Further studies to investigate the relationship between morbid obesity and accuracy of implant positioning are required. Second, the pelvic coordinate system constructed on the navigation system used for surgery and the one constructed on the Zed Hip software used for postoperative measurement may not completely match. However, because both coordinate systems were constructed based on the pelvic bony surface and we uniformly used the radiographic angle for the measurements, the discrepancies should be minimized. With these limitations considered, the result of this study could be beneficial because the acetabular component accuracy in posterolateral THA with CT-navi is retained regardless of a patient’s BMI. Page 6/11 In conclusion, posterolateral THA with CT-navi could offer precise placement of the acetabular cup irrespective of a patient’s BMI In conclusion, posterolateral THA with CT-navi could offer precise placement of the acetabular cup irrespective of a patient’s BMI Competing Interests The authors declare that they have no competing interests. References Accuracy of acetabular cup placement using CT-based navigation in total hip arthroplasty: Comparison between obese and non- obese patients. J Orthop Sci. 2019;24:482-7. 10. Imai N, Takubo R, Suzuki H, Shimada H, Miyasaka D, Tsuchiya K, et al. Accuracy of acetabular cup placement using CT-based navigation in total hip arthroplasty: Comparison between obese and non- obese patients. J Orthop Sci. 2019;24:482-7. 11. Takada R, Jinno T, Miyatake K, Hirao M, Yagishita K, Yoshii T, et al. Supine versus lateral position for accurate positioning of acetabular cup in total hip arthroplasty using the modified Watson-Jones approach: A randomized single-blind controlled trial. Orthop Traumatol Surg Res. 2019;105:915-22. 11. Takada R, Jinno T, Miyatake K, Hirao M, Yagishita K, Yoshii T, et al. Supine versus lateral position for accurate positioning of acetabular cup in total hip arthroplasty using the modified Watson-Jones approach: A randomized single-blind controlled trial. Orthop Traumatol Surg Res. 2019;105:915-22. 12. Arai N, Nakamura S, Matsushita T, Suzuki S. Minimal radiation dose computed tomography for measurement of cup orientation in total hip arthroplasty. J Arthroplasty. 2010;25:263-7. 12. Arai N, Nakamura S, Matsushita T, Suzuki S. Minimal radiation dose computed tomography for measurement of cup orientation in total hip arthroplasty. J Arthroplasty. 2010;25:263-7. 13. Mukaka MM. Statistics corner: A guide to appropriate use of correlation coefficient in medical research. Malawi Med J. 2012;24:69-71. 13. Mukaka MM. Statistics corner: A guide to appropriate use of correlation coefficient in medical research. Malawi Med J. 2012;24:69-71. 14. Asai Y, Tsutsui S, Oka H, Yoshimura N, Hashizume H, Yamada H, et al. Sagittal spino-pelvic alignment in adults: The Wakayama Spine Study. PloS One. 2017;12:e0178697. 14. Asai Y, Tsutsui S, Oka H, Yoshimura N, Hashizume H, Yamada H, et al. Sagittal spino-pelvic alignment in adults: The Wakayama Spine Study. PloS One. 2017;12:e0178697. 15. Sugano N, Takao M, Sakai T, Nishii T, Miki H. Does CT-based navigation improve the long-term survival in ceramic-on-ceramic THA? Clin Orthop Relat Res. 2012;470:3054-9. 15. Sugano N, Takao M, Sakai T, Nishii T, Miki H. Does CT-based navigation improve the long-term survival in ceramic-on-ceramic THA? Clin Orthop Relat Res. 2012;470:3054-9. 16. Iwana D, Nakamura N, Miki H, Kitada M, Hananouchi T, Sugano N. Accuracy of angle and position of the cup using computed tomography-based navigation systems in total hip arthroplasty. Comput Aided Surg. 2013;18:187-94. 16. Iwana D, Nakamura N, Miki H, Kitada M, Hananouchi T, Sugano N. References 1. Lewinnek GE, Lewis JL, Tarr R, Compere CL, Zimmerman JR. Dislocations after total hip-replacement arthroplasties. J Bone Joint Surg Am. 1978;60:217-20. 1. Lewinnek GE, Lewis JL, Tarr R, Compere CL, Zimmerman JR. Dislocations after total hip-replacement arthroplasties. J Bone Joint Surg Am. 1978;60:217-20. 1. Lewinnek GE, Lewis JL, Tarr R, Compere CL, Zimmerman JR. Dislocations after total hip-replacement arthroplasties. J Bone Joint Surg Am. 1978;60:217-20. Page 7/11 Page 7/11 2. Kennedy JG, Rogers WB, Soffe KE, Sullivan RJ, Griffen DG, Sheehan LJ. Effect of acetabular component orientation on recurrent dislocation, pelvic osteolysis, polyethylene wear, and component migration. J Arthroplasty. 1998;13:530-4. 3. Leslie IJ, Williams S, Isaac G, Ingham E, Fisher J. High cup angle and microseparation increase the wear of hip surface replacements. Clin Orthop Relat Res. 2009;467:2259-65. 3. Leslie IJ, Williams S, Isaac G, Ingham E, Fisher J. High cup angle and microseparation increase the wear of hip surface replacements. Clin Orthop Relat Res. 2009;467:2259-65. 4. Little NJ, Busch CA, Gallagher JA, Rorabeck CH, Bourne RB. Acetabular polyethylene wear and acetabular inclination and femoral offset. Clin Orthop Relat Res. 2009;467:2895-900. 4. Little NJ, Busch CA, Gallagher JA, Rorabeck CH, Bourne RB. Acetabular polyethylene wear and acetabular inclination and femoral offset. Clin Orthop Relat Res. 2009;467:2895-900. 5. Abdel MP, von Roth P, Jennings MT, Hanssen AD, Pagnano MW. What Safe Zone? The Vast Majority of Dislocated THAs Are Within the Lewinnek Safe Zone for Acetabular Component Position. Clin Orthop Relat Res. 2016;474:386-91. 6. Widmer KH, Zurfluh B. Compliant positioning of total hip components for optimal range of motion. J Orthop Res. 2004;22:815-21. 7. Yoshimine F. The safe-zones for combined cup and neck anteversions that fulfill the essential range of motion and their optimum combination in total hip replacements. J Biomech. 2006;39:1315-23. 7. Yoshimine F. The safe-zones for combined cup and neck anteversions that fulfill the essential range of motion and their optimum combination in total hip replacements. J Biomech. 2006;39:1315-23. 8. Barrack RL, Krempec JA, Clohisy JC, McDonald DJ, Ricci WM, Ruh EL, et al. Accuracy of acetabular component position in hip arthroplasty. J Bone Joint Surg Am. 2013;95:1760-8. 9. Tsukada S, Wakui M. Decreased accuracy of acetabular cup placement for imageless navigation in obese patients. J Orthop Sci. 2010;15:758-63. 10. Imai N, Takubo R, Suzuki H, Shimada H, Miyasaka D, Tsuchiya K, et al. References Accuracy of angle and position of the cup using computed tomography-based navigation systems in total hip arthroplasty. Comput Aided Surg. 2013;18:187-94. 17. Tsutsui T, Goto T, Wada K, Takasago T, Hamada D, Sairyo K. Efficacy of a computed tomography- based navigation system for placement of the acetabular component in total hip arthroplasty for 17. Tsutsui T, Goto T, Wada K, Takasago T, Hamada D, Sairyo K. Efficacy of a computed tomography- based navigation system for placement of the acetabular component in total hip arthroplasty for Page 8/11 Page 8/11 developmental dysplasia of the hip. J Orthop Surg (Hong Kong). 2017;25:2309499017727954. 18. Kalteis T, Handel M, Bathis H, Perlick L, Tingart M, Grifka J. Imageless navigation for insertion of the acetabular component in total hip arthroplasty: is it as accurate as CT-based navigation? J Bone Joint Surg Br. 2006;88:163-7. 19. Goodnough LH, Finlay AK, Huddleston JI, 3rd, Goodman SB, Maloney WJ, Amanatullah DF. Obesity Is Independently Associated With Early Aseptic Loosening in Primary Total Hip Arthroplasty. J Arthroplasty. 2018;33:882-6. 20. Triantafyllopoulos GK, Soranoglou VG, Memtsoudis SG, Sculco TP, Poultsides LA. Rate and Risk Factors for Periprosthetic Joint Infection Among 36,494 Primary Total Hip Arthroplasties. J Arthroplasty. 2018;33:1166-70. 21. Purcell RL, Parks NL, Cody JP, Hamilton WG. Comparison of Wound Complications and Deep Infections With Direct Anterior and Posterior Approaches in Obese Hip Arthroplasty Patients. J Arthroplasty. 2018;33:220-3. 22. Russo MW, Macdonell JR, Paulus MC, Keller JM, Zawadsky MW. Increased Complications in Obese Patients Undergoing Direct Anterior Total Hip Arthroplasty. J Arthroplasty. 2015;30:1384-7. 23. Ramachandran A, Chamukuttan S, Shetty SA, Arun N, Susairaj P. Obesity in Asia--is it different from rest of the world. Diabetes Metab Res Rev. 2012;28 Suppl 2:47-51. Figures Page 9/11 Page 9/11 Figure 1 Relationship between RI error and the patient’s BMI. RI: radiographic inclination; BMI: body mass index Figure 1 Relationship between RI error and the patient’s BMI. RI: radiographic inclination; BMI: body mass index Relationship between RI error and the patient’s BMI. RI: radiographic inclination; BMI: body mass index Relationship between RI error and the patient’s BMI. RI: radiographic inclination; BMI: body mass index Page 10/11 Figure 2 Relationship between RA error and the patient’s BMI. RA: radiographic anteversion; BMI: body mass index Figure 2 Relationship between RA error and the patient’s BMI. RA: radiographic anteversion; BMI: body mass index Relationship between RA error and the patient’s BMI. RA: radiographic anteversion; BMI: body mass index Relationship between RA error and the patient’s BMI. RA: radiographic anteversion; BMI: body mass index Page 11/11
https://openalex.org/W4380185160	https://ojs.ecologiaaustral.com.ar/index.php/Ecologia_Austral/article/download/2156/1387	Spanish; Castilian	null	Management of grassland/savannas encroached by woody plants in South American Semiarid Chaco: Ecological restoration vs. African grasses introduction	Ecología austral/Ecología austral	2,023	cc-by	12,155	Management of grassland/savannas encroached by woody plants in South American Semiarid Chaco: Ecological restoration vs. African grasses introduction RѢяѼћ D. Cќџію₁,; CюџљќѠ R. KѢћѠѡ₂; AћюљҌю AћџіўѢђѧ₃ ӕ Sюћёџю J. Bџюѣќ₄ Instituto Nacional de Tecnología Agropecuaria (INTA), Estación Experimental Agropecuaria Santiago del Estero. Santiago el Estero, Argentina. 2 Colegio de Ingenieros Agrónomos de Santiago del Estero. 3 Facultad de Agronomía y Agroindustrias, niversidad Nacional de Santiago del Estero. 4 Facultad de Ciencias Forestales, Universidad Nacional de Santiago del Estero. AяѠѡџюѐѡ. In Semiarid Chaco, two management approaches for woody plant-encroached grassland/savannas were evaluated: ecological restoration and the African grass introduction. An experiment with five treatments was carried out: 1) reference community (Ref_Eco; a grassland/savanna in good state, used as standard of comparison); 2) grassland/savanna encroached by woody plants (Woody_Encr; the woody state of grassland/ savannas); 3) roller chopping of the encroached ecosystem, followed by grazing suppression and prescribed fire (Roll_Fire; aimed at inducing grassland/savanna ecological restoration); 4) roller chopping of the encroached ecosystem, with instantaneous seeding of Megathyrsus maximus cv. GaĴon Panic and grazing suppression (Roll_GaĴon Panic), and 5) roller chopping of the encroached ecosystem, with instantaneous seeding of Cenchrus ciliaris cv. Buffel Texas and grazing suppression (Roll_Buffel Texas). The last two treatments aimed to obtain new highly productive grassy states for ranching through the introduction of these African grasses. The results suggested that Ref_Eco (average herbaceous biomass [HERB]=7196.9 kg DM/ha) was dominated by shade intolerant and fire tolerant grasses, and it had good soil quality and good utilitarian range condition. Regarding Ref_Eco: a) in Woody_Encr (HERB=2192 kg DM/ha), the vegetation structure, plant composition and the utilitarian range condition were altered, but it still had good soil quality and abundant native grass populations; b) in Rol_Fire (HERB=6591.6 kg DM/ha), both the vegetation structure and good utilitarian range condition were restored, plant composition was not restored and the good soil quality was kept, and c) Roll_GaĴon Panic and Roll_Buffel Texas (HERB=10972.1 and 7450.8 kg DM/ha, respectively) resulted in new grassy states integrated by native and exotic grasses with good utilitarian range condition and good soil quality. In this study case, the ecological restoration of the encroached ecosystems would be both ecologically and productively viable. So, probably the introduction of African grasses to improve ranching was not necessary. [Keywords: prescribed fire, roller chopping, grazing suppression, plant species composition, vegetation structure, utilitarian range condition] RђѠѢњђћ. Manejo de pastizales/sabanas lignificados del Chaco Semiárido Sudamericano: Restauración ecológica vs. implantación de pasturas africanas. ₄₈₉ ₄₈₉ WќќёѦ ѝљ Ecología Austral 33:489-506 Agosto 2023 Asociación Argentina de Ecología https://doi.org/10.25260/EA.23.33.2.0.2156 WќќёѦ ѝљ Ecología Austral 33:489-506 Agosto 2023 Asociación Argentina de Ecología https://doi.org/10.25260/EA.23.33.2.0.2156 IћѡџќёѢѐѡіќћ Arid and semiarid grassland/savannas are a source of multiple ecosystem services highly valued for society, both at local and global scale (Easdale 2021). These ecosystems are under grazing and are not fit for intensive agricultural practices due to an inappropriate rainfall regime or poor soil quality. Usually, they have been overgrazed and that fact has deeply altered them (Aronson et al. 1993; Gibson et al. 1995; Archer et al. 2017). Therefore, initiatives oriented to regulate the use and restore these ecosystems are desirable. Ecological restoration is the process of assisting the recovery of an ecosystem that has been degraded, damaged or destroyed (Society for Ecological Restoration International Science and Policy Working Group 2004). Normally, it aims to stop threats and to reestablish vegetation structure, species composition and ecological, productive and sociological ecosystem functions (McDonald et al. 2016). In arid and semiarid regions of the southern hemisphere, the complete ecological restoration of historic native ecosystems (those that existed before the intensification of disturbances) may not be achieved, due to: 1) it is rarely possible to precisely determine which original ecosystems were there and what was the complete list of native species, and 2) often, the alteration of the ecosystem evolved so far that the irreversibility thresholds were crossed (e.g., seed bank of key species finished, soil A horizon severely altered or eliminated). Concomitantly, the complete ecological restoration may have prohibitive costs or be practically impossible (Aronson et al. 1993; Allen 1995; Bestelmeyer 2006; Archer et al. 2017; Coppock et al. 2017; Evans et al. 2017). Two types of ecological restoration have been proposed: a) the sensu stricto ecological restoration that aims at complete ecosystem recovery; and b) the sensu lato ecological restoration that simply aims to halt the degradation and to redirect the disturbed ecosystem in a trajectory resembling those presumed to have prevailed prior to the start of disturbances (Aronson et al. 1993). Despite these differences, the primary goal of both types of ecological restoration is to conserve native biodiversity and ecosystem structure and dynamics (Aronson et al. 1993). In South American Semiarid Chaco, overgrazing and the concurrent reduction/ suppression of grassland fires (intensified by drought) have been identified as the main causes of a grassland/savanna massive woody plant encroachment occurring since the early 1900s (Morello and Adamoli 1974; Adamoli et al. 1990). Management of grassland/savannas encroached by woody plants in South American Semiarid Chaco: Ecological restoration vs. African grasses introduction Dos enfoques de manejo de pastizales/sabanas lignificados fueron evaluados: la restauración ecológica y la implantación de pasturas africanas. Se realizó un experimento con cinco tratamientos: 1) comunidad de referencia (Ref_Eco; pastizal/sabana en buen estado, usado como estándar de comparación); 2) pastizal/sabana lignificado (Woody_Encr; estado lignificado de los pastizales/ sabanas); 3) rolado del ecosistema lignificado, seguido por supresión del pastoreo y fuego prescripto (Roll_Fire; objetivo: inducir la restauración ecológica del pastizal/sabana); 4) rolado del ecosistema lignificado con siembra instantánea de Megathyrsus maximus cv. GaĴon Panic y supresión del pastoreo (Roll_GaĴon Panic), y 5) rolado del ecosistema lignificado, con siembra instantánea de Cenchrus ciliaris cv. Buffel Texas y supresión del pastoreo (Roll_Buffel Texas). Los dos últimos tratamientos buscaron obtener nuevos estados graminosos muy productivos para la ganadería mediante la implantación de esas pasturas africanas. Los resultados sugirieron que Ref_Eco (biomasa herbácea promedio [HERB]=7196.9 kg MS/ha) estuvo dominado por gramíneas intolerantes a la sombra y tolerantes al fuego, y tuvo calidad del suelo buena y condición utilitaria del pastizal buena. Comparado con a Ref_Eco: a) en Woody_Encr (HERB=2192 kg MS/ha), la estructura de la vegetación, la composición de plantas y la condición utilitaria del pastizal fueron alteradas, pero éste todavía tuvo calidad del suelo buena y poblaciones de gramíneas nativas abundantes; b) en Roll_Fire (HERB=6591.6 kg MS/ha), la estructura de la vegetación y la condición utilitaria del pastizal fueron restaurados, la composición de plantas no fue restaurada y la calidad del suelo buena fue preservada, y c) Roll_GaĴon Panic y Roll_Buffel Texas (HERB=10972.1 y 7450.8 kg MS/ha, respectivamente) resultaron en nuevos estados graminosos integrados por pastos nativos y exóticos con calidad del suelo buena y condición utilitaria del pastizal buena. En este estudio, la restauración ecológica del ecosistema lignificado sería ecológica y productivamente viable. Por lo tanto, probablemente la implantación de pasturas africanas para mejorar la producción ganadera no fue necesaria. [Palabras clave: fuego prescripto, rolado, supresión del pastoreo, composición de especies de plantas, estructura de la vegetación, condición utilitaria del pastizal] Recibido: 10 de Enero de 2023 Aceptado: 17 de Abril de 2023 rudacoria@yahoo com ar Editor asociado: Ricardo Grau RD Cќџію ђѡ юљ Ecología Austral 33:489-506 ₄₉₀ communities (Aronson et al. 1993; McDonald et al. 2016), and it normally coincides with the historic native ecosystem (Aronson et al. 1993). Management of grassland/savannas encroached by woody plants in South American Semiarid Chaco: Ecological restoration vs. African grasses introduction Often, it is assumed that the reference ecosystem maximizes the ecosystem services, the biodiversity and soil conservation and the management options (Bestelmeyer et al. 2003, 2010). In the Americas, historic native ecosystems can be established as those the European colonizers found at their arrival, and its definition requires to consider the natural variation of both the biotic community and ecosystem functioning (Bestelmeyer et al. 2003, 2010). However, the historic native ecosystems commonly disappeared and cannot be reliably estimated from historic records. As a consequence, they are usually estimated under the assumption that the most palatable plant species dominated on them (Bestelmeyer et al. 2003). IћѡџќёѢѐѡіќћ Usually, it is observed: 1) a substantial increase in shrub cover (a substantial increase in tree cover occurs with overgrazing for more than 50 years); 2) an impoverishment of the utilitarian range condition due to the reduction of perennial forage grasses and the excessive woody cover that complicates accessibility and animal management, and 3) alterations in plant diversity and functional types (Morello and Saravia Toledo 1959; Coria et al. 2021). In contrast, woody plant diversity is less affected by woody plant encroachment (Coria et al. 2021). In extreme alteration cases, there is a severe soil erosion and perennial grasses communities are replaced by annual broadleaf herbs or annual grasses with little forage value and very low productivity (Morello and Saravia Toledo 1959; Adamoli et al. 1972; Díaz 2015). In the Semiarid Chaco area of Argentina, the main approach of woody plant encroachment management aims to improve ranching production and consists in reducing/ eliminating the woody cover by means of roller chopping, and to simultaneously seed African forage grasses. Roller chopping consists in woody plants crushing using a roller chopper (i.e., metallic drum >2 m width and armed with blades) pulled by tractors or bulldozers. Generally, the resulting In ecological restoration, the reference ecosystem is a key concept that represents the comparison standard for altered biotic WќќёѦ ѝљюћѡ ђћѐџќюѐѕњђћѡ њюћюєђњђћѡ ₄₉₁ and stimulate the re-sprout, germination and flowering of grasses (Kunst et al. 2003a,b, 2008; Coria et al. 2021). herbaceous stratum has a mix of African grasses with variable proportions of native grasses, and the increase in the stocking rate is notable (up to 400-500%) (Fumagalli and Kunst 2002). Low/medium intensity roller chopping preserves both a significant part of native vegetation and soil properties (Kunst et al. 2016; Rejžek et al. 2017). This intensity is achieved by using relatively small machinery (e.g., small tractors and roller chopper <2.5 m width) and concentrating the treatment on shrubs (height <3 m). herbaceous stratum has a mix of African grasses with variable proportions of native grasses, and the increase in the stocking rate is notable (up to 400-500%) (Fumagalli and Kunst 2002). Low/medium intensity roller chopping preserves both a significant part of native vegetation and soil properties (Kunst et al. 2016; Rejžek et al. 2017). This intensity is achieved by using relatively small machinery (e.g., small tractors and roller chopper <2.5 m width) and concentrating the treatment on shrubs (height <3 m). IћѡџќёѢѐѡіќћ The objective of this study was to compare the effects on vegetation structure, plant composition and utilitarian range condition of two management approaches on woody plant- encroached grassland/savannas: 1) the sensu lato ecological restoration, and 2) the introduction of the African grasses Megathyrsus maximus cv. Gatton Panic and Cenchrus ciliaris cv. Buffel Texas. The hypothesis stated that returning from a woody to a grassy state is effectively promoted by an appropriate disturbance regime when soil and native grass populations are not too altered. The predictions (P) were that if the encroached ecosystem still has a good superficial soil quality and abundant native grass populations, then: P1) a new grassy state with native species and in better utilitarian range condition (it means, the sensu lato ecological restoration) will occur in the short term by a sequential utilization of low/ medium intensity roller chopping, grazing suppression and prescribed fire, and P2) the application of low/medium intensity roller chopping with both simultaneous seeding of African grasses and grazing suppression, in the short term will result in new grassy states with native and exotic grasses and in good utilitarian range condition. Figure 1 schematizes both predictions. In the region, the sensu stricto ecological restoration of grassland/savannas may not be possible in most of the cases. Instead, when irreversibility thresholds in superficial soil quality and native grass populations have not been crossed (Aronson et al. 1993; Allen 1995; Bestelmeyer 2006; Coppock et al. 2017; Puthod et al. 2020), a sensu lato ecological restoration would be a more realistic goal. So, in the last scenario, a new grassy estate and in a better utilitarian range condition would be achieved through a management that utilizes in sequence: 1) low/medium intensity roller chopping to reduce woody plant competition; 2) grazing suppression during one or two growing seasons to favor the herbaceous biomass productivity (fine fuel), and 3) the application of prescribed fire to reduce woody plant competition again, Figure 1. Management of woody plant encroachment in grassland/savannas of the Semiarid Chaco. If the encroached ecosystem still has good superficial soil quality and abundant native grass populations, predictions P1 and P2 can occur. Figura 1. Manejo de la lignificación en pastizales/sabanas del Chaco Semiárido. Si el pastizal/sabana lignificado todavía conserva una calidad del suelo superficial buena y poblaciones de gramíneas nativas abundantes, entonces las predicciones P1 y P2 pueden ocurrir. Figure 1. Study area Study area The study was conducted at F. Cantos Experimental Ranch, belonging to Santiago del Estero Agricultural and Livestock Experimental Station, Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina (coordinates of the ranch vertices: 27°59’6.94’’ S - 64°24’9.38’’ W; 28°1’23.62’’ S - 64°24’41.25’’ W; 28°3’18.64’’ S - 64°14’15.14’’ W; 28°1’0.19’’ S - 64°13’53.01’’ W) (Figure 2). The site belong to the ecoregion of Dry Chaco, Semiarid Chaco sub-region (Morello et al. 2012). The altitude is ~200 m a. s. l. The climate is subtropical semiarid with warm and wet summers and dry and cold winters (Boletta et al. 2006). The average annual precipitation is 587.1 mm and the average temperature is 20.1 °C. Most of the study area is located within the geomorphological unit Bajada Distal del Piedemonte Subandino Pampeano and the soils are Haplustolls (Prieto Angueira 2016). The first treatment (reference ecosystem; from now on, Ref_Eco), was an area covered by grassland/savannas considered in good state based on the gathered empirical and scientific information. Its objective was to estimate the historic native grassland/savanna and serve as standard of comparison. Ref_Eco was located in a patch of lowland ecological site almost entirely covered by grassland/savannas (1259 ha). The available satellite images show that the grassy state existed at least since 1960 until today (previous to 1960, there are not available images) (Figure 2a and 2b). Inside this patch, Ref_Eco (yellow polygon in Figure 2b) was an area of 6 ha historically isolated from livestock grazing (more than 1850 m away from the watering point). At local scale (200 m - a few kilometers), the soft topographic gradient defines three characteristic ecological sites: 1) highland ecological sites, at high topographic positions, originally occupied by tall hardwood forests; 2) midland ecological sites, at medium topographic positions, originally occupied by open tall forests, and 3) lowland ecological sites, at low topographic positions, originally occupied by grassland/savannas, where this study focused on. The current grassland/ savannas have isolated trees of Schinopsis lorentzii and Aspidosperma quebracho-blanco, and abundant shade intolerant and fire tolerant grasses such as Elionurus muticus, Schizachyrium tenerum, Heteropogon contortus and Pappophorum pappiferum. In woody plant- encroached grassland/savannas, Vachellia aroma, Prosopis nigra, Geoffroea decorticans and Schinus sp. are abundant. Soils are Typic Haplustolls (Anriquez et al. 2005). The rest of the treatments were located in a patch of lowland ecological site originally covered by grassland/savannas and currently completely encroached by woody plants (586 ha). IћѡџќёѢѐѡіќћ Management of woody plant encroachment in grassland/savannas of the Semiarid Chaco. If the encroach ecosystem still has good superficial soil quality and abundant native grass populations, predictions P1 and P2 c occur Figura 1. Manejo de la lignificación en pastizales/sabanas del Chaco Semiárido. Si el pastizal/sabana lignificado todavía conserva una calidad del suelo superficial buena y poblaciones de gramíneas nativas abundantes, entonces las predicciones P1 y P2 pueden ocurrir. RD Cќџію ђѡ юљ RD Cќџію ђѡ юљ Ecología Austral 33:489-506 ₄₉₂ Experimental design This study was carried out at lowland ecological sites in the Bajada Distal del Piedemonte Subandino Pampeano geomorphological unit of the F. Cantos Experimental Ranch. Between December 2016 and April 2019 (that includes three growing seasons: 2016/2017, 2017/2018 and 2018/2019), an experiment with five treatments was performed. Each treatment had eight experimental units (plots of 20x20 m each). Once the experiment was completed, all vegetation and soil variables were measured between April and May 2019. Study area Available remote images show that the grassy state existed at least since 1960 (previous to this year there are no images available) until 1988/2000, when woody plant encroachment was consolidated. Woody state is likely the result of both overgrazing and fire suppression since 1989, intensified by a severe drought occurring during 1988/99 (Kunst et al. 2012; Ledesma et al. 2018). Within the selected area, two smaller areas (area 17.5 ha and 3 ha, respectively) were used due to the presence of roads and fences that ensure grazing suppression (light-blue polygon in Figure 2b). Inside these fenced areas, the rest four treatments were: a) Woody_Encr: it was a woody plant-encroached grassland/ savanna which did not receive any disturbance during the experiment (included grazing). Its objective was to represent the woody state of native grassland/savannas; b) Roll_Fire: it was a woody plant-encroached grassland/ Figure 2a corresponds to the study area and it was obtained from air photographs in 1960. It can be seen that grassland/savannas (light- colored areas) were abundant in lowland ecological sites (red polygons). Figure 2b corresponds to an actual satellite image (August 2019). By comparing both figures, it can be observed that, in the central part of the study area, large surfaces of grassland/ savannas have been encroached by woody plants (dark green areas in Figure 2b). WќќёѦ ѝљюћѡ ђћѐџќюѐѕњђћѡ њюћюєђњђћѡ ₄₉₃ Figure 2. Management of woody plant encroachment in grassland/savannas in Semiarid Chaco. Study area, F. Cantos Experimental Ranch. a) Ancient air photographs (1960). It is appreciated that grassland/savannas (light areas) predominated in lowland ecological sites (red polygons). b) SENTINEL satellite image from August 2019. It is appreciated that in the central part, grassland/savannas were encroached by woody plants (dark green areas). The yellow polygon contains the Ref_Eco treatment. The light-blue polygon contains the Woody_Encr, Roll_Fire, Roll_Gatton Panic and Roll_Buffel Texas treatments. Figura 2. Manejo de la lignificación en pastizales/sabanas del Chaco Semiárido. Área de estudio, Campo Experimental F. Cantos. a) Mosaico de fotografías aéreas antiguas (1960). Se aprecia que los pastizales/sabana (áreas claras) predominaron en los sitios ecológicos bajos (polígonos rojos). b) Imagen satelital SENTINEL de agosto de 2019. Se ₄₉₃ WќќёѦ ѝљюћѡ ђћѐџќюѐѕњђћѡ њюћюєђњђћѡ ₄₉₃ Figure 2. Management of woody plant encroachment in grassland/savannas in Semiarid Chaco. Study area, F. Cantos Experimental Ranch. a) Ancient air photographs (1960). It is appreciated that grassland/savannas (light areas) predominated in lowland ecological sites (red polygons). Study area b) SENTINEL satellite image from August 2019. It is appreciated Figure 2. Management of woody plant encroachment in grassland/savannas in Semiarid Chaco. Study area, F. Cantos Experimental Ranch. a) Ancient air photographs (1960). It is appreciated that grassland/savannas (light areas) predominated in lowland ecological sites (red polygons). b) SENTINEL satellite image from August 2019. It is appreciated that in the central part, grassland/savannas were encroached by woody plants (dark green areas). The yellow polygon contains the Ref_Eco treatment. The light-blue polygon contains the Woody_Encr, Roll_Fire, Roll_Gatton Panic and Roll_Buffel Texas treatments. Figure 2. Management of woody plant encroachment in grassland/savannas in Semiarid Chaco. Study area, F. Cantos Experimental Ranch. a) Ancient air photographs (1960). It is appreciated that grassland/savannas (light areas) predominated in lowland ecological sites (red polygons). b) SENTINEL satellite image from August 2019. It is appreciated that in the central part, grassland/savannas were encroached by woody plants (dark green areas). The yellow polygon contains the Ref_Eco treatment. The light-blue polygon contains the Woody_Encr, Roll_Fire, Roll_Gatton Panic and Roll_Buffel Texas treatments. Figura 2. Manejo de la lignificación en pastizales/sabanas del Chaco Semiárido. Área de estudio, Campo Experimental F. Cantos. a) Mosaico de fotografías aéreas antiguas (1960). Se aprecia que los pastizales/sabana (áreas claras) predominaron en los sitios ecológicos bajos (polígonos rojos). b) Imagen satelital SENTINEL de agosto de 2019. Se aprecia que los pastizales/sabanas del área central se lignificaron (color verde oscuro). El polígono amarillo contiene al tratamiento Ref_Eco. El polígono celeste contiene a los tratamientos Woody_Encr, Roll_Fire, Roll_Gatton Panic y Roll_Buffel Texas. RD Cќџію ђѡ юљ Ecología Austral 33:489-506 ₄₉₄ savanna that received a specific management inducing the sensu lato ecological restoration of the historic native grassland/savanna. Initially, in December 2016 a roller chopping concentrated on shrubs (height ≤3 m) was applied using small machinery (100 hp tractor, roller chopper width=2.5 m and weight ~900 kg). The machine was passed in the same place four times, and the intensity of the disturbance was considered medium since the soil was superficially moved and the shrub cover was almost completely crushed. After this initial disturbance, the herbaceous biomass was left to accumulate during two growing seasons without grazing, and, afterwards, an intense prescribed fire was applied. Prescription was as it follows: average herbaceous biomass >3000 kg DM/ha, air temperature 20-30 °C, wind speed 12-25 km/h, air humidity 25-35% and length flame >3 m (Kunst et al. 2003b). Study area After the fire, grazing exclusion continued on one more growing season; c) Roll_Gatton Panic: it was a woody plant-encroached grassland/ savanna that received a specific management to obtain a new highly productive grassy state through the introduction of the African grass Megathyrsus maximus cv. Gatton Panic (from now on Gatton Panic). This exotic grass is highly productive, shade and fire tolerant and adapts to an annual precipitation >500 mm (Ledesma et al. 2017). In December 2016, a roller chopping was applied with a simultaneous seeding of 12 kg/ha of Gatton Panic seed. This roller chopping had the same features as the one applied in Roll_Fire. However, this seeding failed probably due to the high temperatures and lack of rain during January/February 2017. So, in March 2018 the treatment was roller chopped (two passes at the same place) and 12 kg/ha of Gatton Panic seed were simultaneously seeded again. Grazing was all-time excluded, and d) Roll_Buffel Texas: this treatment had the same features as Roll_Gatton Panic, with the unique difference that the African grass Cenchrus ciliaris cv. Buffel Texas (from now on Buffel Texas) was seeded instead of Gatton Panic. The Buffel Texas is moderately productive, shade intolerant and fire tolerant, growing with an annual precipitation >300 mm (Ledesma et al. 2017). In this experiment, both the roller chopping for herbaceous plants. Livestock grazing suppression had the objective of maximizing the herbaceous biomass productivity. In order to better understand the experiment, Table 1 summarizes the treatments and Supplementary Material-Figure S1 shows photographs of the experiment. Vegetation attributes The following measurements were made in each experimental plot, avoiding the first 2 m from the border. Herbaceous stratum and litter. The initial grass density (it means, prior to the beginning of the experiment) was measured by counting the observed plants in a metallic frame of 0.25 m2. The number obtained was multiplied by four to refer to plants/m2. At the end of the experiment, total and per species herbaceous biomass and litter biomass were obtained by the gravimetric method (collection and drying of materials at 60 °C until constant weight) (Bonham 1989), with four subsamples per experimental plot (the same former metallic frame). The presence-absence of each species was also registered using 10 subsamples per experimental plot (same former metallic frame). The sampling was performed at the end of the growing season (April) when it occurs the maximum expression of herbaceous productivity and diversity (Aronson et al. 1993). Woody stratum. At the end of the experiment, the woody stratum was measured by the modified point quadrat method (Passera et al. 1983). In each experimental plot, two transect with length=15 m each were installed. In each transect, 50 sample points (separated each by 30 cm) were evaluated using a telescopic cane with length=5 m. In each point, it was registered: 1) the number of touches between the cane in vertical position with plant organs, discriminating between shrubs (height ≤3 m) and trees (height >3 m), and 2) the species names and the highest touches (m). Later, the following attributes were obtained: a) total woody cover (touches/transect), counting the total number of touches; b) shrub and tree cover (%), from the percentage of sample points where there were touches with each type of cover; c) woody stratum height (m), averaging the highest touches, and d) woody cover per species (touches/transect), counting the respective touches. In this experiment, both the roller chopping and prescribed fire had the objective of reducing woody plant competition and increasing environmental resource availability (space, sunlight, soil nutrient and water) WќќёѦ ѝљюћѡ ђћѐџќюѐѕњђћѡ њюћюєђњђћѡ ₄₉₅ Shorten name Main characteristics Objective/interpretation Ref_Eco Grassland/savanna in good state. It did not receive any disturbances during the experiment It was the reference ecosystem (Figure 1). It estimated the historic native grassland/ savannas and served as standard of comparison Woody_Encr Grassland/savanna encroached by woody plants. Vegetation attributes It did not receive any disturbances during the experiment To represent the woody state of historic native grassland/savannas (Figure 1) Roll_Fire Woody plant-encroached grassland/savanna that received medium intensity roller chopping, prescribed fire after two growing seasons and grazing suppression during the entire experiment To induce the sensu lato ecological restorati of the grassland/savanna encroached by woody plants (Figure 1, P1) Roll_GaĴon Panic Woody plant-encroached grassland/savanna that received medium intensity roller chopping, seeding of GaĴon Panic grass and grazing suppression during the entire experiment From a grassland/savanna encroached by woody plants, to obtain a new highly productive grassy state by means of the introduction of GaĴon Panic exotic grass (Figure 1, P2) Roll_Buffel Texas Woody plant-encroached grassland/savanna that received medium intensity roller chopping, seeding of Buffel Texas grass and grazing suppression during the entire experiment From a grassland/savanna encroached by woody plants, to obtain a new highly productive grassy state by means of the introduction of Buffel Texas exotic grass (Figure 1, P2) Table 1. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Synthesi treatments. Tabla 1. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Síntesis de tratamientos. To induce the sensu lato ecological restoration of the grassland/savanna encroached by woody plants (Figure 1, P1) From a grassland/savanna encroached by woody plants, to obtain a new highly productive grassy state by means of the introduction of GaĴon Panic exotic grass (Figure 1, P2) From a grassland/savanna encroached by woody plants, to obtain a new highly productive grassy state by means of the introduction of Buffel Texas exotic grass (Figure 1, P2) HFB = PHB + (LPHB x 0.5) Equation 1 HFB = PHB + (LPHB x 0.5) Equation 1 Initial grass density. To assess the initial situation of grass populations (before the beginning of the experiment, P1 y P2), a nonparametric estimation of the initial grass density (plants/m2) and its confidence interval at 95% was performed. Resampling with the replacement bootstrap procedure (n=250) was utilized (Di Rienzo et al. 2013). The estimation obtained was compared with the reference value of 4-5 plants/m2,, where equal or bigger values mean that grass populations are relatively abundant and anticipate quick and effective grass biomass responses to the management (Fumagalli and Kunst 2002). where PHB is the palatable herbaceous biomass (kg DM/ha) and LPHB is the low palatable herbaceous biomass (kg DM/ha) (Kunst et al. 2006). The information of species palatability was obtained from the literature (Morello and Saravia Toledo 1959; Kunst et al. 1986, 1998; Díaz 2015; Ledesma et al. 2017). Significant differences in vegetation structure variables, herbaceous forage biomass and total woody cover (environmental variable), were checked using one factor covariance analysis (factor=treatment), and the Bonferroni statistical for a posteriori tests (α=0.05). Before the analysis, in order to keep normality and homogeneity of residual variances, tree and shrub covers (%) were transformed using the arcsine method (suitable when the variable is a proportion), while the rest of variables were transformed by logarithmic method (Fowler and Cohen 1990). The pH, EC, OM, TN, BD and BWD variables were incorporated to the covariance analysis as co-variables. The INFOSTAT Software 2013 version was utilized (Di Rienzo et al. 2013). Plant composition. To verify modifications in plant composition (P1 and P2), the species presence/absence was utilized to obtain the Chao-Jaccard nonparametric estimator of similarity in broadleaf herb, grass and woody plant composition among pairs of treatments (Colwell 2013). Values were multiplied by 100 to refer to the percentage of similarity. The similarity classes were: 0-39.9% (low), 40-69.9% (moderate) and 70-100% (high). The analyses were performed using the EstimateS 9.10.0 Software (Colwell 2013). Superficial soil quality. To evaluate superficial soil quality (P1 and P2), pH, OM and TN values were interpreted using the standards of Bono et al. (2012), while CE and BD, using the standards of Lorenz (2016). These standards are in Supplementary Material-Table S2. Environmental variables Average fire focuses (AFF) (fire focusses/ year). It is the number of fire focusses per year/total number of years. It was estimated in the period 2003-2019 for both: a) the patch of lowland ecological site covered by grassland/savannas, and b) the patch of lowland ecological site covered by woody plant-encroached grassland/savannas (see ‘Study area’ and ‘Experimental design’). The number of fire focuses was obtained from the ‘Fire Database’ which, by means of images from the Satellite AQUA M-T (Sensor MODIS), forms temporal data series of fire focusses throughout the years (INPE 2020). For each fire focus, the day and time of occurrence and geographic location with an average precision ~400 m, are provided. The detection threshold is determined by fire focuses of 30 m width and 1 m long, or larger. Only those fire focusses, whose location and radius of 400 m were completely inside the studied patches, were considered (arbitrary criterion based on the average precision of locations). Annual precipitation (mm/year). It was obtained for the 2016/2019 period through the meteorological database of F. Cantos Experimental Ranch, which has provided a complete monthly data series since 1988. Annual precipitations were weighted as ‘below normal’, ‘normal’ or ‘above normal’ depending on whether their values correspond respectively to the first, second or third tercile of the data series (SMN 2019). Superficial soil quality. It was evaluated for the first 20 cm of soil, which is the A soil horizon thickness in the study area (Angueira et al. 2007). The following semiarid region relevant indicators were measured (Bono et al. 2012; Lorenz 2016; Taleisnik and Lavado 2017): pH and electric conductivity (EC; dS/ m) (Jackson 1976), organic matter (OM; %) (Norma IRAM- 29571-2-SAGPyA 2011), total nitrogen (TN; %) (Norma IRAM- 29572-1- SAGPyA 2011) and bulk density (BD; kg/dm3) (Lorenz 2016). Per each experimental plot, it was taken: 1) a compound soil sample (four subsamples) in a plastic bag to measure pH, EC, OM and TN, and 2) one soil sample using a metallic cylinder (100 cm3) to measure BD. Total woody cover (touches/transect). This variable was obtained by means of the modified point quadrat method (see ‘Woody stratum’), and it estimates the capacity of the woody cover to intercept sunlight and RD Cќџію ђѡ юљ Ecología Austral 33:489-506 ₄₉₆ decrease the availability of this environmental resource for the herbaceous stratum; more touches, more sunlight interception (Kunst et al. 2006). HFB = PHB + (LPHB x 0.5) Equation 1 To evaluate the participation of exotic and native grasses in Roll_Gatton Panic and Roll_Buffel Texas, the average and standard deviation of grass biomass was obtained grouped according to the species status (i.e., native, naturalized and exotic) (Instituto de Botánica Darwinion 2017). RђѠѢљѡѠ Vegetation structure, utilitarian range condition and total woody cover. The vegetation structure refers to the vegetation aspect or physiognomy. Its modification (P1 and P2) was evaluated through descriptor Environmental variables variables of several vegetal strata: shrub and tree cover (%), woody stratum height, and herbaceous and litter biomass. Changes in pastoral suitability was evaluated using the utilitarian range condition concept (P1 and P2), understood as a description of the resources of the site with respect to livestock grazing (Muir and McClaran 1997). Three condition classes (good, fair and poor) were obtained using the following two valuable attributes for bovine production (Kunst et al. 2006; Díaz 2015): herbaceous forage biomass (kg DM/ha) (food estimator) and shrub cover (%) (accessibility estimator). The herbaceous forage biomass (HFB) was estimated by Burned woody debris biomass (BWD) (t DM/ ha). This variable was estimated only for the treatment that received prescribed fire (Roll_ Fire) by means of the fallen woody material inventory method (Brown 1974), and by deducting the load of woody debris biomass pre- and post-fire. For each experimental plot, the woody debris was evaluated in three transect with length=5 m, prior and posterior to the fire. Environmental variables Annual precipitations. They were considered normal in 2016 (565.9 mm), 2017 (534.5 mm) and 2019 (636.6 mm), and below normal in WќќёѦ ѝљюћѡ ђћѐџќюѐѕњђћѡ њюћюєђњђћѡ ₄₉₇ 2018 (447.8 mm). The precipitation deficit in 2018 was not extreme because the registered value represented 76.3% of the historic annual average (587.1 mm). Therefore, during this study, precipitation availability was considered relatively favorable for herbaceous plant productivity. Average fire focuses. In the lowland ecological site covered by grassland/savannas, between 2003 and 2019, an AFF=1.24 fire focuses/year was obtained, SD=1.89. In the patch of lowland ecological site covered by grassland/savannas encroached by woody plants, and during the studied period, there were no fires (AFF=0 fire focuses/year). Total woody cover. Regarding Ref_Eco, first, this environmental variable was significantly and substantially larger in Woody_Encr, and second, it was not significantly different in Roll_Fire, Roll_Gatton Panic and Roll_Buffel Texas (Figure 3). Figure 3. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Bars graphic (average+SD) for the total woody cover. The result of the covariance analysis is presented. Df=degrees of freedom, F=F statistical, α=0.05. Averages with common letters are not significantly different (Bonferroni Test). Superficial soil quality. Ref_Eco had good superficial soil quality (optimal values of pH, EC, OM and BD, and medium content of TN) (Table 2). Coherently with P1 and P2, Woody_Encr had a good superficial soil quality (optimal values of all indicators) (Table 2). Finally, Roll_Fire, Roll_Gatton Panic and Roll_Buffel Texas maintained the good superficial soil quality after the disturbance application (optimal values of every indicator) (Table 2). See the interpretation of soil quality indicators in Supplementary Material-Table S2. Figura 3. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Gráfico de barra (media+desvío estándar) para la variable cobertura leñosa total. Se presenta el resultado del análisis de la covarianza. Df=grados de libertad, F= estadístico de la prueba F, α=0.05. Medias con letras comunes no son significativamente diferentes (prueba de Bonferroni). rest of the treatments, the variable had value=0 because they were not burned. rest of the treatments, the variable had value=0 because they were not burned. RD Cќџію ђѡ юљ ₄₉₈ P1 and P2, in Roll_Fire, Roll_Gatton Panic and Roll_Buffel Texas the initial grass populations were abundant. Vegetation structure There was a significant effect of the treatment factor (P<0.05) for all vegetation structure were abundant. factor (P<0.05) for all vegetation structur were abundant. factor (P<0.05) for all vegetation structure Figure 4. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Bar graphics (average+SD) for vegetation structural variables. The results of the covariance analysis are presented. Df=degrees of freedom, F=F statistical, α=0.05. Averages with common letters are not significantly different (Bonferroni Test). Figura 4. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Gráficos de barras (media+desvío estándar) para las variables de estructura de la vegetación. Se presentan los resultados de los análisis de covarianza. Df=grados de libertad, F=estadístico de la prueba F, α=0.05. Medias con letras comunes no son significativamente diferentes (prueba de Bonferroni). Figure 4. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Bar graphics (average+SD) for vegetation structural variables. The results of the covariance analysis are presented. Df=degrees of freedom, F=F statistical, α=0.05. Averages with common letters are not significantly different (Bonferroni Test). Figura 4. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Gráficos de barras (media+desvío estándar) para las variables de estructura de la vegetación. Se presentan los resultados de los análisis de covarianza. Df=grados de libertad, F=estadístico de la prueba F, α=0.05. Medias con letras comunes no son significativamente diferentes (prueba de Bonferroni). Figure 4. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Bar graphics (average+SD) for vegetation structural variables. The results of the covariance analysis are presented. Df=degrees of freedom, F=F statistical, α=0.05. Averages with common letters are not significantly different (Bonferroni Test). Figura 4. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Gráficos de barras (media+desvío estándar) para las variables de estructura de la vegetación. Se presentan los resultados de los análisis de covarianza. Df=grados de libertad, F=estadístico de la prueba F, α=0.05. Medias con letras comunes no son significativamente diferentes (prueba de Bonferroni). Figure 4. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Bar graphics (average+SD) for vegetation structural variables. The results of the covariance analysis are presented. Df=degrees of freedom, F=F statistical, α=0.05. Averages with common letters are not significantly different (Bonferroni Test). Figura 4. Experimento en sitios ecológicos bajos, Campo Experimental F. Initial grass density Burned woody debris biomass. In Roll_Fire, the average and standard deviation of BWD were 21.2 and 15.3 t/ha, respectively. For the In all treatments, the estimated average of the initial grass density was above the reference value of 4-5 plants/m2 (Table 3). So, regarding Table 2. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Superficial soil quality indicators (average+SD) and interpretation according to Bono et al. (2012) and Lorenz (2016) standards. Tabla 2. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Indicadores de calidad del suelo superficial (media+desvío estándar) e interpretación según los estándares de Bono et al. (2012) y Lorenz (2016). Treatment EC (dS/m) pH OM (%) TN (%) BD (kg/dm3) Ref_Eco 0.31±0.07 (not saline, optimal) 6.9±0.08 (optimal) 2.33±0.52 (high, optimal) 0.1±0.02 (medium, sub-optimal) 1.12±0.1 (very low, optimal) Woody_Encr 0.42±0.13 (not saline, optimal) 7.03±0.22 (optimal) 2.81±0.71 (high, optimal) 0.14±0.03 (high, optimal) 0.94±0.09 (very low, optimal) Roll_Fire 0.57±0.09 (not saline, optimal) 7.31±0.2 (optimal) 3.15±0.84 (high, optimal) 0.15±0.03 (high, optimal) 0.98±0.13 (very low, optimal) Roll_GaĴon Panic 0.38±0.09 (not saline, optimal) 7.14±0.2 (optimal) 3.62±1.24 (high, optimal) 0.17±0.05 (high, optimal) 0.98±0.11 (very low, optimal) Roll_Buffel Texas 0.5±0.18 (not saline, optimal) 7.24±0.22 (optimal) 3.41±1.03 (high, optimal) 0.17±0.03 (high, optimal) 0.98±0.1 (very low, optimal) Ecología Austral 33:489-506 RD Cќџію ђѡ юљ Species composition The Chao-Jaccard estimator shows that, regarding the rest of treatments, Ref_Eco had the lowest similarity in broadleaf herb, grass and woody plant composition (Table 4, Table 5 and Table 6). So, Ref_Eco had the most singular species of plant community. Between Ref_Eco and Woody_Encr, the similarities in woody plant, broadleaf herb and grass composition were, moderate, moderate and low, respectively. The species lists are in Supplementary Material 3-Table S3 and Supplementary Material 4-Table S4. Treatment Estimated average (plants/m2) Lower bound (95%) Upper bound (95%) Ref_Eco 27.1 20.8 33.7 Woody_Encr 9.5 6.6 12.8 Roll_Fire 9.7 7.2 11.9 Roll_GaĴon Panic 9.3 7.9 10.7 Roll_Buffel Texas 7.8 6.4 9.4 variables (Figure 4). In Woody_Encr regarding Ref_Eco, first, the shrub and tree cover, woody stratum height and litter biomass were significantly larger, and second, the herbaceous biomass was significantly lower (Figure 4). Coherently with P1 and P2, in Roll_Fire, Roll_Gatton Panic and Roll_Buffel The grass biomass results grouped according to species status are in Table 7. The most part in Ref_Eco corresponded to native species (78.5%), but a noticeable part corresponded to a naturalized species (21.5%, Heteropogon contortus). In Woody_Encr, Table 4. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Similarity in broadleaf herb composition obtained by Chao-Jaccard estimator. Tabla 4. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Porcentajes similitud en la composición de hierbas de hoja ancha obtenidas a partir del estimador Chao-Jaccard. 4. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Porcentajes de ud en la composición de hierbas de hoja ancha obtenidas a partir del estimador Chao-Jaccard. Treatment Treatment Similarity (%) Weighting Ref_Eco Roll_Fire 49.2 Moderate Ref_Eco Roll_Buffel Texas 49.6 Moderate Ref_Eco Roll_GaĴon Panic 57.1 Moderate Ref_Eco Woody_Encr 64 Moderate Roll_Buffel Texas Woody_Encr 88.2 High Roll_Fire Woody_Encr 93 High Roll_Buffel Texas Roll_GaĴon Panic 93.2 High Roll_GaĴon Panic Woody_Encr 95.7 High Roll_Buffel Texas Roll_Fire 99.2 High Roll_GaĴon Panic Roll_Fire 100 High similitud en la composición de hierbas de hoja ancha obtenidas a partir del estimador Chao Jaccard. Table 5. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Similarity in gra composition obtained by Chao-Jaccard estimator. p y Tabla 5. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Porcentajes similitud en la composición gramíneas obtenidas a partir del estimador Chao-Jaccard. RD Cќџію ђѡ юљ Cantos, Chaco Semiárido. Gráficos de barras (media+desvío estándar) para las variables de estructura de la vegetación. Se presentan los resultados de los análisis de covarianza. Df=grados de libertad, F=estadístico de la prueba F, α=0.05. Medias con letras comunes no son significativamente diferentes (prueba de Bonferroni). WќќёѦ ѝљюћѡ ђћѐџќюѐѕњђћѡ њюћюєђњђћѡ ₄₉₉ Texas all vegetation structure variables did not significantly differ from Eco_Ref (a new grassy state was achieved) (Figure 4). Table 3. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Estimated average and lower and upper bounds (95%) of the initial grass density. Tabla 3. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Media estimada y limites inferior y superior (95%) de la densidad inicial de gramíneas. Table 4. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Similarity in broadleaf herb composition obtained by Chao-Jaccard estimator. Species composition The result of the covariance analysis is presented. Df=degrees of freedom, F=F statistical, α=0.05. Averages with common letters are not significantly different (Bonferroni Test). Figura 5. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Gráfico de barra (media+desvío estándar) para la forrajimasa herbácea. Se presenta el resultado del análisis de la covarianza. DF=grados de libertad, F=estadístico de la prueba F, α=0.05. Medias con letras comunes no son significativamente diferentes (prueba de Bonferroni). Table 7. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Grass biomass according to the species status (average+SD). Tabla 7. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Biomasa de gramíneas según el status de las especies (media+desvío estándar). Treatment Exotic average SD Native average SD Naturalized average SD Eco_Ref 0.0 0.0 5446.3 3312.8 1487.5 1410.3 Woody_Encr 0.0 0.0 1835.9 2049.4 0.0 0.0 Roll_Fire 8.8 34.8 3764.3 2550.9 0.0 0.0 Roll_Buffel Texas 2444.8 3209.2 4327.5 3642.5 0.0 0.0 Roll_GaĴon Panic 6685.8 9656.7 3177.9 2837.1 0.0 0.0 Tabla 7. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Biomasa de gramíne según el status de las especies (media+desvío estándar). the 100% corresponded to native species. Consistently with P1 and P2, 1) in Roll_Fire, 99.76% corresponded to native species and a negligible part to an exotic species (0.24%, Eragrostis cilianensis); 2) Roll_Gatton Panic and Roll_Buffel Texas were unique treatments with appreciable participation of exotic grasses (in the first case, 100% corresponded to Gatton Panic and, in the second one, 99.94% corresponded to Buffel Texas), and 3) in both Roll_Gatton Panic and Roll_Buffel Texas there was also a high participation of native grasses (average >3000 kg DM/ha). Species composition Treatment Treatment Similarity (%) Weighting Ref_Eco Roll_GaĴon Panic 17.9 Low Ref_Eco Woody_Encr 27.9 Low Ref_Eco Roll_Fire 33.7 Low Ref_Eco Roll_Buffel Texas 44.2 Moderate Roll_GaĴon Panic Roll_Fire 66.2 Moderate Roll_GaĴon Panic Woody_Encr 67.8 Moderate Roll_Buffel Texas Roll_GaĴon Panic 72.1 High Roll_Buffel Texas Woody_Encr 75.2 High Roll_Fire Woody_Encr 78.6 High Roll_Buffel Texas Roll_Fire 93.8 High p g p J Ecología Austral 33:489-506 Ecología Austral 33:489-506 RD Cќџію ђѡ юљ ₅₀₀ Table 6. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Similarity in wood plant composition obtained by Chao-Jaccard estimator. 6. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Similarity in woody omposition obtained by Chao-Jaccard estimator. bla 6. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Porcentajes de militud en la composición plantas leñosas obtenidos a partir del estimador Chao-Jaccard. Treatment Treatment Similarity (%) Weighting Ref_Eco Roll_GaĴon Panic 37 Low Ref_Eco Roll_Fire 46 Moderate Ref_Eco Roll_Buffel Texas 52 Moderate Ref_Eco Woody_Encr 65 Moderate Roll_GaĴon Panic Woody_Encr 82 High Roll_GaĴon Panic Roll_Fire 85 High Roll_Buffel Texas Woody_Encr 88 High Roll_Fire Woody_Encr 88 High Roll_Buffel Texas Roll_Fire 95 High Roll_Buffel Texas Roll_GaĴon Panic 99 High ble 7. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Grass biomass cording to the species status (average+SD). the 100% corresponded to native species. Consistently with P1 and P2, 1) in Roll_Fire, 99.76% corresponded to native species and a negligible part to an exotic species (0.24%, Eragrostis cilianensis); 2) Roll_Gatton Panic and Roll_Buffel Texas were unique treatments with appreciable participation of exotic grasses (in the first case, 100% corresponded to Gatton Panic and, in the second one, 99.94% corresponded to Buffel Texas), and 3) in both Roll_Gatton Panic and Roll_Buffel Texas there was also a high participation of native grasses (average >3000 kg DM/ha). Utilitarian range condition Ref_Eco had low shrub cover (Figure 4b) and high herbaceous forage biomass (Figure 5), which justify its utilitarian range conditions. In Woody_Encr regarding Ref_Eco, the shrub cover was significantly higher (91.2%) (Figure 4b) and the herbaceous forage biomass was significantly lower (Figure 5). Despite that, in Woody_Encr, the herbaceous forage biomass was moderate (1817.5 kg DM/ha). Woody_ Encr is classified as in poor utilitarian range condition, being its excessive shrub cover its main limitation. Regarding Ref_Eco, in Figure 5. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Bars graphic (average+SD) for the herbaceous forage biomass. The reference ecosystem In this study, several facts reflected that the reference ecosystem utilized (Ref_Eco) represented a well conserved grassland/ savanna and in consequence an acceptable standard of comparison. On one hand, between 2003 and 2019, the patch that contained Ref_Eco had periodic fires (AFF=1.24 fire focuses/year). Additionally, for this same patch, between 1925 and 1996, by means of dendrochronological methods, an average fire frequency of 0.18 fires/year was determined (Bravo et al. 2010). As a consequence, the available data indicate that for the most part between 1925 and 2019 the patch containing Ref_Eco had recurrent fires. This would have maintained the grassy state over time and favored grassland/savanna adapted plants (shade intolerant and fire tolerant) (Ripley et al. 2015; Archer et al. 2017; Coria et al. 2021). Coherently with the former, in Ref_Eco, the three most abundant grasses were shade intolerant and fire tolerant (Elionurus muticus, Heteropogon contortus and Pappophorum pappiferum) (Kunst et al. 1998) and they represented 83.3% of the total herbaceous biomass . On the other hand, in Ref_Eco, the good superficial soil quality, the high herbaceous forage biomass (4959.4 kg DM/ ha) and the good utilitarian range condition would be the results of a low historical grazing intensity (Abril and Bucher 1999; Bestelmeyer et al. 2003; Díaz 2015; Coria et al. 2021). The herbaceous forage biomass even surpassed other grassland/savannas in good utilitarian range condition from nearby areas (1789-3071 kg DM/ha) (Kunst et al. 2006). The low similarity in grass composition between Woody_Encr and Ref_Eco could be due to a prevalence of grasses adapted to shrubland/forests in the first (Digitaria californica, Digitaria insularis, Gouinia latifolia, Gouinia paraguayensis, Setaria lachnea, Setaria sp. and Leptochloa pluriflora represented the 96.2% of grass biomass); while in the second, grasses adapted to grassland/savannas prevailed (see first paragraph of this discussion). It is remarkable that in the patch where Woody_Encr was located, evaluations of the herbaceous stratum done between 1983 and 1988 (i.e., previous to woody plant encroachment) show an herbaceous plant composition adapted to grassland/savannas, where the Elionurus muticus grass was the most abundant species (average cover=67.3%; SD=36.5%) (unpublished data). Globally, this evidence suggests that woody plant encroachment would have promoted the replacement of grassland/savanna adapted grasses by other adapted to shrubland/forests (i.e., shade tolerant) (Archer et al. 2017; Coria et al. 2021). The former analysis also highlights the importance of alterations in fire regimes and sunlight availability on grass composition. The grassland/savanna encroached by woody plants The grassland/savanna encroached by woody plants The grassland/savanna encroached by woody plants Roll_Fire, Roll_Gatton Panic and Roll_Buffel Texas, the shrub cover and the herbaceous forage biomass did not differ significantly (Figure 4b and Figure 5). Therefore, the last three treatments were classified as in a good utilitarian range condition, which agrees with P1 and P2. Despite the lack of significant differences, the very high herbaceous forage biomass average in Roll_Gatton Panic is notable (10213.7 kg DM/ha). For example, it was a little more than twice the average of Roll_Fire (Figure 5). However, at the same time, Roll_Gatton Panic had an herbaceous forage biomass variability (SD=5011.9 kg DM/ha) far above the rest of treatments. Roll_Fire, Roll_Gatton Panic and Roll_Buffel Texas, the shrub cover and the herbaceous forage biomass did not differ significantly (Figure 4b and Figure 5). Therefore, the last three treatments were classified as in a good utilitarian range condition, which agrees with P1 and P2. Despite the lack of significant differences, the very high herbaceous forage biomass average in Roll_Gatton Panic is notable (10213.7 kg DM/ha). For example, it was a little more than twice the average of Roll_Fire (Figure 5). However, at the same time, Roll_Gatton Panic had an herbaceous forage biomass variability (SD=5011.9 kg DM/ha) far above the rest of treatments. The results suggested that the encroachment of woody plants substantially altered the characteristics of grassland/savanna vegetation. In Woody_Encr compared with Ref_Eco, 1) the woody stratum height, shrub and tree cover and litter biomass were significantly higher (1340.0%, 305.6%, 4350.0% and 511.6%, respectively); 2) the herbaceous biomass was significantly lower (69.5%); 3) the similarity in woody plant, broadleaf herb and grass composition were respectively moderate (65%), moderate (64%) and low (27.9%), and 4) the utilitarian range condition was poor. However, in Woody_Encr, the moderate changes in woody plant composition were unexpected, because in a previous study this attribute was little affected by woody plant encroachment (Coria et al. 2021). Utilitarian range condition Ref_Eco had low shrub cover (Figure 4b) and high herbaceous forage biomass (Figure 5), which justify its utilitarian range conditions. In Woody_Encr regarding Ref_Eco, the shrub cover was significantly higher (91.2%) (Figure 4b) and the herbaceous forage biomass was significantly lower (Figure 5). Despite that, in Woody_Encr, the herbaceous forage biomass was moderate (1817.5 kg DM/ha). Woody_ Encr is classified as in poor utilitarian range condition, being its excessive shrub cover its main limitation. Regarding Ref_Eco, in Figure 5. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Bars graphic (average+SD) for the herbaceous forage biomass. The result of the covariance analysis is presented. Df=degrees of freedom, F=F statistical, α=0.05. Averages with common letters are not significantly different (Bonferroni Test). Figure 5. Experiment in lowland ecological sites, F. Cantos Experimental Ranch, Semiarid Chaco. Bars graphic (average+SD) for the herbaceous forage biomass. The result of the covariance analysis is presented. Df=degrees of freedom, F=F statistical, α=0.05. Averages with common letters are not significantly different (Bonferroni Test). Figura 5. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Gráfico de barra (media+desvío estándar) para la forrajimasa herbácea. Se presenta el resultado del análisis de la covarianza. DF=grados de libertad, F=estadístico de la prueba F, α=0.05. Medias con letras comunes no son significativamente diferentes (prueba de Bonferroni). Figura 5. Experimento en sitios ecológicos bajos, Campo Experimental F. Cantos, Chaco Semiárido. Gráfico de barra (media+desvío estándar) para la forrajimasa herbácea. Se presenta el resultado del análisis de la covarianza. DF=grados de libertad, F=estadístico de la prueba F, α=0.05. Medias con letras comunes no son significativamente diferentes (prueba de Bonferroni). WќќёѦ ѝљюћѡ ђћѐџќюѐѕњђћѡ њюћюєђњђћѡ ₅₀₁ The reference ecosystem Normally, woody plant encroachment reduces or suppresses fire events and increases woody cover generating shaded habitats. In this study, the AFF analysis showed that in the patch where Woody_Encr was located there were no fires between 2003 and 2019. Kunst et al. (2012) reported that in this patch, the last RD Cќџію ђѡ юљ Ecología Austral 33:489-506 Ecología Austral 33:489-506 ₅₀₂ fire occurred in the late 1990s. Additionally, compared with Ref_Eco, in Woody_Encr, the total woody cover (touches/transect) significantly higher (1002.5%) suggests the greater capacity of the last treatment to reduce the sunlight availability for grasses (Kunst et al. 2006). The results also suggest that the short-term management applied in Roll_Fire would not restore the plant composition of the encroached ecosystems. This is due to, between Ref_Eco and Roll_Fire, the similarities in grass, broadleaf herb and woody plant composition that were low (33.7%), moderate (49.2%) and moderate (46%), respectively. We believe that to consider these attributes reasonably restored, these similarities should be high (≥70%). Very noticeable and coherent with P1 and P2, the results suggested that, in this specific study, woody plant encroachment did not affect superficial soil quality (optimal values of all soil quality indicators in Woody_Encr) and did not finish native grass populations (initial grass density in Woody_Encr=9.5 plants/m2); which would determine a high potential of sensu lato ecological restoration of the encroached ecosystems. In Woody_Encr, this favorable results would be because, on one hand, the historical overgrazing was not extreme (Morello and Saravia Toledo 1959; Adamoli et al. 1972; Díaz 2015) and, on the other hand, the high woody cover and litter biomass had a beneficial effect on physical, chemical and biologic soil properties (Anriquez and Albanesi 2008; Anriquez et al. 2008). It is interpreted that in Roll_Fire, the grasses that responded to the management were essentially the same shrubland/forest adapted species present in the predecessor encroached ecosystem. It is supported by 1) the prevalence of shrubland/forest adapted grasses in Roll_Fire (Gouinia latifolia, Setaria lachnea and Leptochloa plutiflora represented the 75.5% of the grass biomass), and 2) the high similarity in grass composition between Roll_ Fire and Woody_Encr (78.6%). In Roll_Fire, the high achieved fine fuel load (>3000 kg DM/ha) suggests the feasibility of reconstructing the grassland/savanna normal fire regime (an event each three years, approximately) (Bravo et al. 2010). The reference ecosystem Future studies could check if with long-term practices like these, the abundance of grassland/savanna adapted grasses is increased. Introduction of African grasses to grassland/ savannas encroached by woody plants In the Americas, the C4 African grasses introduced to improve ranching have often expanded and colonized natural areas at great speed, with effects in the ecosystem such as the increase of fire frequency, alterations in energy, nutrients and water fluxes, and the displacement of native species. (D’Antonio and Vitousek 1992; Williams and Baruch 2000; Marshall et al. 2012). In this study, the productivity of Gatton Panic (7044.3 kg DM/ha) and Buffel Texas (2444.8 kg DM/ha) was within the expected values for Semiarid Chaco (4300-8600 and 1500-4500 kg DM/ha, respectively) (De León 1998; Avila et al. 2014). Coherently with P2, a) in both Roll_Gatton Panic and Roll_Buffel Texas there was also a high participation of native grasses (3177.9 and 4327.5 kg DM/ha, respectively), then, there were truly mixes between native and exotic grasses, and b) both treatments achieved a good utilitarian range condition. The results also suggested that in these encroached ecosystems and with a mostly normal annual precipitation, 1) the combined use of medium intensity roller chopping, grazing suppression, and prescribed fire after two growing seasons would produce a new gassy state, where a) both the vegetation structure and good utilitarian range condition would be restored; b) the plant composition would not be restored, especially grasses; c) the good superficial soil quality would be kept, and d) a normal fire regime could be restored by means of prescribed fires. Therefore, this management would be capable of reasonably inducing a sensu lato ecological restoration of encroached ecosystems, and 2) the combined use of medium intensity roller chopping, grazing suppression and seeding of the African grasses Gatton Panic and Buffel Texas would produce new grassy states integrated by exotic and native grasses and in good utilitarian range condition. These new ecosystems would be friendly to both the superficial soil quality and the native plant diversity of the encroached ecosystems. However, this appreciation is valid only for this study and should be taken as preliminary because a precise valuation requires more long-term complex studies. Surprisingly, both Roll_Gatton Panic and Roll_Buffel Texas were quite similar to the treatment designed to induce the encroached ecosystem restoration (i.e., Roll_Fire). Among these treatments, all variables of vegetation structure and the herbaceous forage biomass did not differ significantly, similarities in plant species composition were high in most of the cases and all indicators of superficial soil quality had optimal values. Sensu lato ecological restoration of the grassland/ savanna encroached by woody plants Consistently with P1, in Roll_Fire all vegetation structure variables did not differ significantly from Ref_Eco, suggesting that the management applied was able to restore the grassy state of the encroached ecosystem. This new grassy state would be explained by a conjunction of environmental and management factors. In first place, the high initial grass density (9.7 plants/m2 in Roll_Fire), the good superficial soil quality in the encroached ecosystem, and the annual precipitation mostly normal, would have determined a great capacity of recovery of the herbaceous stratum. Later, the medium intensity roller chopping used would have been effective in reducing woody plant competition and, together with grazing suppression, would have allowed herbaceous plants to substantially increase their biomass (Kunst et al. 2012; Díaz 2015). During two growing seasons, the fine fuel accumulated allowed to applicate the prescribed fire, which reduced even more the competition of woody plants and benefit again herbaceous plants (Allen 2008; Scott et al. 2013). As a final result, in Roll_Fire the balance between woody and herbaceous plants was modified in favor of the latter. Consistent with P1, between Ref_Eco and Roll_Fire there were no significant differences in shrub cover and herbaceous forage biomass, which justified a good utilitarian range condition in Roll_Fire. The high herbaceous forage biomass achieved in Roll_Fire (4010.6 kg DM/ha), representing 80.9% of the herbaceous forage biomass of Ref_Eco, is remarkable. So, the management applied would be beneficial for ranching. In Roll_Fire, it is highlighted that the applied management did not affect the superficial soil quality (optimal values of all soil quality indicators). However, the soil quality indicator types utilized (chemical and physical) often respond slowly to disturbances. Therefore, a long term monitoring on them would be appropriate (Abril 2003; Cardoso et al. 2013; Bünemann et al. 2018). As a global appreciation, the management applied in Roll_Fire was able to restore almost all ecosystem attributes to Ref_Eco levels, with the exception of plant composition (especially of grasses). Thus, in the context of this study, this management would have the potential WќќёѦ ѝљюћѡ ђћѐџќюѐѕњђћѡ њюћюєђњђћѡ ₅₀₃ to reasonably induce a sensu lato ecological restoration of the encroached ecosystems. important changes in vegetation structure and plant composition (in native grasses, there was a replacement of species adapted to grassland/savannas by other adapted to shrubland/forests), and it impoverished the utilitarian range condition. Introduction of African grasses to grassland/ savannas encroached by woody plants This suggests that the introduction of African grasses was friendly with both the superficial soil and native plant diversity of the encroached ecosystems. However, this appreciation is valid only for this study and it should be taken as preliminary because a precise valuation requires more long-term complex studies. On the other hand, due to the good utilitarian range condition achieved in Roll_Fire, the introduction of African grasses to improve ranching would not have been necessary. In Dry Chaco, it was emphasized that the utilization of African grasses must be restricted to the most degraded areas, due to the profitability of well managed natural grasslands (Díaz 2015). Globally, predictions 1 and 2 were supported, thus the hypothesis as well. Regarding management implications, in the studied encroached ecosystems, the sensu lato ecological restoration would be a viable ecological and productive alternative. For that reason, in similar cases, the introduction of African grasses to enhance ranching should be carefully evaluated because it might not be necessary. AѐјћќѤљђёєђњђћѡѠ . We thank: 1) V. Navarrete and J. Godoy (INTA EEA Santiago del Estero) for their help assembling the field experiment; 2) H. Van Meer and N. Dominguez (INTA EEA Santiago del Estero) Sensu lato ecological restoration of the grassland/ savanna encroached by woody plants However, the phenomenon would not have negatively affected the superficial soil quality nor finished the native grass populations, which would have determined a high potential of sensu lato ecological restoration of the encroached ecosystems. 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https://openalex.org/W2115390111	https://www.scielo.br/j/aabc/a/qrz8dpnD8s6Tc8XqgdYhjYk/?lang=en&format=pdf	English	null	Stratigraphy of amethyst geode-bearing lavas and fault-block structures of the Entre Rios mining district, Paraná volcanic province, southern Brazil	Anais da Academia Brasileira de Ciências	2,013	cc-by	6,335	ABSTRACT The Entre Rios mining district produces a large volume of amethyst geodes in underground mines and is part of the world class deposits in the Paraná volcanic province of South America. Two producing basalt flows are numbered 4 and 5 in the lava stratigraphy. A total of seven basalt flows and one rhyodacite flow are present in the district. At the base of the stratigraphy, beginning at the Chapecó river bed, two basalt flows are Esmeralda, low-Ti type. The third flow in the sequence is a rhyodacite, Chapecó type, Guarapuava subtype. Above the rhyodacite flow, four basalt flows are Pitanga, high-Ti type including the two mineralized flows; only the topmost basalt in the stratigraphy is a Paranapanema, intermediate-Ti type. Each individual flow is uniquely identified from its geochemical and gamma-spectrometric properties. The study of several sections in the district allowed for the identification of a fault-block structure. Blocks are elongated NW and the block on the west side of the fault was downthrown. This important structural characterization of the mining district will have significant consequences in the search for new amethyst geode deposits and in the understanding of the evolution of the Paraná volcanic province. Key words: amethyst geode, Entre Rios mining district, Paraná volcanic province, geochemistry, gamma-spectrometry Key words: amethyst geode, Entre Rios mining district, Paraná volcanic province, geochemistry, gamma-spectrometry. Stratigraphy of amethyst geode-bearing lavas and fault-block structures of the Entre Rios mining district, Paraná volcanic province, southern Brazil LÉO A. HARTMANN, LUCAS M. ANTUNES and LEONARDO M. ROSENSTENGEL Instituto de Geociências, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Bairro Agronomia, 91501-970 Porto Alegre, RS, Brasil Instituto de Geociências, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Bairro Agronomia, 91501-970 Porto Alegre, RS, Brasil Manuscript received on November 27, 2012, accepted for publication on October 8, 2013 Manuscript received on November 27, 2012, accepted for publication on October 8, 2013 Correspondence to: Léo Afraneo Hartmann E-mail: leo.hartmann@ufrgs.br Anais da Academia Brasileira de Ciências (2014) 86(1): (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 www.scielo.br/aabc http://dx.doi.org/10.1590/0001-37652014201120004 187-198 INTRODUCTION new deposits. Only preliminary observations were previously made on the district (Juchem et al. 2010), so this is a novel contribution to the geology of the Entre Rios mining district with significant implications for the entire volcanic province. The large-scale amethyst geode production (600 ton/month) from mines in the Paraná volcanic province, mostly Ametista do Sul (Brazil) and Los Catalanes (Uruguay) makes the understanding of the structure of mining districts a major topic in volcanic geology. The Entre Rios mining district in Santa Catarina, Brazil, has a large, unmeasured production of amethyst geodes. The establishment of lava stratigraphy and overall structure of the district is most significant for the discovery of The Paraná volcanic province is a major geological unit (Fig. 1) in southeastern South America (917,000 km2; Frank et al. 2009) and is composed of many (possibly 120) lava flows in each stratigraphic section. The rocks have varied bimodal compositions, mostly basalt and rhyodacite (e.g., Bellieni et al. 1984, Hartmann et al. 2010a, b) and are included in the Serra Geral An Acad Bras Cienc (2014) 86 (1) LÉO A. HARTMANN, LUCAS M. ANTUNES AND LEONARDO M. ROSENSTENGEL 188 Group. The volcanic group is near the top of the Paraná basin (Zalán et al. 1991) and the age of the rocks is close to 135 Ma (e.g., Pinto et al. 2011, Janasi et al. 2011). the sandstones, e.g. Ametista do Sul mining district, Brazil. The Entre Rios mining district (Fig. 2) is also approximately 1,000 m above the contact with the paleo-erg. The district is located in a transitional region between low-Ti basalt flows in the south and high-Ti flows in the north of the Paraná volcanic province (Bellieni et al. 1984, Peate et al. 1992). It is in the depocenter region of the Paraná basin (e.g., Arioli et al. 2008). Figure 1 - Regional distribution of the Paraná volcanic province; location of Entre Rios town indicated. Figure 2 - Satellite image of the Entre Rios mining district. Location of geological section (AB) and analyzed samples shown. Figure 2 - Satellite image of the Entre Rios mining district. Location of geological section (AB) and analyzed samples shown. The Serra Geral Group differs from other continental flood basalt provinces due to its position above the Guarani aquifer (Araújo et al. 1999), which is contained in the Botucatu Formation. An Acad Bras Cienc (2014) 86 (1) INTRODUCTION The fluid supersaturated in silica that caused the hydrotermal alteration, the formation of the silica gossan and the opening and filling of amethyst geodes, originated in this aquifer (Duarte et al. 2009, Hartmann et al. 2010a, b). Figure 1 - Regional distribution of the Paraná volcanic province; location of Entre Rios town indicated. Previous studies in mineralized regions of the Paraná volcanic province led initially to the understanding that the protogeodes were formed by degassing of the basaltic lava at high temperature (1,150°C), e.g. Gilg et al. (2003), Proust and Fontaine (2007) and Morteani et al. (2010). The observation that the basalts attained brittle conditions (130°C) and were intensely altered to smectite and zeolites before Underground mining of amethyst and agate geodes occurs in basal andesite and basaltic andesite flows near the contact with the underlying Botucatu Formation eolian sandstones, e.g. Los Catalanes gemological district, Uruguay, but also in basalts situated 1,000 m above the contact with An Acad Bras Cienc (2014) 86 (1) AMETHYST-BEARING LAVAS FROM ENTRE RIOS, PARANÁ PROVINCE 189 Serra Geral Group is transitional between low-Ti basaltic flows in the south and the high-Ti flows in the northern portion of the group (Peate et al. 1992). The region has an elevation of up to 740 m above sea level (a.s.l.), with the base of the studied stratigraphy at 389 m along the Chapecó River bed. Only recently has the fault-block structure of the Ametista do Sul mining district (100 km to the south of Entre Rios) been recognized (Rosenstengel and Hartmann 2012). The investigation of the structure of mining districts and other portions of the Paraná volcanic province is most significant for the understanding of the processes responsible for the opening of the South Atlantic Ocean. the protogeode formation led to a more accurate hypothesis of low-temperature, epigenetic origin of the cavities (Duarte et al. 2009, 2011, Hartmann et al. 2012a, b, c), also supported by Commin-Fischer et al. (2010). The fluid that formed silica minerals in the geodes originated in the Guarani aquifer (Gilg et al. 2003). Only low-temperature (commonly ˂80°C) minerals are present in the geodes, as shown by oxygen isotopes (Juchem et al. 2010, Morteani et al. 2010, Duarte et al. 2011) and no epidote, chlorite or amphibole are known in the basaltic province, so higher temperature than 130°C should not be considered for the alteration and mineralization. LÉO A. HARTMANN, LUCAS M. ANTUNES AND LEONARDO M. ROSENSTENGEL 190 We presently report field mapping, rock geo chemistry and gamma-spectrometric emission rates of rocks and integrate this evidence to determine the local stratigraphy and overall district structure. The Entre Rios district in the state of Santa Catarina (Fig. 1) offers a rare opportunity for the study of the varied processes related to volcanism, hydrothermal alteration, sand injection, geode mineralization, lava stratigraphy and block-faulting in a context of a major continental flood basalt province. study to determine the geological relationships of the lava flows, the amethyst geode mineralization and the faulting of the rocks in the district. In addition, the gamma-ray emission rate of the outcrops was measured with a portable gamma-ray spectrometer (Exploranium model GR110). Rock samples (n = 21) were collected (location in Fig. 2) for petrographic study and analyzed for their chemical composition in the ACME laboratory, Canada, using methods for groups 4A and 4B. MATERIALS AND METHODS Previous investigations of mineralized and barren portions of the Paraná volcanic province were initially revised (e.g., Peate et al. 1992, Nardy et al. 2008, Duarte et al. 2009, 2011, Hartmann et al. 2010a, b, 2012a, b, c, d, Pinto and Hartmann et al. 2011, Pertille et al. 2013, Rosenstengel and Hartmann 2012). Field work followed satellite image The integrated use of field relationships, geochemistry and gamma-spectrometry led to the identification of the lava flows present, their stratigraphy and the overall fault-block structure of the mining district (Table I). Field and petro graphic characterization support the proposed stratigraphic section. INTRODUCTION Volcanic rocks in the Entre Rios district are nearly horizontal, so the presence of vertical faulting is commonly overlooked. Evidence of large faults is readily seen in satellite images, but these are usually considered transcurrent, similar to the eastern border of the volcanic province (e.g. Jacques et al. 2010). The district of Entre Rios is located in the western region of the state of Santa Catarina, where the Sand injectites are observed in the Serra Geral Group, both in amethyst and agate geode- mineralized and barren portions (Hartmann et al. 2012c, d). In spite of extensive weathering and soil cover of the studied region, silicified sand dikes in basalt and massive sandstones inside geodes are observed in the Entre Rios mining district (Fig. 3). Figure 3 - Photomicrographs and field photos of the eight studied lava flows in the Entre Rios mining district. (a): flow 1; (b) and (c): flow 2; (d), (e) and (f): flow 3; (g): flow 4; (h): flow 5; (i): flow 6 (j): flow 7; (k): microphenocrysts of magnetite in flow 8; (l): satellite image of top of flow 8; arrows indicate gossans; (m): silicified basalt on top of flow 8; (n), (o): silicified sandstone in geodes of flow 5, showing a sequence of chalcedony, fine quartz, coarse quartz and amethyst; (p): sand dikes in flow 5 evolving to chalcedony along fracture. Symbols: b = basalt, c – chalcedony, s = silicified sandstone, q = quartz, am = amethyst. Figure 3 - Photomicrographs and field photos of the eight studied lava flows in the Entre Rios mining district. (a): flow 1; (b) and (c): flow 2; (d), (e) and (f): flow 3; (g): flow 4; (h): flow 5; (i): flow 6 (j): flow 7; (k): microphenocrysts of magnetite in flow 8; (l): satellite image of top of flow 8; arrows indicate gossans; (m): silicified basalt on top of flow 8; (n), (o): silicified sandstone in geodes of flow 5, showing a sequence of chalcedony, fine quartz, coarse quartz and amethyst; (p): sand dikes in flow 5 evolving to chalcedony along fracture. Symbols: b = basalt, c – chalcedony, s = silicified sandstone, q = quartz, am = amethyst. An Acad Bras Cienc (2014) 86 (1) LÉO A. HARTMANN, LUCAS M. ANTUNES AND LEONARDO M. ROSENSTENGEL AMETHYST-BEARING LAVAS FROM ENTRE RIOS, PARANÁ PROVINCE 191 The geochemical analysis of rocks was an essential tool for the characterization of lava flows in the mining district of Entre Rios (Fig. 4), because each flow has a characteristic concentration of major, minor and trace elements, mainly TiO2, P2O5, MgO, K2O, Zr, U, Nb, Sr, Rb, Th, and Y (Fig. 5). The geochemical composition indicates the presence of varied magma types in the district and an overall fault- block structure. Three contrasting basalt magma types (Peate et al. 1992) are present in the mining district, the low-Ti Esmeralda, the high-Ti Pitanga and the intermediate-Ti Paranapanema types, in addition to a Chapecó-type (Guarapuava subtype, Nardy et al. 2008) rhyodacite (SiO2 ~64.5 wt.%). The overall stratigraphy includes eight flows with individual flow thicknesses estimated at 25-60 m. Basalt compositions in the seven flows range from SiO2 = 44.35 to 51.63 wt.%. The amethyst geode mineralization occurs in two Pitanga-type flows (numbered 4 and 5; see Table II). Location and elevation of each analyzed sample are given in Table III. probably a result of hydrothermal alteration (LOI >5 wt.%). It has the highest values of TiO2, Sr and V, and the lowest K2O and Rb. Flow 5 (also mineralized) is enriched in some elements in relation to the other Pitanga-type basalts, particularly SiO2 (50.83-51.63 wt.%), P2O5, K2O, Rb, Th, Zr and Y. Flow 6 is poorer in TiO2, P2O5, Nb, Rb, Sr, Th, and Zr, and slightly enriched in MgO and CaO. Flow 7 has intermediate contents of many of the elements in relation to other Pitanga-type basalts. The Paranapanema-type basalt (flow 8) has intermediate composition between the depleted Esmeralda-type flows 1 and 2 and the enriched Pitanga-type flows 4, 5, 6 and 7. Gamma-spectrometric readings are also characteristic of each flow and can be used as a field guide for the quick correlation of outcrops, particularly the continuous exposures of two or three different flows. The cps values are directly related to the contents of K2O, U and Th of the rock (Fig. 6). The rhyodacite (flow 3) stands out at 135 cps, whereas the basalts vary from 35-80 cps.i The presence of silicified sand as dikes, sills and partly filling geodes seems to be a necessary requirement for the formation of the silica ore. TABLE I Flow Rock Chemical type Thickness, m Mineralogy 1 Basalt Esmeralda 17 Aphanitic, holocristaline, composed mostly of plagioclase and augite. Alteration to smectite. Presence of hematite. 2 Basalt Esmeralda 10 Aphanitic, holocristaline, composed mostly of plagioclase and augite. Alteration to smectite. 3 Rhyodacite Chapecó 65 Porphyritic with phenocrysts of plagioclase and augite. Quartz geodes (size ranging from mm to cm) and amygdales filled with celadonite. Microcrystalline matrix intensely altered. 4 Basalt Pitanga 15 Microporphyritic with phenocrysts of plagioclase. Microcrystalline matrix composed of plagioclase and augite. Intense alteration to smectite and celadonite. Mineralized. 5 Basalt Pitanga 15 Microporphyritic with phenocrysts and matrix of zoned plagioclase. Microcrystalline matrix composed of plagioclase and augite. Alteration to smectite and celadonite. 6 Basalt Pitanga 25 Microporphyritic with phenocrysts and matrix of plagioclase and augite. Alteration to smectite. Mineralized. 7 Basalt Pitanga 40 Microporphyritic with phenocrysts and matrix of plagioclase and augite. Alteration to smectite. 8 Basalt Paranapanema 40 Aphanitic, holocrystalline; plagioclase and clinopyroxene dominant. TABLE I Selected characteristics of the eight lava flows from the Entre Rios district. Flows 5 and 6 are mineralized in amethyst-geodes. TABLE I Selected characteristics of the eight lava flows from the Entre Rios district. Flows 5 and 6 are mineralized in amethyst-geodes. An Acad Bras Cienc (2014) 86 (1) AMETHYST-BEARING LAVAS FROM ENTRE RIOS, PARANÁ PROVINCE LÉO A. HARTMANN, LUCAS M. ANTUNES AND LEONARDO M. ROSENSTENGEL 192 Flow 1 2 3 4 5 6 7 8 Sample 13A 13B 14 12 2A 2B 3 11 7A 7B 4 9A 10A 10B 16 1 5 6 8 17 18 SiO2 49.15 50.45 49.75 50.89 65.42 63.67 64.22 64.27 44.35 44.88 51.11 51.46 51.07 51.63 50.83 48.96 48.85 49.35 49.32 49.16 49.25 TiO2 1.31 1.24 1.26 1.59 1.38 1.44 1.52 1.45 4.10 4.07 3.31 3.29 3.33 3.37 3.25 3.16 3.00 3.72 3.70 3.58 2.22 Al2O3 13.57 13.51 13.61 13.76 13.75 13.57 13.27 13.16 12.62 12.52 12.08 11.96 12.13 12.32 12.21 12.61 12.51 12.58 12.47 12.48 13.16 Fe2O3 12.84 12.19 12.48 12.47 5.61 6.89 7.16 7.04 16.23 15.79 14.54 15.32 15.46 14.17 15.36 15.15 14.66 14.89 15.46 15.44 14.47 MgO 7.16 7.09 7.01 5.57 0.91 1.18 1.21 1.09 4.55 4.89 2.94 2.98 3.13 3.01 3.08 4.61 4.91 4.02 4.28 4.33 5.83 CaO 11.02 11.07 11.22 9.23 2.91 2.78 3.00 2.45 8.56 8.63 7.27 6.90 7.11 7.10 6.99 8.85 9.19 8.32 8.26 8.37 9.67 Na2O 2.40 2.31 2.21 2.71 3.46 3.35 3.45 3.17 2.16 2.18 2.78 2.76 2.73 2.79 2.74 2.46 2.44 2.63 2.56 2.57 2.28 K2O 0.26 0.19 0.23 0.34 4.20 4.54 4.16 4.63 0.82 0.85 1.77 2.16 2.08 2.17 2.18 1.35 1.30 1.58 1.55 1.21 0.94 P2O5 0.12 0.11 0.11 0.15 0.44 0.49 0.48 0.47 0.53 0.52 0.90 0.89 0.89 0.89 0.90 0.38 0.36 0.60 0.57 0.59 0.29 MnO 0.19 0.18 0.20 0.19 0.11 0.12 0.12 0.12 0.17 0.18 0.17 0.21 0.22 0.20 0.21 0.16 0.16 0.20 0.22 0.21 0.21 Cr2O3 0.029 0.029 0.029 0.013 − − − − 0.003 0.002 − − − − − 0.009 0.01 0.004 0.004 0.002 0.016 LOI 1.7 1.4 1.6 2.8 1.5 1.7 1.1 1.9 5.5 5.1 2.8 1.7 1.5 2.0 1.9 2.0 2.3 1.7 1.2 1.7 1.4 Total 99.75 99.77 99.71 99.71 99.69 99.73 99.69 99.75 99.59 99.61 99.67 99.63 99.65 99.65 99.65 99.69 99.69 99.59 99.59 99.64 99.74 Ni 29.7 27.0 26.6 27.2 1.10 1.30 1.50 1.00 15.9 14.2 6.0 5.8 8.3 7.6 6.1 25.0 31.2 14.5 17.4 27.0 70.0 Sc 40 39 39 34 12 12 13 12 32 32 26 26 26 25 25 33 34 33 33 31 37 Ba 77 105 77 211 878 789 853 914 460 472 706 657 663 677 627 436 531 509 539 511 321 Co 43.3 45.0 43.2 36.3 5.7 5.7 6.0 5.4 42.0 43.1 31.3 32.2 31.5 33.1 29.6 39.3 42.3 40.0 44.1 43.8 51.2 Cs 0.2 0.1 0.1 0.2 0.8 0.7 1.4 1.1 0.5 0.3 1.0 0.5 0.7 0.4 0.5 0.5 0.2 0.3 0.3 0.4 0.2 Ga 18.4 18.2 18.6 20.8 25.1 25.2 26.0 24.9 23.2 23.3 24.3 24.4 25.3 25.3 24.9 21.5 20.0 21.8 22.7 21.5 19.4 Hf 2.0 2.2 2.3 2.7 14.5 14.9 14.9 15.5 7.3 7.5 8.8 9.1 9.4 9.5 9.3 5.3 4.7 6.2 6.4 6.3 4.8 Nb 3.3 2.8 2.9 4.1 49.6 52.7 51.1 52.3 26.1 25.6 30.7 32.3 31.6 33.2 31.7 21.6 19.2 25.8 24.8 24.5 13.5 Rb 6.8 3.3 4.8 6.6 100.1 106.3 98.0 104.4 12.0 8.2 50.0 46.7 45.9 44.6 44.0 28.3 28.0 35.9 35.9 25.3 20.7 TABLE II Chemical analyses of studied rocks. AMETHYST-BEARING LAVAS FROM ENTRE RIOS, PARANÁ PROVINCE The sand was probably injected 1,000 m upwards from the buried, non-lithified erg at the time of volcanism, in a process similar to the underground sand mobilization described from the North Sea offshore basins (Hurst et al. 2011). The rhyodacite flow is an important stratigraphic marker in the district, because it is located at the top of the Esmeralda-type basalt flow 2 and therefore below four Pitanga-type basalt flows. A Paranapanema-type basalt occurs on top of the stratigraphic sequence. The Esmeralda-type lavas have low silicon content (SiO2 = 49.15 to 50.89 wt.%), low Ti, P2O5 and K2O, Rb, Sr, Zr, U, Th and Nb. The highest MgO (7.1 wt.%) occurs in lava 1, present in the Chapecó river bed, while MgO varies between 3-5 wt.% in most basalts. Flow 5 has different characteristics from the other basalts, because it is enriched in most incompatible trace elements; e.g., P2O5, Zr, Rb, U and Nb (Fig. 5). As expected, the rhyodacite is much richer in K2O, Nb, Rb, Th, U, Zr, Y and Sr relative to the basalts. The hydrothermal alteration of the rocks in the district is evidenced by the significant presence of smectite in all basalt flows, the occurrence of celadonite in mineralized basalts both filling amygdales and rimming amethyst geodes. Also, the presence of veins of silicified sandstones in minera lized basalt flows and the intense silicification in the flow of the hilltop are evidence for hydrothermal processes. Another evidence is the presence of hydrothermal breccia below flow 8 (Paranapanema, intermediate-Ti type), which has columnar jointing and displays the presence of hydrothermal breccias (Hartmann et al. 2010a, b) at the base. These factors The Pitanga-type basalts are geochemically similar but have small geochemical variations from flow to flow. Flow 4 (mineralized) has the lowest values of SiO2 (44.35 to 44.88 wt.%), which is An Acad Bras Cienc (2014) 86 (1) LÉO A. HARTMANN, LUCAS M. ANTUNES AND LEONARDO M. ROSENSTENGEL Fe2O3 = total iron. Major elements in weight percent, trace elements in ppm; LOI = loss on ignition; (−) = Cr2O3 <0.002. All flows are basalt, except flow 3, a rhyodacite. All samples have ER- as initials. An Acad Bras Cienc (2014) 86 (1) AMETHYST-BEARING LAVAS FROM ENTRE RIOS, PARANÁ PROVINCE AMETHYST-BEARING LAVAS FROM ENTRE RIOS, PARANÁ PROVINCE 193 Flow 1 2 3 4 5 6 7 8 Sample 13A 13B 14 12 2A 2B 3 11 7A 7B 4 9A 10A 10B 16 1 5 6 8 17 18 Sr 166 159 157 272 418 349 372 362 544 523 480 490 500 489 471 425 411 447 434 455 370 Ta 0.3 0.2 0.2 0.3 3.0 3.2 3.1 3.1 1.4 1.4 1.9 2.0 1.9 2.0 2.0 1.2 1.1 1.5 1.5 1.7 0.8 Th 0.8 0.8 0.7 1.5 8.6 8.8 9.9 10.5 3.2 3.4 4.4 4.7 5.1 5.1 4.6 2.8 2.6 4.6 3.6 3.7 2.6 U 0.2 0.2 0.2 0.3 1.9 2.1 1.9 2.3 0.7 0.7 1.0 1.0 1.0 1.1 1.1 0.7 0.6 0.8 0.9 0.8 0.5 V 362 354 362 373 65 41 40 35 511 516 260 271 286 268 261 511 480 466 450 507 488 Zr 73 68 70 98 563 583 590 588 269 266 334 354 351 349 345 212 198 250 245 243 148 Y 26.2 28.7 25.2 53.2 69.5 61.5 62.4 59.3 36.3 35.4 45.4 48.5 47.1 52.1 48.2 32.0 33.6 40.8 38.5 40.5 31.6 La 5.8 5.6 5.2 12.8 82.4 75.7 71.6 71.7 32.5 33.5 45.8 49.6 48.7 49.2 48.7 29.4 27.9 36.6 35.8 36.1 21.6 Ce 14.9 13.6 13.2 23.1 185.8 168.9 159.2 159.3 75.5 76.7 104.2 116.0 111.3 116.2 115.0 66.1 60.9 82.8 79.3 77.1 46.0 Pr 2.07 1.98 1.89 3.66 22.09 20.31 19.23 19.79 9.79 9.82 13.47 14.31 14.22 14.35 14.43 8.21 7.60 10.12 9.77 10.11 6.01 Nd 9.5 10.4 9.4 16.9 90.8 82.8 80.1 81.3 41.6 42.9 59.4 61.4 61.3 60.6 62.4 34.7 32.2 41.6 41.2 41.7 25.3 Sm 3.11 3.17 3.01 4.71 17.34 15.81 15.01 15.78 8.74 8.48 11.38 12.27 11.92 12.65 12.42 6.93 6.46 8.78 8.32 8.73 5.45 Eu 1.14 1.15 1.13 1.65 4.39 4.02 3.85 4.17 2.72 2.78 3.42 3.66 3.68 3.72 3.77 2.18 2.08 2.67 2.61 2.72 1.77 Gd 4.15 4.37 4.08 6.93 16.02 14.37 13.78 14.54 8.50 8.58 11.08 11.62 11.43 12.18 11.86 6.55 6.33 8.38 8.17 8.43 5.62 Tb 0.78 0.79 0.76 1.17 2.46 2.21 2.15 2.19 1.31 1.28 1.69 1.78 1.72 1.83 1.80 1.09 1.04 1.35 1.32 1.29 0.91 Dy 4.77 4.83 4.43 6.84 13.21 11.29 11.10 11.84 6.86 6.54 8.80 9.17 8.90 10.06 9.45 5.70 5.67 7.20 6.96 7.17 5.17 Ho 0.96 0.98 0.92 1.44 2.29 2.13 2.15 2.19 1.26 1.28 1.64 1.74 1.66 1.85 1.78 1.09 1.10 1.39 1.35 1.36 1.05 Er 2.64 2.81 2.56 4.09 6.33 5.82 5.64 5.73 3.32 3.29 4.33 4.58 4.32 4.93 4.76 3.00 3.07 3.84 3.85 3.95 3.05 Tm 0.37 0.41 0.37 0.55 0.93 0.86 0.84 0.83 0.48 0.50 0.62 0.65 0.63 0.67 0.64 0.45 0.44 0.56 0.55 0.55 0.43 Yb 2.33 2.46 2.29 3.15 5.97 5.11 5.24 5.04 2.84 3.03 3.82 3.99 3.98 4.08 4.07 2.87 2.71 3.66 3.30 3.29 2.70 Lu 0.35 0.38 0.34 0.50 0.79 0.70 0.73 0.73 0.39 0.42 0.55 0.55 0.54 0.60 0.57 0.39 0.38 0.49 0.49 0.49 0.42 Cu 148 208 152 219 8 8 7 7 178 200 177 205 198 207 238 231 211 163 180 93 233 TABLE II (continuation) An Acad Bras Cienc (2014) 86 (1) 194 LÉO A. An Acad Bras Cienc (2014) 86 (1) AMETHYST-BEARING LAVAS FROM ENTRE RIOS, PARANÁ PROVINCE HARTMANN, LUCAS M. ANTUNES AND LEONARDO M. ROSENSTENGEL TABLE III Location and elevation of analyzed samples. UTM coordinates, Datum SIRGAS2000. Samples Coordinates Elevation East North a.s.l., m ER-1 342641 7044028 536 ER-2 341594 7044199 463 ER-3 342218 7043266 455 ER-4 342947 7043146 512 ER-5 343208 7043214 541 ER-6 343425 7043187 566 ER-7 345237 7043180 610 ER-8 345403 7043976 639 ER-9 345557 7044041 616 ER-10 344404 7042809 573 ER-11 341376 7047919 481 ER-12 340335 7047489 410 ER-13 340209 7047519 390 ER-14 342181 7048250 436 ER-16 343100 7046265 549 ER-17 345641 7044263 668 ER-18 345977 7044060 712 Figure 4 - Chemical classification of the eight lava flows from the Entre Rios mining district, based on (a) Na2O + K2O x SiO2 (Le Bas et al. 1986), (b) R1 x R2 (De La Roche et al. 1980), (c) Zr/TiO2 x SiO2 (Winchester and Floyd 1977). Flow number indicated by number (e.g. 4). Abbreviations: bas. trachy-and. = basaltic trachyandesite, rhyo = rhyolite, l-b = lati-basalt, l-a = lati-andesite, trach = trachyte, trach-and = trachy-andesite, ab = alkaline basalt, sub-ab = subalkaline basalt. 194 LÉO A. HARTMANN, LUCAS M. ANTUNES AND LEONARDO M. ROSENSTENGEL TABLE III Location and elevation of analyzed samples. UTM coordinates, Datum SIRGAS2000. Samples Coordinates Elevation East North a.s.l., m ER-1 342641 7044028 536 ER-2 341594 7044199 463 ER-3 342218 7043266 455 ER-4 342947 7043146 512 ER-5 343208 7043214 541 ER-6 343425 7043187 566 ER-7 345237 7043180 610 ER-8 345403 7043976 639 ER-9 345557 7044041 616 ER-10 344404 7042809 573 ER-11 341376 7047919 481 ER-12 340335 7047489 410 ER-13 340209 7047519 390 ER-14 342181 7048250 436 ER-16 343100 7046265 549 ER-17 345641 7044263 668 ER-18 345977 7044060 712 TABLE III Location and elevation of analyzed samples. UTM coordinates, Datum SIRGAS2000. Samples Coordinates Elevation East North a.s.l., m ER-1 342641 7044028 536 ER-2 341594 7044199 463 ER-3 342218 7043266 455 ER-4 342947 7043146 512 ER-5 343208 7043214 541 ER-6 343425 7043187 566 ER-7 345237 7043180 610 ER-8 345403 7043976 639 ER-9 345557 7044041 616 ER-10 344404 7042809 573 ER-11 341376 7047919 481 ER-12 340335 7047489 410 ER-13 340209 7047519 390 ER-14 342181 7048250 436 ER-16 343100 7046265 549 ER-17 345641 7044263 668 ER-18 345977 7044060 712 Figure 4 - Chemical classification of the eight lava flows from the Entre Rios mining district, based on (a) Na2O + K2O x SiO2 (Le Bas et al. AMETHYST-BEARING LAVAS FROM ENTRE RIOS, PARANÁ PROVINCE 1986), (b) R1 x R2 (De La Roche et al. 1980), (c) Zr/TiO2 x SiO2 (Winchester and Floyd 1977). Flow number indicated by number (e.g. 4). Abbreviations: bas. trachy-and. = basaltic trachyandesite, rhyo = rhyolite, l-b = lati-basalt, l-a = lati-andesite, trach = trachyte, trach-and = trachy-andesite, ab = alkaline basalt, sub-ab = subalkaline basalt. TABLE III Location and elevation of analyzed samples. UTM coordinates, Datum SIRGAS2000. Figure 4 - Chemical classification of the eight lava flows from the Entre Rios mining district, based on (a) Na2O + K2O x SiO2 (Le Bas et al. 1986), (b) R1 x R2 (De La Roche et al. 1980), (c) Zr/TiO2 x SiO2 (Winchester and Floyd 1977). Flow number indicated by number (e.g. 4). Abbreviations: bas. trachy-and. = basaltic trachyandesite, rhyo = rhyolite, l-b = lati-basalt, l-a = lati-andesite, trach = trachyte, trach-and = trachy-andesite, ab = alkaline basalt, sub-ab = subalkaline basalt. Figure 4 - Chemical classification of the eight lava flows from the Entre Rios mining district, based on (a) Na2O + K2O x SiO2 (Le Bas et al. 1986), (b) R1 x R2 (De La Roche et al. 1980), (c) Zr/TiO2 x SiO2 (Winchester and Floyd 1977). Flow number indicated by number (e.g. 4). Abbreviations: bas. trachy-and. = basaltic trachyandesite, rhyo = rhyolite, l-b = lati-basalt, l-a = lati-andesite, trach = trachyte, trach-and = trachy-andesite, ab = alkaline basalt, sub-ab = subalkaline basalt. with downthrow to the west (Figs. 7, 8). This is similar to the structure identified by Rosenstengel and Hartmann (2012) in the Ametista do Sul mining district situated 300 km to the south. The faulting is usually interpreted as a consequence of the opening of the South Atlantic Ocean in the Cretaceous. characterize a silica gossan (Pertille et al. 2013) which is an important structure for the geological exploration of amethyst geodes. Silica gossans are present at the top of the uppermost flow.l The distribution of the lava flows in the mining district displays a marked structure of fault-blocks AMETHYST-BEARING LAVAS FROM ENTRE RIOS, PARANÁ PROVINCE 195 Figure 5 - (a)-(f) Chemical variation diagrams displaying the characteristic composition (TiO2, MgO, P2O5, Zr, Rb, U and Nb) of each lava flow in the district. Figure 5 - (a)-(f) Chemical variation diagrams displaying the characteristic composition (TiO2, MgO, P2O5, Zr, Rb, U and Nb) of each lava flow in the district. AMETHYST-BEARING LAVAS FROM ENTRE RIOS, PARANÁ PROVINCE Figure 6 - Diagram displaying the positive correlation between the contents of K2O, U, Th and the gamma-ray emission rate. Cps values indicated (e.g., 75). Figure 6 - Diagram displaying the positive correlation between the contents of K2O, U, Th and the gamma-ray emission rate. Cps values indicated (e.g., 75). An Acad Bras Cienc (2014) 86 (1) LÉO A. HARTMANN, LUCAS M. ANTUNES AND LEONARDO M. ROSENSTENGEL 196 Figure 7 - Geological section of the Entre Rios mining district. Inset photo shows the entrance to underground mine. Figure 7 - Geological section of the Entre Rios mining district. Inset photo shows the entrance to underground mine. Figure 8 - Correlation chart of the lava flows in the Entre Rios mining district. Fault-block structures caused downthrow of the flows to the west. Figure 8 - Correlation chart of the lava flows in the Entre Rios mining district. Fault-block structures caused downthrow of the flows to the west. An Acad Bras Cienc (2014) 86 (1) RESUMO O distrito mineiro de Entre Rios produz um grande volume de geodos de ametista em minas subterrâneas e faz parte das jazidas de classe mundial da província vulcânica Paraná na América do Sul. Dois derrames de basalto são os produtores e têm números 4 e 5 na estratigrafia das lavas. Há sete derrames e um de riodacito no distrito. Na base da estratigrafia situada no leito do rio Chapecó, há dois derrames Esmeralda do tipo baixo- Ti. O terceiro derrame na sequência é um riodacito do tipo Chapecó, subtipo Guarapuava. Acima do derrame de riodacito, há 4 derrames de basalto Pitanga do tipo alto-Ti e que incluem os dois derrames mineralizados; somente o derrame do topo da estratigrafia é do tipo Paranapanema, médio-Ti. Cada derrame é identificado individualmente pelas suas propriedades geoquímicas e gama-espectrométricas. O estudo de várias seções no distrito levou à identificação de uma estrutura de blocos de falha. Os blocos estão alinhados NO, sendo que o bloco situado no lado oeste da falha foi rebaixado. Esta importante caracterização estrutural do distrito mineiro terá consequências significativas na busca de novas jazidas de geodos de ametista e no entendimento da evolução da província vulcânica Paraná. De La Roche H, Leterrier J, GrandClaude P and Marchal M. 1980. A classification of volcanic and plutonic rocks using R1,R2-diagrams and major element analysis ‒ its relationships with current nomenclature. Chem Geol 29: 183-210. Duarte LC, Hartmann LA, Ronchi LH, Berner Z, Theye T and Massonne HJ. 2011. Stable isotope and mineralogical investigation of the genesis of amethyst geodes in the Los Catalanes gemological district, Uruguay, southernmost Paraná volcanic province. Mineralium Dep 46: 239-255. Duarte LC, Hartmann LA, Vasconcellos MAZ, Medeiros JTN and Theye T. 2009. Epigenetic formation of amethyst-bearing geodes from Los Catalanes gemological district, Artigas, Uruguay, southern Paraná Magmatic Province. Jour Volc Geoth Res 184: 427-436. Frank HT, Gomes MEB and Formoso MLL. 2009. Review of the areal extent and the volume of the Serra Geral Formation, Paraná Basin, South America. Pesquisas Geociências (UFRGS) 36: 49-57. Gilg HA, Morteani G, Kostitsyn Y, Preinfalk C, Gatter I and Strieder AJ. 2003. Genesis of amethyst geodes in basaltic rocks of the Serra Geral Formation (Ametista do Sul, Rio Grande do Sul, Brazil): a fluid inclusion, REE, oxygen, carbon, and Sr isotope study on basalt, quartz, and calcite. Mineralium Dep 38: 1009-1025. Hartmann LA, Arena KR and Duarte SK. 2012a. REFERENCES Araújo LM, França AB and Potter PE. 1999. Hydrogeology of the Mercosul aquifer system in the Paraná and Chaco- Paraná Basins, South America, and comparison with the Navajo-Nugget aquifer system, USA. Hydrogeol Jour 7: 317-336. CONCLUSIONS with seven basalt flows and these belong to the Esmeralda, Pitanga and Paranapanema chemical types. This is a result of intense variation in the mantellic and crustal processes responsible for the generation of the volcanism present in the district. The ore was generated by intense hydrothermal alteration of the basalts at low temperature, after Most significant conclusions are reached regarding the geological constraints of the amethyst geodes in the Entre Rios mining district. Among the eight flows present, only flows 4 and 5 are currently mined, but marked silica gossans occur at the top of the eighth flow. One rhyodacite occurs intercalated An Acad Bras Cienc (2014) 86 (1) AMETHYST-BEARING LAVAS FROM ENTRE RIOS, PARANÁ PROVINCE 197 Palavras-chave: geodo de ametista, distrito mineiro de Entre Rios, província vulcânica Paraná, geoquímica, gama-espectrometria. the injection of sand from the deeply buried paleo- erg. The distribution of the lava flows indicates a fault-block structure for the mining district, and this is of major significance in the search for additional deposits. ACKNOWLEDGMENTS Financial support for the study was provided by Programa de Apoio a Núcleos de Excelência (PRONEX)-Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS)/ Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) on “Strategic minerals from southern Brazil” coordinated by the first author. The Geological Survey of Brazil Companhia de Pesquisa de Recursos Minerais (CPRM) allowed the use of a portable field gamma-spectrometer. Arioli EE, Licht OAB, Vasconcellos EMG, Bonnet KL and Santos EM. 2008. Faciologia vulcânica da Formação Serra Geral na região de Guarapuava, Paraná. IV Simpósio de Vulcanismo e Ambientes Associados, Foz do Iguaçu; Sociedade Brasileira de Geologia, Anais, 6 p. Bellieni G, Vomin-Chiaramonti P, Marques LS, Melfi AJ, Nardy AJR, Piccirillo EM and Roisenberg A. 1984. High and Low TiO2 flood basalts from the Paraná plateau (Brazil): petrology and geochemical aspects bearing on their mantle origin. Neues Jahrb Mineral Petrol 150: 273-306. Commin-Fischer A, Berger G, Polvé M, Dubois M, Sardini P, Beaufort D and Formoso MLL. 2010. Petrography and chemistry of SiO2 filling phases in the amethyst geodes from the Serra Geral Formation deposit, Rio Grande do Sul, Brazil. Jour South Am Earth Sci 29: 751-760. LÉO A. HARTMANN, LUCAS M. ANTUNES AND LEONARDO M. ROSENSTENGEL LÉO A. HARTMANN, LUCAS M. ANTUNES AND LEONARDO M. ROSENSTENGEL 198 Hartmann LA, Arena KR, Duarte SK and Pertille J. 2012b. Long distance lava correlation in the Lower Cretaceous Paraná volcanic province along the Serra Geral cuesta, southeastern Brazil. Intern Jour Earth Sci 102: 1655-1669. Morteani G, Kostitsyn Y, Preinfalk C and Gilg HA. 2010. The genesis of the amethyst geodes at Artigas (Uruguay) and the paleohydrology of the Guaraní aquifer: structural, geochemical, oxygen, carbon, strontium isotope and fluid inclusion study. Intern Jour Earth Sci 99: 927-947. Nardy AJR, Machado FB and Oliveira MAF. 2008. As rochas vulcânicas mesozóicas ácidas da Bacia do Paraná: litoestratigrafia e considerações geoquímico- estratigráficas. Rev Bras Geocien 38: 178-195. Hartmann LA, Baggio SB and Duarte SK. 2012c. Decoding geochemical and gamma-spectrometric signatures from lavas and sand injectites at the base of the Paraná volcanic province, Novo Hamburgo, Brazil. Intern Geol Rev 55: 510-524. Peate DW, Hawkesworth CJ and Mantovani MSM. 1992. Chemical stratigraphy of Paraná lavas (South America): classification of magma types and their spatial distribution. Bull Volc 55: 119-139. Hartmann LA et al. 2012d. Sequential opening and filling of cavities forming vesicles, amygdales and giant amethyst geodes in lavas from the southern Paraná volcanic province, Brazil and Uruguay. Intern Geol Rev 54: 1-14. Pertille J, Hartmann LA, Duarte SK, Arena KR, Rosa MLCC and Barboza EG. 2013. Gossan characterization in the Quaraí and Los Catalanes amethyst geode districts (Brazil and Uruguay), Paraná volcanic province, using rock geochemistry and gamma-spectrometry. Jour Geoch Expl 124: 127-139. Hartmann LA, Duarte SK and Pertille J. 2010a. Roteiro de Campo. Geologia dos geodos de ametista de Quaraí, Brasil – Uma imersão no vulcanismo, hidrotermalismo e na exploração mineral no Grupo Serra Geral. Porto Alegre, Gráfica da UFRGS, p. 13-71. Pinto VM and Hartmann LA. 2011. Flow-by-flow chemical stratigraphy and evolution of thirteen Serra Geral Group basalt flows from Vista Alegre, southernmost Brazil. An Acad Bras Cienc 83: 425-440. Hartmann LA, Wildner W, Duarte LC, Duarte SK, Pertille J, Arena KR, Martins LC and Dias NL. 2010b. Geochemical and scintillometric characterization and correlation of amethyst geode-bearing Paraná lavas from the Quaraí and Los Catalanes districts, Brazil and Uruguay. Geol Mag 147: 954-970. Pinto VM, Hartmann LA, Santos JOS, McNaughton NJ and Wildner W. 2011. Zircon U-Pb geochronology from the Paraná bimodal volcanic province support a brief eruptive cycle at ~135 Ma. Chem Geol 281: 93-102. RESUMO Geological relationships of basalts, andesites and sand injectites at the base of the Paraná volcanic province, Torres, Brazil. Jour Volc Geoth Res 237-238: 97-111. An Acad Bras Cienc (2014) 86 (1) An Acad Bras Cienc (2014) 86 (1) LÉO A. HARTMANN, LUCAS M. ANTUNES AND LEONARDO M. ROSENSTENGEL Hurst A, Scott A and Vigorito M. 2011. Physical charac teristics of sand injectites. Earth-Sci Rev 106: 215-246. Proust D and Fontaine C. 2007. Amethyst-bearing lava flows in the Paraná basin (Rio Grande do Sul, Brazil): cooling, vesiculation and formation of the geodic cavities. Geol Mag 144: 53-65. Jacques PD, Machado R and Nummer AR. 2010. Lineamentos estruturais na borda leste da Bacia do Paraná em Santa Catarina: análise multiescala com base em imagens LANDSAT e SRTM. Pesquisas Geociências (UFRGS) 37: 117-131. Rosenstengel LM and Hartmann LA. 2012. Geochemical stratigraphy of lavas and fault-block structures in the Ametista do Sul geode mining district, Paraná volcanic province, southern Brazil. Ore Geol Rev 48: 332-348. Janasi VA, Freitas VA and Heaman LH. 2011. The onset of flood volcanism, Northern Paraná Basin, Brazil: A precise U-Pb baddeleyite/zircon age for a Chapecó-type dacite. Earth Planet Sci Lett 302: 147-153. Winchester JA and Floyd PA. 1977. Geochemical discrimination of different magma series and their differentiation products using immobile elements. Chem Geol 20: 325-343. Juchem PL, Chodur NL and Liccardo A. 2010. Depósitos de ametista em Santa Catarina, sul do Brasil. In: 45º Congresso Brasileiro de Geologia, Belém, 1 p. Zalán PV, Wolff S, Astolfii MAM, Vieira IS, Conceição JCJ, Appi VT, neto EVS, Cerqueira JR and Marques A. 1991. The Paraná Basin, Brazil. In: Leighton MW, Kolata DR, Oltz DF and Eidel JJ (Eds), Interior Cratonic Basins. AAPG Mem 51: 681-708. Le Bas MJ, Le Maitre RW, Streckeisen A, Zanettin B and Iugs Sub Commission on the Systematic of Igneous Rocks. 1986. A chemical classification of volcanic rocks based on the total alkali-silica diagram. Jour Petrol 27: 745-750. An Acad Bras Cienc (2014) 86 (1)
https://openalex.org/W3164244566	https://nottingham-repository.worktribe.com/preview/5572710/s41598-021-91397-8.pdf	English	null	BSE can propagate in sheep co-infected or pre-infected with scrapie	Scientific reports	2,021	cc-by	13,171	BSE can propagate in sheep co‑infected or pre‑infected with scrapie Angela Chong1, James D. Foster1, Wilfred Goldmann1, Lorenzo Gonzalez2, Martin Jeffrey2, Matthew J. O’Connor3, Keith Bishop4, Ben C. Maddison4, E. Fiona Houston1, Kevin C. Gough 3 & Nora Hunter1 OPEN To understand the possible role of mixed-prion infections in disease presentation, the current study reports the co-infection of sheep with bovine spongiform encephalopathy (BSE) and scrapie. The bovine BSE agent was inoculated subcutaneously into sheep with ARQ/ARQ or VRQ/ARQ PRNP genotypes either at the same time as subcutaneous challenge with scrapie, or three months later. In addition, VRQ/VRQ sheep naturally infected with scrapie after being born into a scrapie-affected flock were challenged subcutaneously with BSE at eight or twenty one months-of-age. Sheep were analysed by incubation period/attack rate, and western blot of brain tissue determined the presence of BSE or scrapie-like PrPSc. Serial protein misfolding cyclic amplification (sPMCA) that can detect very low levels of BSE in the presence of an excess of scrapie agent was also applied to brain and lymphoreticular tissue. For VRQ/ARQ sheep challenged with mixed infections, scrapie-like incubation periods were produced, and no BSE agent was detected. However, whilst ARQ/ARQ sheep developed disease with BSE-like incubation periods, some animals had a dominant scrapie western blot phenotype in brain, but BSE was detected in these sheep by sPMCA. In addition, VRQ/VRQ animals challenged with BSE after natural exposure to scrapie had scrapie-like incubation periods and dominant scrapie PrPSc in brain, but one sheep had BSE detectable by sPMCA in the brain. Overall, the study demonstrates for the first time that for scrapie/BSE mixed infections, VRQ/ARQ sheep with experimental scrapie did not propagate BSE but VRQ/VRQ sheep with natural scrapie could propagate low levels of BSE, and whilst BSE readily propagated in ARQ/ARQ sheep it was not always the dominant PrPSc strain in brain tissue. Indeed, for several animals, a dominant scrapie biochemical phenotype in brain did not preclude the presence of BSE prion. Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative conditions that occur naturally in various mammalian species, for example sheep (scrapie), deer (chronic wasting disease, CWD) and humans (e.g. Creutzfeldt Jacob Disease, CJD). TSEs are protein misfolding diseases but are unusual in that they are also transmissible between individuals, For instance, some TSEs can be transmitted through contact with bodily fluids1, via environmental contamination2, via contaminated feed/food3 or by experimental inoculation4, 5. With scrapie, the infectious agent, or prion, is readily detected in excreta and secreta6–10 and is widespread in the environment of such infected animals11, 12. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports/ TSEs are frequently studied experimentally in mice and sheep models from which considerable information has been generated over many decades on the transmission properties and characteristics of multiple prion strains. Strains can be defined in terms of incubation period (time between infection and disease)16, 17, interac- tion with particular host PrP (PRNP) genotype5, pathology, immunohistochemical (IHC) detection of patterns of PrPSc distribution18 and the appearance of the extracted PrPSc protein on western blots19–21. Strains are more usually studied singly in isolation where their characteristics are reproducible and predictable and therefore allow the study of particular aspects of early infection, pathology and pathogenesis. However, natural scrapie infections in sheep are not always due to a single strain and can be the result of mixtures of strains22, 23. Multiple passage in mice of isolates of natural scrapie can produce several different strains depending on the mouse lines used at each passage22, 24, 25. This has been interpreted as selection, from the original sheep classical scrapie infec- tion, of particular strains that replicate more quickly or more efficiently in specific mouse lines, both inbred and transgenic22, 24, 25. Similar findings for CWD transmission in transgenic mice also suggest that deer infection might consist of more than one strain per outbreak26. Direct evidence for mixed infection within single sheep has been reported for classical scrapie and atypical scrapie27, 28, and the report of PrPSc in natural sheep scrapie with features akin to both bovine spongiform encephalopathy (BSE) and classical scrapie could be the result of mixed infection or a hybrid strain phenotype29. y p yp Compared with the considerable literature on the biology of single TSE strain infections, much less has been reported on the effects of deliberately mixing strains. Because of the indirect methods used to identify strains, it is necessary to use two strains with easily identifiable features, for example short and long incubation periods, different patterns of brain vacuolation (lesion profiles) and those producing PrPSc with distinguishable patterns on western blots and/or differential antibody binding. In this way it has been established that a strain with a long incubation period can interfere with the ability of a short incubation period strain to produce disease30, 31. The strength of the interference is dependent on route of infection and precise timing but complete blockage of one strain by another can occur32. www.nature.com/scientificreports/ These studies gave rise to the idea that the number of replication sites is finite and that the binding of one strain will preclude binding of a second superinfecting strain. If large enough amounts of the first strain are used, the replication sites were postulated to be completely occupied and the superinfecting strain cannot then replicate32. Additionally, strain interference was also demonstrated using the oral route suggesting it can occur in natu- ral infections33 and evidence points strongly at PrPC being the limiting resource (replication site) for which the strains compete34. Further studies in hamsters have also shown that although following some mixed infections all clinical disease and pathology seem to result from the faster replicating strain, more sophisticated tests (PrPSc stability) suggest that the slower replicating strain is still present, perhaps at low levels23. y gg g Studies have shown that certain combinations of strain do not compete, and animals have the pathology and PrPSc of the shorter incubation strain without delay in incubation periods35–37. In addition, the two coin- fecting strains can replicate independently, where an animal has the pathology of the shorter incubation strain without delay, but PrPSc is a mixture of both strains23, 36–38. Surprisingly, some animals can display a dominant PrPSc phenotype of a blocking strain yet the incubation period and clinical signs indicative of the faster strain23. In addition, the use of certain combinations of a non-transmissible strain with a highly pathogenic strain for coinfections can cause delay in the disease caused by the pathogenic strain. This suggests that either the non- pathogenic strain can replicate to some extent or that agent replication is not required for strain interference36. Overall, studies suggest that coinfections can lead to an inhibitory interference of one strain over another or independent replication of one or both strains.i Here, we present for the first time a study of mixed TSE infection in sheep. We used natural scrapie, SSBP/1 experimental scrapie and cattle BSE; the latter can be distinguished from the scrapie strains by incubation period, IHC and western blotting39, 40. Furthermore, a specific in vitro replication assay (sPMCA) that can detect BSE PrPSc even in the presence of a large excess of scrapie PrPSc was also applied41, 42. www.nature.com/scientificreports/ Sheep of different PRNP genotypes underwent co-challenge with BSE and scrapie (SSBP/1) or pre-infection with scrapie (either SSBP/1 or natural scrapie43) followed by BSE, and incubation periods were indicative of either scrapie or BSE disease. For selected animals, brain was analysed using western blotting to detect the dominant PrPSc type. Furthermore, brain and lymphoid tissue were analysed by a high sensitivity BSE-specific sPMCA assay. The objectives were to ascertain whether BSE will cause clinical disease when in competition with scrapie, and also to determine whether sheep displaying a scrapie phenotype in brain by conventional analysis (western blotting) could still harbour BSE prions. BSE can propagate in sheep co‑infected or pre‑infected with scrapie Angela Chong1, James D. Foster1, Wilfred Goldmann1, Lorenzo Gonzalez2, Martin Jeffrey2, Matthew J. O’Connor3, Keith Bishop4, Ben C. Maddison4, E. Fiona Houston1, Kevin C. Gough 3 & Nora Hunter1 OPEN The prion protein (PrP) is central to the pathogenesis of TSEs and exists in two main isoforms. PrPC is a normal host protein which is expressed in many cell types especially in brain, and misfolded PrPSc which is so closely associated with TSE infectivity that it is believed to form part, or all, of the infectious agent13. Incoming PrPSc in an inoculum is believed to convert endogenous PrPC into more PrPSc and hence to spread and replicate. PrPSc generated during disease progression is deposited in brain and lymphoid tissues and neuronal vacuolation is usually found at post mortem in infected brain. The protein conversion process (PrPC to PrPSc) can be mimicked in vitro including, in some cases, the creation of de novo infectivity, in a process termed serial protein misfolding cyclic amplification (sPMCA)14, 15. 1The Roslin Institute, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK. 2Animal and Plant Health Agency Lasswade, Pathology Department, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK. 3School of Veterinary Medicine and Science, The University of Nottingham, College Road, Sutton Bonington, Loughborough, Leicestershire LE12 5RD, UK. 4ADAS Biotechnology, School of Veterinary Medicine and Science, The University of Nottingham, College Road, Sutton Bonington, Loughborough, Leicestershire LE12 5RD, UK. *email: Fiona.houston@roslin.ed.ac.uk; Kevin.gough@nottingham.ac.uk \| https://doi.org/10.1038/s41598-021-91397-8 Scientific Reports \| (2021) 11:11931 Results b i Incubation periods of sheep infected with scrapie and/or BSE. Cheviot sheep with VRQ/ARQ and ARQ/ARQ PRNP genotypes (referring to the three codons at positions 136, 154, 171) were injected subcutane- ously with: SSBP/1 alone (S), BSE alone (B), SSBP/1 and BSE administered on the same day (S/B) or SSBP/1 followed after 3 months by BSE (S + B3m).hi t The results of VRQ/ARQ sheep challenges are presented in Table 1 and Fig. 1. For SSBP/1 alone, all five inoculated sheep developed TSE clinical signs. The mean incubation period was 234 ± 41 days post injection (dpi). When challenged with BSE alone, only two out of six sheep developed TSE clinical signs with incubation periods of 1008 and 1229 dpi. Of the four sheep that did not develop clinical signs, two survived to the end of the study at 2001 dpi and two were culled with intercurrent disease (not TSE) at 774 and 1379 dpi. y p p All VRQ/ARQ sheep challenged with both SSBP/1 and BSE developed TSE clinical signs. S/B sheep (n = 17) had mean incubation period 233 ± 32 dpi. In the S + B3m group (n = 17), the data is presented in two ways. Firstly, the incubation period dating from the SSBP/1 inoculation, and secondly dating from the BSE inocula- tion. Mean incubation period for the whole group (n = 17) was 245 ± 42 from scrapie challenge (161 ± 42) dpi Scientific Reports \| (2021) 11:11931 \| https://doi.org/10.1038/s41598-021-91397-8 www.nature.com/scientificreports/ Table 1. Incubation periods of TSE inoculated sheep. a S: time between scrapie inoculation and culling. Results b i d This animal had the P168L polymorphisms associated with longer incubation times for BSE. e This animal had the M112T polymorphisms associated with longer incubation times for BSE. Table 1. Incubation periods of TSE inoculated sheep. a S: time between scrapie inoculation and culling. b B: time between BSE inoculation and culling. c NS: time between birth (assumed exposure to scrapie) and culling. d This animal had the P168L polymorphisms associated with longer incubation times for BSE. e This animal had the M112T polymorphisms associated with longer incubation times for BSE. from BSE challenge). Comparing these results to those of the single inocula, the mixed infection groups had incubation periods indistinguishable from SSBP/1 infection (p > 0.99) and significantly different from those for BSE only (p < 0.001). from BSE challenge). Comparing these results to those of the single inocula, the mixed infection groups had incubation periods indistinguishable from SSBP/1 infection (p > 0.99) and significantly different from those for BSE only (p < 0.001). y (p ) For VRQ/ARQ animals challenged with SSBP/1 only, there appeared to be shorter incubation periods for sheep with PRNP codon 141 genotype LF compared to LL141. However, numbers were low and inconsistent effects of this codon on incubation periods have previously been reported in similarly small cohorts of sheep challenged subcutaneously with SSBP/15. Indeed, with the much larger mixed infections groups, incubation period did not appear to be influenced by the codon at 141 (p > 0.99 and p = 0.70 within S/B and S + B3m groups, respectively). The incubation period results for the sheep of ARQ/ARQ PRNP genotype were in general much longer than those of VRQ/ARQ sheep (Table 1 and Fig. 1). For SSBP/1 alone, four out of the six sheep developed TSE clinical signs, with a mean incubation period of 1541 ± 392 dpi. There were two survivors at the end of the study at 2002 dpi. Although numbers are low, there was no evidence that codon 141 had any influence on incubation period (p = 0.89) as the four clinically positive animals included two each of FF141 (1245 and 1763 dpi) and LF141 (1169 and 1987 dpi). For BSE alone, all six challenged sheep developed TSE clinical signs, with mean incubation period of 909 ± 289 dpi. Results b i b B: PRNP genotype 136/154/171 Challenge group Codon 141 subgroup Number challenged Clinically positive and PrPSc positive Clinically negative and PrPSc negative Number Mean incubation period in days (± SD)a,b,c Number Survival times, range in days VRQ/ARQ SSBP/1 alone All 5 5 234 (± 41) 0 LL 2 2 276, 280d 0 LF 3 3 204 (± 7) 0 BSE alone All 6 2 1008, 1229 4 774–2001 LL 5 2 1008, 1229 3 774–2001 LF 1 0 1 2001 S/B All 17 17 233 (± 32) 0 LL 6 6 232 (± 41) 0 LF 11 11 233 (± 28) 0 S + B3m All 17 17 S: 245 (± 42) B:161 (± 42) 0 LL 6 6 S: 239 (± 43) B: 155 (± 43) 0 LF 11 11 S: 248 (± 43) B: 164 (± 43) 0 ARQ/ARQ SSBP/1 alone All 6 4 1541 (± 398) 2 2002, 2002 LL 0 FF 3 2 1245, 1763 1 2002 LF 3 2 1169, 1987 1 2002 BSE alone All 6 6 909 (± 289) 0 LL 1 1 516 0 FF 2 2 678, 851 0 LF 3 3 1136 (± 159) 0 S/B All 17 15 918 (± 195) 2 1960e, 1975 LL 0 FF 9 9 791 (± 83) 0 LF 8 6 1107 (± 153) 2 1960, 1975 S + B3m All 17 17 S: 998 (± 206) B: 914 (± 206) 0 LL 1 1 S: 643 B: 559 0 FF 8 8 S: 866 (± 79) B:782 (± 79) 0 LF 8 8 S: 1130 (± 213) B: 1046 (± 213) 0 VRQ/VRQ NS All 6 6 NS: 793 (± 74) 0 NS + B8m All 9 9 NS: 777 (± 50) 0 NS + B21m All 9 9 NS: 803 (± 68) 0 Table 1. Incubation periods of TSE inoculated sheep. a S: time between scrapie inoculation and culling. b B: time between BSE inoculation and culling. c NS: time between birth (assumed exposure to scrapie) and culling. d This animal had the P168L polymorphisms associated with longer incubation times for BSE. e This animal had the M112T polymorphisms associated with longer incubation times for BSE. Table 1. Incubation periods of TSE inoculated sheep. a S: time between scrapie inoculation and culling. b B: time between BSE inoculation and culling. c NS: time between birth (assumed exposure to scrapie) and culling. Results b i In this case the codon 141 genotype appeared to have an influence on incubation period length with the LF141 genotype having longer incubation periods (p = 0.03; Table 1, Fig. 1b). For VRQ/ARQ animals challenged with SSBP/1 only, there appeared to be shorter incubation periods for sheep with PRNP codon 141 genotype LF compared to LL141. However, numbers were low and inconsistent effects of this codon on incubation periods have previously been reported in similarly small cohorts of sheep challenged subcutaneously with SSBP/15. Indeed, with the much larger mixed infections groups, incubation period did not appear to be influenced by the codon at 141 (p > 0.99 and p = 0.70 within S/B and S + B3m groups, respectively).h ppl y p p g p p y The incubation period results for the sheep of ARQ/ARQ PRNP genotype were in general much longer than those of VRQ/ARQ sheep (Table 1 and Fig. 1). For SSBP/1 alone, four out of the six sheep developed TSE clinical signs, with a mean incubation period of 1541 ± 392 dpi. There were two survivors at the end of the study at 2002 dpi. Although numbers are low, there was no evidence that codon 141 had any influence on incubation period (p = 0.89) as the four clinically positive animals included two each of FF141 (1245 and 1763 dpi) and LF141 (1169 and 1987 dpi). For BSE alone, all six challenged sheep developed TSE clinical signs, with mean incubation period of 909 ± 289 dpi. In this case the codon 141 genotype appeared to have an influence on incubation period length with the LF141 genotype having longer incubation periods (p = 0.03; Table 1, Fig. 1b). https://doi.org/10.1038/s41598-021-91397-8 Scientific Reports \| (2021) 11:11931 \| www.nature.com/scientificreports/ Figure 1. Incubation periods of individual VRQ/ARQ, ARQ/ARQ and VRQ/VRQ sheep. Sheep were inoculated with SSBP/1 scrapie, BSE or mixtures (A,B) or were infected with natural scrapie and then superinfected with BSE (C). The codon 141 PRNP genotype is indicated: LL141 (grey fill); LF141 (black fill); FF141 (no fill). Circles are clinical and pathology positive sheep, triangles are non-TSE animals. In A and B, lines represent SSBP/1, BSE, S/B, S + B3m dating from SSBP/1 inoculation (S), and S + B3m dating from BSE inoculation (B). Results b i In C, lines represent natural scrapie (NS) and NS superinfected with BSE at 8 months of age (NS + B8m) or 21 months of age (NS + B21m). For natural scrapie incubation periods are calculated from birth (exposure to scrapie). Figure 1. Incubation periods of individual VRQ/ARQ, ARQ/ARQ and VRQ/VRQ sheep. Sheep were inoculated with SSBP/1 scrapie, BSE or mixtures (A,B) or were infected with natural scrapie and then superinfected with BSE (C). The codon 141 PRNP genotype is indicated: LL141 (grey fill); LF141 (black fill); FF141 (no fill). Circles are clinical and pathology positive sheep, triangles are non-TSE animals. In A and B, lines represent SSBP/1, BSE, S/B, S + B3m dating from SSBP/1 inoculation (S), and S + B3m dating from BSE inoculation (B). In C, lines represent natural scrapie (NS) and NS superinfected with BSE at 8 months of age (NS + B8m) or 21 months of age (NS + B21m). For natural scrapie incubation periods are calculated from birth (exposure to scrapie). In ARQ/ARQ sheep (n = 18) receiving a mixed inoculation (S/B), 15 developed clinical TSE signs with mean incubation period 918 ± 195 dpi indicative of a BSE incubation period. Although this group included no LL141 sheep, there was evidence that the FF141 homozygotes had shorter incubation periods than LF141 heterozygotes (mean 791 ± 83 and mean 1107 ± 153, respectively), although a slight overlap is present between groups (Fig. 1b; p = 0.002). Two further LF141 sheep remained healthy until they were culled at 1960 and 1975 dpi; one of these was heterozygous for the M112T PRNP polymorphism, which has previously been associated with resistance to TSEs44. A further animal was culled due to intercurrent disease at 421 days was also not included in the analysis. y y In the S + B3m (n = 17), all sheep developed TSE clinical signs. Mean incubation period was 998 ± 206 days post scrapie challenge and 914 ± 206 days post BSE challenge, suggestive of BSE pathology. Again, the LF141 animals had longer mean incubation periods than LL141 and FF141 animals (p = 0.006), but again there was some overlap (Fig. 1b). Across all ARQ/ARQ animals that produced a BSE like incubation period, TSE disease incubation period was strongly influenced by the genotype at codon 141, with heterozygote LF141 sheep having extended duration to clinical disease (p < 0.001). Results b i In the group that received an inoculation of BSE at 21 m of age (NS + B21m, n = 9), incubation time was 803 ± 68 days. These incubation times are very similar to those of the control group showing no effect of the BSE super-infection (p = 0.69). Times of death following the BSE inoculation were 532 ± 53 days and 182 ± 68 days for NS + B8m and NS + B21m groups, respectively, and these times are likely to be too short to be BSE incubation periods or for the clinical signs to be the result of BSE rather than natural scrapie. We do not have data on subcutaneous inoculation of VRQ/VRQ sheep with BSE alone, however after intracerebral inoculation of VRQ/VRQ sheep with BSE, the average incubation period was > 1000 days5. and animals were culled with incubation time of 777 ± 50 days. In the group that received an inoculation of BSE at 21 m of age (NS + B21m, n = 9), incubation time was 803 ± 68 days. These incubation times are very similar to those of the control group showing no effect of the BSE super-infection (p = 0.69). Times of death following the BSE inoculation were 532 ± 53 days and 182 ± 68 days for NS + B8m and NS + B21m groups, respectively, and these times are likely to be too short to be BSE incubation periods or for the clinical signs to be the result of BSE rather than natural scrapie. We do not have data on subcutaneous inoculation of VRQ/VRQ sheep with BSE alone, however after intracerebral inoculation of VRQ/VRQ sheep with BSE, the average incubation period was > 1000 days5. Differential western blot analysis of PrPSc in brain from inoculated sheep. The determination of the presence of BSE or scrapie-like PrPSc in brain was carried out by western blot analysis of proteinase K (PK) digested samples (Fig. 2, Supplementary Fig. S1, Table 2 for individual animals). BSE can be distinguished from scrapie on western blot by the slightly lower molecular weight of the unglycosylated PrPSc band (19 kDa vs 20 kDa). This reflects an altered PK cleavage site resulting in reduced staining by monoclonal antibody P4 in comparison to 6H4 in BSE samples, due to loss of the P4 epitope39. Results b i Shaded samples indicate sheep inoculated/infected with a single strain, all of these samples conformed to established molecular phenotypes of BSE or scrapie PrPSc and so provided within-blot controls for these phenotypes. Where samples were subjected to repeat analysis, the assay gave consistent results (examples shown for Z107, 70 × 71 and Z13). Figure 2. PrPSc phenotype in sheep brain. Brain homogenates were digested with PK and analysed on western blots probed with 6H4 (A) or P4 (B). Representative blots are shown. Approximate molecular weight (MW) indicated at 30 kDa and 20 kDa. Individual sheep references (e.g. Z107 or 70 × 71 formats) are indicated. A relatively low MW for the unglycosylated PrP band with 6H4 staining or a relatively low P4 signal (a ratio of 6H4/P4 signal of > 2) indicated the PrPSc had a BSE phenotype; strong P4 staining (a ratio of 6H4/P4 signal of 2 or less) and a relatively high MW unglycosylated band indicated a scrapie phenotype. These criteria and strain phenotypes are indicated. Shaded samples indicate sheep inoculated/infected with a single strain, all of these samples conformed to established molecular phenotypes of BSE or scrapie PrPSc and so provided within-blot controls for these phenotypes. Where samples were subjected to repeat analysis, the assay gave consistent results (examples shown for Z107, 70 × 71 and Z13). Figure 2. PrPSc phenotype in sheep brain. Brain homogenates were digested with PK and analysed on western blots probed with 6H4 (A) or P4 (B). Representative blots are shown. Approximate molecular weight (MW) indicated at 30 kDa and 20 kDa. Individual sheep references (e.g. Z107 or 70 × 71 formats) are indicated. A relatively low MW for the unglycosylated PrP band with 6H4 staining or a relatively low P4 signal (a ratio of 6H4/P4 signal of > 2) indicated the PrPSc had a BSE phenotype; strong P4 staining (a ratio of 6H4/P4 signal of 2 or less) and a relatively high MW unglycosylated band indicated a scrapie phenotype. These criteria and strain phenotypes are indicated. Shaded samples indicate sheep inoculated/infected with a single strain, all of these samples conformed to established molecular phenotypes of BSE or scrapie PrPSc and so provided within-blot controls for these phenotypes. Where samples were subjected to repeat analysis, the assay gave consistent results (examples shown for Z107, 70 × 71 and Z13). and animals were culled with incubation time of 777 ± 50 days. Results b i (p ) Comparing the results of mixed infections to those of the single inocula in ARQ/ARQ sheep, the mixed infection groups had incubation periods similar to BSE infection (p = 0.91 and p = 0.95 for S/B and S + B3m, respectively) and significantly different from those for scrapie (p < 0.001). Incubation times of natural scrapie sheep super‑infected with BSE. Incubation time of VRQ/ VRQ natural scrapie (NS) Roslin sheep (n = 6) was 793 d ± 74 until scrapie clinical signs developed. Of those animals which also received an inoculation of BSE at 8 m of age (NS + B8m, n = 9) clinical TSE signs developed Scientific Reports \| (2021) 11:11931 \| https://doi.org/10.1038/s41598-021-91397-8 www.nature.com/scientificreports/ Figure 2. PrPSc phenotype in sheep brain. Brain homogenates were digested with PK and analysed on western blots probed with 6H4 (A) or P4 (B). Representative blots are shown. Approximate molecular weight (MW) indicated at 30 kDa and 20 kDa. Individual sheep references (e.g. Z107 or 70 × 71 formats) are indicated. A relatively low MW for the unglycosylated PrP band with 6H4 staining or a relatively low P4 signal (a ratio of 6H4/P4 signal of > 2) indicated the PrPSc had a BSE phenotype; strong P4 staining (a ratio of 6H4/P4 signal of 2 or less) and a relatively high MW unglycosylated band indicated a scrapie phenotype. These criteria and strain phenotypes are indicated. Shaded samples indicate sheep inoculated/infected with a single strain, all of these samples conformed to established molecular phenotypes of BSE or scrapie PrPSc and so provided within-blot controls for these phenotypes. Where samples were subjected to repeat analysis, the assay gave consistent results (examples shown for Z107, 70 × 71 and Z13). Figure 2. PrPSc phenotype in sheep brain. Brain homogenates were digested with PK and analysed on western blots probed with 6H4 (A) or P4 (B). Representative blots are shown. Approximate molecular weight (MW) indicated at 30 kDa and 20 kDa. Individual sheep references (e.g. Z107 or 70 × 71 formats) are indicated. A relatively low MW for the unglycosylated PrP band with 6H4 staining or a relatively low P4 signal (a ratio of 6H4/P4 signal of > 2) indicated the PrPSc had a BSE phenotype; strong P4 staining (a ratio of 6H4/P4 signal of 2 or less) and a relatively high MW unglycosylated band indicated a scrapie phenotype. These criteria and strain phenotypes are indicated. Results b i As expected, VRQ/ARQ sheep infected with SSBP/1 alone (n = 4) showed the typical scrapie-like PrPSc western blot profile, whilst the two VRQ/ARQ sheep infected with BSE alone displayed the BSE western blot profile (n = 2). VRQ/ARQ sheep infected with S/B or S + B3m (n = 10 in total) all produced a scrapie-like PrPSc pattern on western blot, in agreement with their incubation periods, which were as expected for SSBP/1 in this genotype. Similarly, ARQ/ARQ sheep infected with SSBP/1 alone (n = 2) showed scrapie-like PrPSc whereas those infected with BSE alone (n = 6) showed typi- cal BSE-like PrPSc profiles. ARQ/ARQ sheep infected with S/B (n = 8) showed PrPSc patterns that were BSE-like, again in accordance with the incubation period data, which was typical of ARQ/ARQ sheep infected with BSE rather than SSBP/1. Six out of eight ARQ/ARQ sheep infected with S + B3m, gave a BSE-like PrPSc pattern on western blot. However, in two animals (Z73 and Z115) with the LF141 genotype, western blotting of PrPSc gave a scrapie-like signature.i p g In the natural scrapie sheep groups (n = 9), the PrPSc profile on western blot were all indicative of scrapie, regardless of whether or not they were also challenged with BSE. https://doi.org/10.1038/s41598-021-91397-8 Scientific Reports \| (2021) 11:11931 \| www.nature.com/scientificreports/ Table 2. BSE-specific sPMCA test results compared with BSE and scrapie PrPSc determination by west blot analysis for individual sheep. a Animal codes are given (e.g. Z75). b SC: Scrapie. c NEG: negative for B by PMCA, –: not done, A: sample not analysed due to autolysis. d PSLN: prescapular lymph node e − 8 o indicate superinfection with BSE was carried out at 8 or 21 months of age, respectively. Table 2. BSE-specific sPMCA test results compared with BSE and scrapie PrPSc determination by western blot analysis for individual sheep. a Animal codes are given (e.g. Z75). b SC: Scrapie. c NEG: negative for BSE by PMCA, –: not done, A: sample not analysed due to autolysis. d PSLN: prescapular lymph node e − 8 or − 21 indicate superinfection with BSE was carried out at 8 or 21 months of age, respectively. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. sPMCA amplification of BSE prions in sheep brain and LRS tissues. Following amplification of samples by sPMCA, PK-resistant PrPSc was detected with either SHa31 (top panels) or P4 (lower panels). Representative blots are shown. Br brain, Sp spleen, Ln PSLN tissue. Scrapie (S+) and BSE (B+) brain controls were also analysed on each blot. Z37 and Z43 were ARQ/ARQ sheep inoculated with BSE only; Z97 was an ARQ/ARQ sheep inoculated with S/B; Z90, Z115 and Z73 were ARQ/ARQ sheep inoculated with S + B3m; Z94 was a VRQ/ARQ sheep inoculated with SSBP/1 only; Z65 and Z48 were VRQ/ARQ sheep inoculated with BSE only; sheep 75 × 44 had natural scrapie and was superinfected, NS + B8m. Molecular weight markers (M) are shown at 20, 30 and 40 kDa and positions are also indicated on each blot. Figure 3. sPMCA amplification of BSE prions in sheep brain and LRS tissues. Following amplification of samples by sPMCA, PK-resistant PrPSc was detected with either SHa31 (top panels) or P4 (lower panels). Representative blots are shown. Br brain, Sp spleen, Ln PSLN tissue. Scrapie (S+) and BSE (B+) brain controls were also analysed on each blot. Z37 and Z43 were ARQ/ARQ sheep inoculated with BSE only; Z97 was an ARQ/ARQ sheep inoculated with S/B; Z90, Z115 and Z73 were ARQ/ARQ sheep inoculated with S + B3m; Z94 was a VRQ/ARQ sheep inoculated with SSBP/1 only; Z65 and Z48 were VRQ/ARQ sheep inoculated with BSE only; sheep 75 × 44 had natural scrapie and was superinfected, NS + B8m. Molecular weight markers (M) are shown at 20, 30 and 40 kDa and positions are also indicated on each blot. sPMCA analysis to detect BSE in inoculated sheep. During the study, a sPMCA method became available that can detect very low amounts of BSE PrPSc in a background of an excess of scrapie41, 42. The assay does not detect scrapie PrPSc. Previous reports use the method with brain tissues, here it was applied to both brain, spleen and prescapular lymph node (PSLN) material. Initial analysis demonstrated that the use of LRS tissue did not have any matrix effects on the assay and the lowest level of ovine BSE brain spike that was analysed (a 10,000 × dilution of a BSE infected brain seed) was detected when diluted into brain or the LRS healthy tissues (data not shown). www.nature.com/scientificreports/ ( ) Selected samples from each challenge group or control groups were analysed by sPMCA using both LRS and brain tissues; each animal had also been analysed by a differentiating western blot that could define a dominant scrapie or BSE PrPSc type in brain (Table 2, Fig. 3, Supplementary Fig. S2). The sPMCA assay did not detect BSE in any scrapie-only experimental challenge controls, as expected (4 × each of brain, spleen and PSLN samples). All samples from BSE-only challenged animals were positive for BSE with the exception of two spleen samples (from 8 × brain, 8 × spleen and 7 × PSLN samples). This may indicate that the BSE agent does not accumulate consistently within the spleen at clinical stages of disease. For experimental mixed infections (S/B, S + B3m) in sheep with VRQ/ARQ genotype, the sPMCA correlated with the western blot and incubation period data, in that all sheep examined (n = 6) had no detectable BSE PrPSc in brain, spleen or PSLN. For ARQ/ARQ sheep challenged with S/B (n = 5), BSE was detected in all brains and all but one animal had BSE present in the LRS samples analysed. The exception was Z97 that was negative for BSE in spleen and PSLN. In ARQ/ARQ sheep challenged with S + B3m, BSE was present in the brain and /or PSLN and spleen for all four animals analysed. For Z115, the sPMCA data showed BSE present in brain and LRS tissues and this was discordant with a dominant scrapie western blot in the brain. Similarly, Z73 was previously shown to have a dominant scrapie brain PrPSc profile in western blots and whilst the brain was also negative for BSE by sPMCA, BSE could be detected in spleen and PSLN. g y p For NS infection, no BSE was found in scrapie-only controls (n = 2), as expected. In the animals superinfected NS + B21m (n = 2), no BSE was present in brain or LRS. When BSE superinfection was NS + B8m (n = 3), all samples examined were negative for BSE, except the brain sample from animal 75 × 44. The western blot profile for this brain indicated scrapie PrPSc was dominant. Immunohistochemistry analysis of tissue from sheep demonstrating a dominant scrapie western blot profile in brain but also propagating BSE in the LRS. www.nature.com/scientificreports/ Previous work has shown that BSE and scrapie can be distinguished on IHC by including certain antibodies (e.g. 12B2) that label intracellular PrPd (intraneuronal in brain, in tingible body macrophages of lymphoid tissues) in scrapie-infected, but not BSE-infected sheep40. This method was therefore applied to samples from the two animals in the S + B3m group that demonstrated discordant data in western blot and sPMCA, Z115 and Z73. Both demonstrated a dominant scrapie phenotype in brain by western blot but the presence of BSE prion in brain and/or LRS by sPMCA. Tissues for animal Z73 could not be reliably analysed due to autolysis. IHC data for Z115 confirmed that the dominant TSE type in brain was scrapie whilst that in LRS (tonsil) was BSE (Fig. 4). Results b i Prnp genotype Inoculation group Animal ID (codon 141 subgroup)a Western blotb BSE-specific sPMCAc Brainc Brain Spleen PSLNd VRQ/ARQ SSBP/1 Z75 (LF) SC NEG NEG NEG Z94 (LF) SC NEG NEG NEG Z26 (LF) SC – – – Z45 (LL) SC – – – BSE Z48 (LL) BSE BSE NEG BSE Z65 (LL) BSE BSE BSE BSE S/B Z92 (LL) SC NEG NEG NEG Z114 (LF) SC NEG NEG NEG S + B3m Z66 (LF) SC NEG NEG NEG Z146 (LL) SC NEG NEG NEG Z161 (LF) SC NEG NEG NEG Z172 (LF) SC NEG NEG NEG Z121 (LF) SC – – – Z136 (LL) SC – – – Z49 (LL) SC – – – Z20 (LF) SC – – – ARQ/ARQ SSBP/1 Z10 (LF) SC NEG NEG NEG Z35 (FF) SC NEG NEG NEG BSE Z13 (FF) BSE BSE BSE BSE Z28 (LF) BSE BSE NEG BSE Z37 (LL) BSE BSE BSE BSE Z43 (LF) BSE BSE BSE A Z53 (LF) BSE BSE BSE BSE Z58 (FF) BSE BSE BSE BSE S/B Z79 (FF) BSE BSE BSE BSE Z91 (FF) BSE BSE BSE BSE Z97 (FF) BSE BSE NEG NEG Z113 (FF) BSE BSE BSE BSE Z116 (FF) BSE BSE BSE BSE Z88 (LF) BSE – – – Z107 (LF) BSE – – – Z63 (FF) BSE – – – S + B3m Z54 (FF) BSE BSE NEG NEG Z90 (FF) BSE BSE BSE BSE Z115 (LF) SC BSE BSE BSE Z73 (LF) SC NEG BSE BSE Z64 (FF) BSE – – – Z44 (FF) BSE – – – Z59 (LF) BSE – – – Z139 (FF) BSE – – – VRQ/VRQ NS 74 × 66 (LL) SC NEG NEG NEG 75 × 17 (LL) SC NEG NEG NEG 70 × 71 (LL) SC – – – NS + Be 70 × 64 (LL)-21 SC NEG NEG NEG 72 × 35 (LL)-21 SC NEG NEG NEG 74 × 44 (LL)-8 SC NEG NEG NEG 75 × 09 (LL)-8 SC NEG NEG NEG 75 × 44 (LL)-8 SC BSE NEG NEG 74 × 90 (LL)-8 SC – – – https://doi.org/10.1038/s41598-021-91397-8 Scientific Reports \| (2021) 11:11931 \| Discussionh There are relatively few reports on the study of mixed prion infections. Rodent models have demonstrated that experimental infection with two prion strains can result in the dominance of the strain with the most favourable replication properties such as a shorter incubation time. In these circumstances this dominant strain can cause pathology with correlating PrPSc phenotype accumulation at the end stage of disease36, 38, 45. However, models have also shown that the slower replicating strain can delay or completely block the disease of the faster strain33, 35, 46. https://doi.org/10.1038/s41598-021-91397-8 Scientific Reports \| (2021) 11:11931 \| www.nature.com/scientificreports/ Figure 4. Discriminatory immunohistochemistry of sheep brain and tonsil from a mixed infection sheep with scrapie signal in brain and BSE in periphery. Sections of medulla [lateral cuneate nucleus; (A, B); × 20] and tonsil [(C,D); × 10) from VRQ/ARQ sheep Z115 were stained with antibody R145 in (A) and (C); or 12B2 in (B) and (D). Results show labelling of intraneuronal PrPd with both R145 and 12B2 in brain (A,B), whereas in tonsil (C,D) labelling of tingible body macrophages within germinal centre was seen only with R145, the 12B2 reaction being negative. Figure 4. Discriminatory immunohistochemistry of sheep brain and tonsil from a mixed infection sheep with scrapie signal in brain and BSE in periphery. Sections of medulla [lateral cuneate nucleus; (A, B); × 20] and tonsil [(C,D); × 10) from VRQ/ARQ sheep Z115 were stained with antibody R145 in (A) and (C); or 12B2 in (B) and (D). Results show labelling of intraneuronal PrPd with both R145 and 12B2 in brain (A,B), whereas in tonsil (C,D) labelling of tingible body macrophages within germinal centre was seen only with R145, the 12B2 reaction being negative. The delayed disease then usually has the clinical presentation and PrPSc of the faster strain. Alternatively, mixed nfections can result in independent replication of both strains37.hih This is the first study on experimentally mixed infections in sheep. There has been considerable historical concern that the BSE agent may have propagated in sheep and been masked by the scrapie agent. This was a particular concern in the UK where endemic scrapie existed and it was considered that sheep were probably exposed to BSE-contaminated feed during the bovine BSE epidemic (Statement by Spongiform Encephalopathy Advisory Committee: “Experimental transmission of BSE to sheep and risk of exposure to BSE through feed (10/07/1996). Archived at: https://webarchive.nationalarchives.gov.uk/20070308092252/http://www.seac.gov. uk/statements/state10jul96.htm). www.nature.com/scientificreports/ two survivors at the end of the study at 2002 dpi. This prolonged incubation period is similar to a previous study that challenged ARQ/ARQ sheep with SSBP/1 and reported no clinical disease at 1108 days48. For BSE alone, all six challenged sheep developed clinical disease with mean incubation period of 909 ± 289 dpi. For challenge with both TSEs at the same time, the incubation periods were more reflective of BSE infection but the attack rate was incomplete. Western blot and sPMCA showed that the PrPSc present in brain and LRS was BSE. Combined, data indicates the animals in this cohort likely all died from the BSE agent that was the dominant PrPSc in the tissues analysed. For delayed challenge with BSE after scrapie inoculations, the outcomes were more complex. The incubation periods were relatively rapid and with high attack rates indicating BSE infections. Biochemical analysis of tissues showed that most sheep displayed dominant BSE PrPSc in all tissues analysed. However, two sheep had a dominant scrapie PrPSc/PrPd phenotype in brain (examined by western blot and/or IHC) despite displaying incubation periods most indicative of BSE infection. These animals also had BSE agent present in LRS tissues and/or brain when analysed by sPMCA. This data correlates with a small number of individual animals in a rodent model of prion superinfections where they displayed a dominant PrPSc phenotype of the slower blocking strain yet the incubation period and clinical signs were indicative of the faster strain23. There is no assay available that can detect very low levels of scrapie PrPSc in the presence of high levels of BSE PrPSc and so it is not known whether the ARQ/ARQ sheep with BSE pathology and a dominant BSE PrPSc phenotype harboured low levels of scrapie PrPSc.l p When considering the polymorphism at position 141, this did not appear to have any influence on the incubation periods for VRQ/ARQ sheep where scrapie pathology was dominant. In contrast, in ARQ/ARQ sheep where BSE-like incubation periods were observed, the LF141 genotype had significantly longer incubation periods than homozygous genotypes. A similar effect has been previously reported for BSE infections in ARQ/ ARQ sheep49. The small number of ARQ/ARQ sheep infected with SSBP/1 only did not show any evidence that codon 141 influenced incubation period. l For natural scrapie infections followed by BSE challenge, all animals displayed scrapie like PrPSc in brain by western blot. Methods Sh i Sheep were challenged with either SSBP/1 or BSE by injection of two separate 1 ml doses (2 ml total dose) into each hind leg. The mixed injection groups, herein described as S/B, were each injected with 2 ml of a 1:1 by volume mixture of SSBP/1 and BSE homogenates in each hind leg. The separate injection groups, herein described as S + B3m, were first injected with 2 × 1 ml SSBP/1, and 3 months (84 days) later with 2 × 1 ml BSE in each hind leg. All sheep were between 789 and 878 days old at the primary challenge. To investigate the interaction of BSE and natural scrapie, Cheviots were sourced from the Roslin Scrapie Flock (NPU Cheviot Flock). Natural scrapie is endemic in these sheep affecting 100% of VRQ/VRQ sheep at around 2 years of age. Infection is assumed to occur in these animals at or around the time of lambing but the timing is not known precisely. Incubation times for disease in these animals is calculated from birth. It is likely that by the time the sheep were exposed to BSE, natural scrapie infection would have been well established. This part of the study aimed to replicate more closely a realistic situation whereby a sheep incubating natural scrapie might become superinfected with BSE by peripheral infection. A control group of VRQ/VRQ sheep was left to develop natural scrapie with no additional challenge and are described as NS sheep. Additional animals were inoculated subcutaneously with BSE as above (2 × 1 ml of 10% BSE cattle brain homogenate into the hind limbs). g p y p y g To investigate the interaction of BSE and natural scrapie, Cheviots were sourced from the Roslin Scrapie Flock (NPU Cheviot Flock). Natural scrapie is endemic in these sheep affecting 100% of VRQ/VRQ sheep at around 2 years of age. Infection is assumed to occur in these animals at or around the time of lambing but the timing is not known precisely. Incubation times for disease in these animals is calculated from birth. It is likely that by the time the sheep were exposed to BSE, natural scrapie infection would have been well established. This part of the study aimed to replicate more closely a realistic situation whereby a sheep incubating natural scrapie might become superinfected with BSE by peripheral infection. www.nature.com/scientificreports/ No amplifiable BSE PrPSc could be detected in spleen or PSLN by sPMCA. However, one of five brain samples analysed by sPMCA showed the presence of BSE even though the dominant western blot signature was scrapie-like. Again, this demonstrates the replication of multiple prion strains within a ruminant host and the masking of the presence of BSE prion by a dominant scrapie pathology. Whilst this was not an exhaustive study of ovine genotypes and their susceptibility to mixed infections, the study demonstrates that in sheep, a rapid TSE strain usually ‘out competes’ a slower propagating strain and causes the pathology/clinical disease. However, the data also shows that the dominant PrPSc does not always cor- relate with this pathology, for instance scrapie can be the most dominant PrPSc in brain even though incubation periods would indicate a BSE disease. Furthermore, the dominant PrPSc strain found in brain does not preclude the presence of another co-infecting strain. Here, of thirteen co-infected sheep that displayed a scrapie-like biochemical phenotype in brain by western blot analysis and were also analysed by sPMCA, three had detect- able BSE in brain and/or LRS. This study therefore highlights that surveillance for specific TSE strains requires analyses of distinct tissues with a suite of biochemical tests to fully elucidate their presence or likely absence. This is significant as strains present at low levels within mixed infections could potentially become the dominant, disease-causing agent due to changes in the replication environment, for example sub-passage into a host with a different PRNP genotype. Discussionh However, considerable surveillance has failed to detect any BSE cases in sheep and only two from goats, despite sheep being susceptible to bovine BSE through experimental oral challenge47. This raises the questions of how coinfection of scrapie and BSE compete or co-exist within a ruminant host. To investigate this, we carried out mixed infection studies in sheep with scrapie and ovine BSE. The study looked at sheep challenged with both TSEs simultaneously and also when challenged with BSE three months after scra- pie. In addition, sheep naturally infected with scrapie were challenged with BSE at eight and 21 months of age. h h b d d VRQ/ARQ sheep propagate SSBP/1 scrapie with a mean incubation period was 234 ± 41 days post injection (dpi), which compares well with previous studies48. This disease progression is more effective and more rapid (approximately 5 × shorter incubation period) than BSE. Challenge with BSE alone resulted in only two out of six sheep developing TSE clinical signs with incubation periods of 1008 and 1229 dpi. In contrast, in a previous study, VRQ/ARQ (LL141) sheep infected with BSE by the intracerebral route had average incubation periods of 875 ± 77 dpi and a 100% attack rate5. Data illustrates that infection with BSE by subcutaneous inoculation is less efficient than intracerebral inoculation. Mixed infections of such sheep with both strains resulted in a rapid progression to clinical disease and only scrapie PrPSc could be detected in brain or LRS by western blotting in animals that were tested, and sPMCA failed to detect low levels of BSE PrPSc. These data indicate that the VRQ/ ARQ sheep died from scrapie infections before the BSE agent could accumulate to a detectable level and that the BSE agent did not compete with the scrapie agent when animals were infected with both strains. This supports the conclusion that challenge of sheep with BSE did not interfere with scrapie disease progression in a genotype where scrapie is the faster replicating strain.hf p p g The propagation of SSBP/1 and BSE in ARQ/ARQ sheep presents a different scenario, where BSE propagates more efficiently and slightly more rapidly (~ 1.7 × shorter incubation period) than scrapie. For SSBP/1 alone, four out of the six sheep developed TSE clinical signs, with a mean incubation period of 1541 ± 392 dpi, with https://doi.org/10.1038/s41598-021-91397-8 Scientific Reports \| (2021) 11:11931 \| www.nature.com/scientificreports/ Methods Sh i Sheep inoculations. For experimental infections, Cheviot sheep were sourced from the Defra flock of New Zealand origin that was free of classical scrapie. The sheep genotypes were confirmed as previously described47 (eg VRQ/ARQ). After the project was initiated, and animal groups chosen, additional sequencing detected vari- ation at other codons (112, 141 and 168) on the ARQ alleles encoded by the sheep. Numbers of animals with codon 112 or 168 variants were too small to be informative (single animals), however codon 141 variation (L replaced with F) was much greater, although in uneven proportions, and is presented here as a genotype at codon 141 alone (LL, FF or LF in Tables, or e.g. LL141 in text). The other relevant sheep PRNP allele is VRQ, which shows no variation at codons 112, 141 or 168. Sheep of genotypes VRQ/ARQ and ARQ/ARQ were challenged with the well characterised scrapie brain pool, SSBP/148 and/or with cattle BSE, an inoculum termed BBB/2/02 made by pooling six cattle brains sup- plied by the Animal and Plant Health Agency (APHA) and used previously in other sheep challenge studies50. Animals were injected subcutaneously in the medial thigh with 10% (w/v) homogenates of SSBP/1 or BSE alone or together in mixed inoculations. Sheep were challenged with either SSBP/1 or BSE by injection of two separate 1 ml doses (2 ml total dose) into each hind leg. The mixed injection groups, herein described as S/B, were each injected with 2 ml of a 1:1 by volume mixture of SSBP/1 and BSE homogenates in each hind leg. The separate injection groups, herein described as S + B3m, were first injected with 2 × 1 ml SSBP/1, and 3 months (84 days) later with 2 × 1 ml BSE in each hind leg. All sheep were between 789 and 878 days old at the primary challenge. h f d l h d f h l Sheep of genotypes VRQ/ARQ and ARQ/ARQ were challenged with the well characterised scrapie brain pool, SSBP/148 and/or with cattle BSE, an inoculum termed BBB/2/02 made by pooling six cattle brains sup- plied by the Animal and Plant Health Agency (APHA) and used previously in other sheep challenge studies50. Animals were injected subcutaneously in the medial thigh with 10% (w/v) homogenates of SSBP/1 or BSE alone or together in mixed inoculations. www.nature.com/scientificreports/ When comparing coinfection groups to BSE only challenge, the incubation times from BSE challenge were used; when compar- ing coinfection groups to scrapie only challenge, the incubation times from scrapie challenge were used. When comparing incubation periods between sheep homozygote or heterozygote for codon 141, an unpaired Student’s T test was used. Detection of BSE or scrapie PrPSc by western blotting. Selected samples were subjected to PrPSc analysis by PK digestion and western blot analysis as previously described39, 40. Samples were run on 16% (w/v) Tris–glycine gels (Invitrogen, Paisley, UK) and immunoblotted onto polyvinylidene difluoride (PVDF) mem- branes. For detection of sheep PrPSc, two monoclonal antibodies were used: 6H4 (epitope 144–152 in human PrP, Prionics, Schlieren, Switzerland, 2 mg/ml, diluted 1:5000), and P4 (epitope 93–99 in ovine PrP; R-Biopharm, Darmstadt, Germany, 1 mg/ml, diluted 1:2500). The "visualisation" process used a chemiluminescence substrate (Roche, Lewes, UK) and Lumi-film (Roche). For re-probing a membrane with a second antibody, the 6H4 anti- body was removed using Restore (Thermo Fisher Scientific, Loughborough, UK) and then the membrane was re-probed with the P4 antibody. Each blot was imaged at 3 different exposure times (30 s, 3 min and 10 min) to allow visualisation of the unglycosylated band. The molecular weight of this band was determined by com- parison to the MW markers and was assigned as relatively low or high with a difference of ~ 2 kDa. Densitom- etry analysis (using Image J) of blots allowed identification of the ratio of PrPSc signals after probing with each antibody, a ratio of 6H4/P4 signal greater than 2 indicated BSE, analysis criteria are as previously described39. Samples were defined as BSE or scrapie when they consistently met both MW and relative P4 reactivity criteria. However, some ovine BSE samples could not be resolved to determine the MW of the unglycosylated band (due to very low levels of this form of PrPSc), in these cases the definition of BSE was confirmed by the P4 reactivity criteria alone. Serial PMCA to detect BSE PrPSc. Selected sheep tissues were examined using sPMCA optimised for the specific detection of BSE as previously described41. This technique scores samples as positive or negative for BSE and does not detect scrapie. www.nature.com/scientificreports/ These animals are described as NS + B. Animals were either challenged with BSE at 8 months of age (NS + B8m; age at challenge was between 234 and 258 days) or were challenged at 21 months of age (NS + B21m; age at chal- lenge was between 614 and 633 days), the latter time point is just prior to the expected development of natural scrapie clinical signs. These animals are described as NS + B. Animals were either challenged with BSE at 8 months of age (NS + B8m; age at challenge was between 234 and 258 days) or were challenged at 21 months of age (NS + B21m; age at chal- lenge was between 614 and 633 days), the latter time point is just prior to the expected development of natural scrapie clinical signs. p g All of the animals were observed daily for clinical signs of TSE and culled humanely once such signs were clearly seen at agreed end-points. Any animals with intercurrent illness were treated by a veterinary surgeon and culled humanely if recovery was not apparent. All animals were subject to post mortem collection of brain and lymphoid tissues, for subsequent analysis. Times between inoculation and culling were recorded as follows. An incubation period was assigned when an animal had both TSE clinical signs and positive PrPd detection by IHC analysis of brain. A survival time was assigned when an animal was negative for one or both of these features (TSE signs and PrPd). Sections of the brainstem at the level of the obex and the thalamus from all sheep, including intercurrent deaths, were labelled by IHC using the monoclonal antibody BG4 (binding epitopes at 47–57 and 89–99 in bovine PrP), in order to achieve a positive or negative confirmation of disease status (data not shown but outcome is implicit in the reported incubation periods). All sheep studies were reviewed and approved by Animal Welfare and Ethical Review Committees at IAH Compton and the Roslin Institute and carried out in accordance with the Animals (Scientific Procedures) Act 1986, under the authority of UK Home Office Project and Personal Licenses (and complying with the ARRIVE guidelines). Statistical analysis of incubation period data was carried out using one way ANOVA followed by Tukey’s multiple comparison analysis when comparing incubation times between challenge groups. www.nature.com/scientificreports/ In summary, tissue (100–150 mg) was homogenised in lysis buffer (PBS containing 0.5% (w/v) sodium deoxycholate and 0.5% (v/v) NP40 to give a final 10% (w/v) tissue) using a mini beadbeater set at 4800 rpm for 60 s (brain and spleen) or 180 s (PSLN). Homogenised samples were aliquoted and frozen at − 80 °C. sPMCA amplification was carried out for 5 rounds in total using alternating AHQ/AHQ and VRQ/ VRQ sheep brain substrates for each round. For amplifications, two different sPMCA sonicator horns were each used to analyse a replicate of the same sample. A sample was noted as being BSE positive if either replicate pro- duced PrPSc with a BSE western blot phenotype. During the sPMCA, samples were diluted 1:3 every 24 h with the substrate, as previously described41. An equal number of test samples and scrapie only (negative control) samples were amplified on each sPMCA run. No negative controls produced any PrPSc signal (data not shown). Following sPMCA, samples were digested with 50 µg/ml PK at 37 °C for 1 h in the presence of 0.5% (w/v) SDS. An equivalent to 5 µl of the original PMCA reaction was then subjected to SDS PAGE using a 12% (w/v) NuPAGE electrophoresis system with MOPs buffer. The separated proteins were electroblotted to PVDF membrane and blocked with 3% (w/v) milk protein overnight. Duplicate blots were probed with the monoclonal antibody SHa31 (epitope 145–151 in hamster PrP) or P4. Densitometry analysis of blots probed with SHa31 allowed identification of samples that produced a PrPSc signal above the background produced with control scrapie samples. Samples were scored positive for BSE when producing a SHa31 signal above background and a ratio of their SHa31/P4 signal greater than 2.9, analysis criteria are as previously described41, 42. We have previously demonstrated the limit of detection of BSE by sPMCA in the presence of scrapie-infected brain41. The assay gave 100% sensitivity at 1/50 dilution of BSE in scrapie, falling to 90% and 80% sensitivity at 1/225 and 1/500 dilutions, respectively. Here, a demonstration of the assay limit of detection was again performed, to further evaluate any effects of the spleen and PSLN matrix with respect to the amplification of BSE. These experiments were carried out twice using two different sPMCA sonicators. Due to delays in the availability of spleen tissue from TSE-free sheep, Serial PMCA to detect BSE PrPSc. Methods Sh i A control group of VRQ/VRQ sheep was left to develop natural scrapie with no additional challenge and are described as NS sheep. Additional animals were inoculated subcutaneously with BSE as above (2 × 1 ml of 10% BSE cattle brain homogenate into the hind limbs). Scientific Reports \| (2021) 11:11931 \| https://doi.org/10.1038/s41598-021-91397-8 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Selected sheep tissues were examined using sPMCA optimised for the specific detection of BSE as previously described41. This technique scores samples as positive or negative for BSE and does not detect scrapie. In summary, tissue (100–150 mg) was homogenised in lysis buffer (PBS containing 0.5% (w/v) sodium deoxycholate and 0.5% (v/v) NP40 to give a final 10% (w/v) tissue) using a mini beadbeater set at 4800 rpm for 60 s (brain and spleen) or 180 s (PSLN). Homogenised samples were aliquoted and frozen at − 80 °C. sPMCA amplification was carried out for 5 rounds in total using alternating AHQ/AHQ and VRQ/ VRQ sheep brain substrates for each round. For amplifications, two different sPMCA sonicator horns were each used to analyse a replicate of the same sample. A sample was noted as being BSE positive if either replicate pro- duced PrPSc with a BSE western blot phenotype. During the sPMCA, samples were diluted 1:3 every 24 h with the substrate, as previously described41. An equal number of test samples and scrapie only (negative control) samples were amplified on each sPMCA run. No negative controls produced any PrPSc signal (data not shown). F ll i PMCA l di d i h 50 / l PK 37 °C f 1 h i h f 0 5% ( / ) SDS p pi g p y g Following sPMCA, samples were digested with 50 µg/ml PK at 37 °C for 1 h in the presence of 0.5% (w/v) SDS. An equivalent to 5 µl of the original PMCA reaction was then subjected to SDS PAGE using a 12% (w/v) NuPAGE electrophoresis system with MOPs buffer. The separated proteins were electroblotted to PVDF membrane and blocked with 3% (w/v) milk protein overnight. Duplicate blots were probed with the monoclonal antibody SHa31 (epitope 145–151 in hamster PrP) or P4. Densitometry analysis of blots probed with SHa31 allowed identification of samples that produced a PrPSc signal above the background produced with control scrapie samples. Samples were scored positive for BSE when producing a SHa31 signal above background and a ratio of their SHa31/P4 signal greater than 2.9, analysis criteria are as previously described41, 42. We have previously demonstrated the limit of detection of BSE by sPMCA in the presence of scrapie-infected brain41. The assay gave 100% sensitivity at 1/50 dilution of BSE in scrapie, falling to 90% and 80% sensitivity at 1/225 and 1/500 dilutions, respectively. www.nature.com/scientificreports/ Here, a demonstration of the assay limit of detection was again performed, to further evaluate any effects of the spleen and PSLN matrix with respect to the amplification of BSE. These experiments were carried out twice using two different sPMCA sonicators. Due to delays in the availability of spleen tissue from TSE-free sheep, https://doi.org/10.1038/s41598-021-91397-8 Scientific Reports \| (2021) 11:11931 \| www.nature.com/scientificreports/ these experiments were carried out in a background of healthy ovine brain and PSLN samples and the spleens of tg338 mice (mice expressing ovine VRQ PrP)51. these experiments were carried out in a background of healthy ovine brain and PSLN samples and the spleens of tg338 mice (mice expressing ovine VRQ PrP)51. Detection of BSE or scrapie PrPd by immunohistochemistry. The IHC methods do not result in detection of PrPC but as no PK is used in the process, the PrP protein detected by IHC is referred to here as disease-associated PrP (PrPd)40. The term PrPSc is reserved for the protein detected in PK treated samples. h Brain and lymphoid tissues were analysed using a method that discriminates between BSE and scrapie and has been described in detail several times previously18, 40. When antibodies that recognise specific epitopes within the N terminus of PrP are used, epitopes to the sequence 93–99 of the PrP protein are preserved in SSBP/1 but lost in BSE40. Labelling was with R145 (epitope 221–232 in bovine PrP) and the N-terminal discriminatory antibody 12B2 (epitope 93–97 in bovine PrP). References 81, 10532–10539 (2007).h 2. Thackray, A. M., Hopkins, L., Lockey, R., Spiropoulos, J. & Bujdoso, R. Propagation of ovine prions from “poor” transmitter scrapie isolates in ovine PrP transgenic mice. Exp. Mol. Pathol. 92, 167–174 (2012). g p 3. 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Competition between different scrapie agents in mice. Nature 237 244–245 (1972).f 32. Dickinson, A. G. References Scrapie infectivity correlates with converting activity, protease resist- ance and aggregation of scrapie associated prion protein in guanidine denaturation studies J Virol 71 4107 4110 (1997) 3. Caughey, B., Raymond, G. J., Kocisko, D. A. & Lansbury, P. T. Scrapie infectivity correlates with converting activity, protease resist- ance, and aggregation of scrapie-associated prion protein in guanidine denaturation studies. J. Virol. 71, 4107–4110 (1997).i 4. Saborio, G. P., Permanne, B. & Soto, C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfold- ing. Nature 411, 810–813 (2001).i 5. Morales, R., Duran-Aniotz, C., Diaz-Espinoza, R., Camacho, M. V. & Soto, C. Protein misfolding cyclic amplification of infectious prions. Nat. Protoc. 7, 1397–1409 (2012).hf p 16. Bruce, M. E., McConnell, I., Fraser, H. & Dickinson, A. G. 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Comparative susceptibility of sheep of different origin, breeds and PRNP genotypes to challenge with bovine spongiform encephalopathy and scrapie. PLoS One 10, e0143251 (2015). y g 5. Houston, F. et al. Data availabilityh The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Received: 2 February 2021; Accepted: 24 May 2021 Received: 2 February 2021; Accepted: 24 May 2021 www.nature.com/scientificreports/ Molecular analysis of the abnormal prion protein during coinfection of mice by bovine spongiform encephalopathy and a scrapie agent. J. Virol. 75, 107–114 (2001). l f d d fi S l l l ( ) p p y p g 46. Bartz, J. C. et al. Prion interference is due to a reduction in strain-specific PrPSc levels. J. Virol. 81, 689–697 (2007). 4 H N l S ibili f h l i f i i h b i if h l h d i 47. Hunter, N. et al. Susceptibility of young sheep to oral infection with bovine spongiform encephalopathy decreases significantly after weaning. J. Virol. 86, 11856–11862 (2012).f p y y g p p g p p y gi y after weaning. J. Virol. 86, 11856–11862 (2012). 48. Houston, E. F., Halliday, S. I., Jeffrey, M., Goldmann, W. & Hunter, N. New Zealand sheep with scrapie-susceptible PrP genotype succumb to experimental challenge with a sheep-passaged scrapie isolate (SSBP/1). J. Gen. Virol. 83, 1247–1250 (2002).if t g ( ) 48. Houston, E. F., Halliday, S. I., Jeffrey, M., Goldmann, W. & Hunter, N. New Zealand sheep with scrapie-susceptible PrP genotype succumb to experimental challenge with a sheep-passaged scrapie isolate (SSBP/1). J. Gen. Virol. 83, 1247–1250 (2002).if p g p p g p 9. Tan, B. C. et al. Significant differences in incubation times in sheep infected with bovine spongiform encephalopathy result from variation at codon 141 in the PRNP gene. J. Gen. Virol. 93, 2749–2756 (2012). g 0. Foster, J. D., Parnham, D., Chong, A., Goldmann, W. & Hunter, N. Clinical signs, histopathology and genetics of experimenta transmission of BSE and natural scrapie to sheep and goats. Vet. Rec. 148, 165–171 (2001). 51. Vilotte, J. L. et al. Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine PrP. J. Virol. 75, 5977–5984 (2001). Acknowledgementsh g This work was supported by Defra [Grant number SE1436]; and the Biotechnology and Biological Sciences Research Council [Grant number BB/J014508/1]. The authors thank members of large animal research ser- vices at the IAH, Compton and the Roslin Institute for excellent care of the sheep and technical assistance with experimental procedures. We would also like to thank Suzanne Beckett, Dawn Drummond and David Parnham for contributing to IHC analysis, and Paula Stewart for PRNP genotyping. We acknowledge the contribution of Hugh Simmons and colleagues from the Animal and Plant Health Agency (APHA) and ADAS in breeding and provision of sheep for the experiments. BSE-infected cattle brain homogenates were provided by the Biological Archive Group (formerly TSE Archive) at APHA Weybridge. Author contributions N.H., J.D.F. and E.F.H. designed the sheep study and western blot experiments. M.J. designed the IHC experi- ments. B.C.M. and K.C.G. designed the PMCA experiments. E.F.H. and J.D.F. carried out the sheep study, A.C. performed western blot experiments, K.B. and M.J.O’C. carried out the PMCA experiments, W.G. carried out the genotyping, L.G. carried out the IHC experiments. N.H., E.F.H., B.C.M. and K.C.G. analysed the data and wrote the paper. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 4. Shikiya, R. A., Ayers, J. I., Schutt, C. R., Kincaid, A. E. & Bartz, J. C. Coinfecting prion strains compete for a limiting cellular resource. J. 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Prion strain interactions are highly selective. J. Neurosci. 30, 12094– 12102 (2010). 9. Stack, M. J. et al. Monitoring for bovine spongiform encephalopathy in sheep in Great Britain, 1998–2004. J. Virol. 87, 2099–2107 (2006).f ( ) 0. Jeffrey, M. & Gonzalez, L. Classical sheep transmissible spongiform encephalopathies: Pathogenesis, pathological phenotypes and clinical disease. Neuropathol. Appl. Neurobiol. 33, 373–394 (2007). 41. Gough, K. C., Bishop, K. & Maddison, B. C. Highly sensitive detection of small ruminant bovine spongiform encephalopathy within transmissible spongiform encephalopathy mixes by serial protein misfolding cyclic amplification. J. Clin. Microbiol. 52, 3863–3868 (2014). 42. Simmons, M. M. et al. Does the presence of scrapie affect the ability of current statutory discriminatory tests to detect the presence of bovine spongiform encephalopathy?. J. Clin. Microbiol. 53, 2593–2604 (2015).li p g p p y 3. Hunter, N. et al. Natural scrapie in a closed flock of Cheviot sheep occurs only in specific PrP genotypes. Arch. Virol. 141, 809–924 (1996).f 4. Saunders, G. C. et al. Protective effect of the T112 PrP variant in sheep challenged with bovine spongiform encephalopathy. J. Gen Virol. 90, 2569–2574 (2009). 45. Baron, T. & Biacabe, A. G. References et al. Extraneural competition between different scrapie agents leading to loss of infectivity. Nature 253, 556 (1975). ) chutt, C. R. & Bartz, J. C. Prion interference with multiple prion isolates. Prion 2, 61–63 (2008). ( ) 33. Schutt, C. R. & Bartz, J. C. Prion interference with multiple prion isolates. Prion 2, 61–63 (2008). https://doi.org/10.1038/s41598-021-91397-8 Scientific Reports \| (2021) 11:11931 \| Competing interests h p g The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-91397-8. 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https://openalex.org/W4221050977	https://bibliotecadigital.ipb.pt/bitstream/10198/25245/1/Brancaliao-2022-Systematic-mapping-literature-revie.pdf	English	null	Systematic Mapping Literature Review of Mobile Robotics Competitions	Sensors	2,022	cc-by	24,999	Citation: Brancalião, L.; Gonçalves, J.; Conde, M.Á.; Costa, P. Systematic Mapping Literature Review of Mobile Robotics Competitions. Sensors 2022, 22, 2160. https:// doi.org/10.3390/s22062160 Academic Editor: Jesús Ureña Received: 1 February 2022 Accepted: 8 March 2022 Published: 10 March 2022 Citation: Brancalião, L.; Gonçalves, J.; Conde, M.Á.; Costa, P. Systematic Mapping Literature Review of Mobile Robotics Competitions. Sensors 2022, 22, 2160. https:// doi.org/10.3390/s22062160 Academic Editor: Jesús Ureña Received: 1 February 2022 Accepted: 8 March 2022 Published: 10 March 2022 Citation: Brancalião, L.; Gonçalves, J.; Conde, M.Á.; Costa, P. Systematic Mapping Literature Review of Mobile Robotics Competitions. Sensors 2022, 22, 2160. https:// doi.org/10.3390/s22062160 Systematic Review Systematic Mapping Literature Review of Mobile Robotics Competitions Laiany Brancalião 1,2,3, José Gonçalves 1,2,4,* , Miguel Á. Conde 3 and Paulo Costa 4,5 1 Polytechnic Institute of Bragança, 5300-253 Bragança, Portugal; laiany@ipb.pt 1 Polytechnic Institute of Bragança, 5300-253 Bragança, Portugal; laiany@ipb.pt 2 Research Centre in Digitalization and Intelligent Robotics (CeDRI), 5300-253 Bragança, Portugal 3 Robotics Group, Engineering School, University of Léon, Campus de Vegazana s/n, 24071 Léon, Spain; mcong@unileon.es 4 Institute for Systems and Computer Engineering (INESC TEC), 4200-465 Porto, Portugal; paco@fe.up.pt 5 Faculty of Engineering, University of Porto, 4200-465 Porto, Portugal * Correspondence: goncalves@ipb.pt 1 Polytechnic Institute of Bragança, 5300-253 Bragança, Portugal; laiany@ipb.pt 2 Research Centre in Digitalization and Intelligent Robotics (CeDRI), 5300-253 Bragança, Portugal 3 Robotics Group, Engineering School, University of Léon, Campus de Vegazana s/n, 24071 Léon, Spain; mcong@unileon.es 4 I tit t f S t d C t E i i (INESC TEC) 4200 465 P t P t l @f t y g g g y p p 2 Research Centre in Digitalization and Intelligent Robotics (CeDRI), 5300-253 Bragança, Port 3 Robotics Group, Engineering School, University of Léon, Campus de Vegazana s/n, 24071 Léon, Spain; mcong@unileon.es 4 Institute for Systems and Computer Engineering (INESC TEC), 4200-465 Porto, Portugal; paco@fe.up.pt 5 Faculty of Engineering, University of Porto, 4200-465 Porto, Portugal 4 Institute for Systems and Computer Engineering (INESC TEC), 4200-465 Porto, Portugal; paco@fe.up.pt 5 Faculty of Engineering, University of Porto, 4200-465 Porto, Portugal * C d l @i b * Correspondence: goncalves@ipb.pt Abstract: This paper presents a systematic mapping literature review about the mobile robotics competitions that took place over the last few decades in order to obtain an overview of the main objectives, target public, challenges, technologies used and ﬁnal application area to show how these competitions have been contributing to education. In the review we found 673 papers from 5 different databases and at the end of the process, 75 papers were classiﬁed to extract all the relevant information using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) method. More than 50 mobile robotics competitions were found and it was possible to analyze most of the competitions in detail in order to answer the research questions, ﬁnding the main goals, target public, challenges, technologies and application area, mainly in education. Keywords: robotic competitions; challenges; evaluation; benchmarking; robotic applications sensors sensors sensors 1. Introduction Robotics technology is increasingly present in our daily life and even more in industry. Inside this context, the emergence of robotics competitions around the world has provided great beneﬁts for society. Robotics competitions are an excellent tool for the development of new solutions and innovations, to push the state of the art in several ﬁelds, benchmark- ing and even to motivate students to participate in science, technology, engineering and mathematics (STEM) areas and to encourage them to join engineering careers [1,2]. g j g g The concept of robotics competitions begins in 1977 when IEEE Spectrum magazine had the intention to make an Amazing Micromouse Competition. The ﬁrst event took place in New York in 1979 where the goal was for a mobile robot to complete a maze as fast as possible. Later, Micromouse became very popular in Europe, Japan and USA until nowadays. Due to the success of Micromouse, Dean Kamen founded FIRST (For Inspiration and Recognition of Science and Technology) Association in 1989 and then the FIRST Robotic Competition season happened in 1992 in which high school students had to build and program a robot to complete a challenge [3,4]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Attached to the growth of the digital world other robotics competitions were being created, including different types of robots, themes, challenges and scenarios. Currently, it is possible to ﬁnd many competitions related to mobile robots like humanoid robots, automated guided vehicles (AGV), unmanned aerial vehicles (UAV) and even underwater robots. The themes and scenarios can vary from rescue, dance, domestic service, logistics and manufacturing, marine services, virtual robots to soccer games. The challenges range from the simplest to the most complex and the goals facing industry, domestic tasks, education, natural disaster and benchmarking [4]. 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/sensors Sensors 2022, 22, 2160. https://doi.org/10.3390/s22062160 Sensors 2022, 22, 2160 2 of 38 Among the types of robots and robotics competitions, the most common robots found are the mobile robots which are growing over the last years. Mobile robots applications have been widely implemented in industry and even in the domestic context. 1. Introduction For some industrial tasks like transportation from one place to another, the AGVs can be useful once they are able to move in a dynamic environment with unexpected obstacles [5]. The service robots, which are mobile robots too, are designed for domestic tasks and are useful to assist people with disabilities. A famous competition related to this theme is RoboCup@Home, which started in 2006 as a new league of the RoboCup competition, which includes many other leagues [6]. The autonomous navigation of mobile robots also contributes to applications like autonomous cars [7]. pp According to advances in robotics and the contributions that robotics competitions have been providing, these competitions have been gaining attention in the education area, as a way to encourage students to STEM concepts, attracting them to pursue a career in the ﬁelds of technologies, promote the values of the engineering profession and also assist in teaching several multidisciplinary engineering topics and disciplines at universities [8,9]. Some of the most popular robotics competitions with a focus on education is the FIRST Robotics Competition, BotBall and RoboCupJunior [10]. The objective of this work is to present a systematic mapping literature review about the mobile robotics competitions which took place over the last few decades. The intention is to ﬁnd many topics related to each competition like the target public and its age, the main places where the competitions take place, the different types of challenges, technologies applied and ﬁnal application area. Finally, it is intended to have an overview of all the types of mobile robotics competitions with detailed descriptions, the different goals, the results that have been found and how the competitions can contribute positively to education. This paper is structured as follows: Section 2 explains the systematic mapping litera- ture review process and describes the planning done for this theme and how to conduct the review. Section 3 shows all the numbers related to the papers found and details the conducting process of the review that was done. Section 4 presents, in a detailed form, all the mobile robot competitions found and discussed the answers to the research questions. Finally, Section 5 ends our review. 2.1.1. Planning the Research Questions The ﬁrst step to start the systematic mapping literature review is to elaborate the research questions, which are focused on a theme and the points to be discovered, under- stood or studied must be found in its answers. These questions must deﬁne clearly the problem to be solved. It is important to emphasize that the research questions of mapping are broader than those created for the SLR [11]. Taking into account that the main context of this work is mobile robotics competitions, the research questions are: • RQ1: What type of mobile robotics competitions exist in the last few decades and with what aim? • RQ1: What type of mobile robotics competitions exist in the last few decades and with what aim? • RQ2: Where do the mobile robotics competitions take place currently who and is their target public? g p • RQ3: What type of robotics challenges are addressed by the mobile robotics competi- tions? • RQ4: What type of technologies are used in mobile robotics competitions? • RQ5: What is the ﬁnal application area of the mobile robotics competitions? • RQ6: How have these competitions been contributing positively to education? 2. Methodology This paper followed the systematic mapping literature review methodology, also called literature mapping, which is useful at the beginning of research for the contextualization of ideas. literature mapping aims to seek all the knowledge available about an idea and ﬁnd, at the end of the survey, the most relevant papers according to your research questions. It is also used to complement a systematic literature review (SLR), which is another methodology of evaluating all available research and evaluating the relevant papers related to the main idea. The difference between SLR and literature mapping is that the last one is wide and SLR is more speciﬁc, but when the both are used together the best results are found. Commonly, ﬁrst one develops literature mapping to have an overview of the theme and then an SLR is obtained in order to obtain more speciﬁc and detailed results [11–15]. The literature mapping process follows some steps which are practically the same as the SLR steps that will be described below. Before starting any SLR or literature mapping, the ﬁrst step is to make a search on the Internet in order to verify if there already is a literature mapping about the intended topic. If there is a literature mapping or even an SLR, it is not necessary to conduct another one, however, if no results were found for the speciﬁc idea selected, the literature mapping or SLR can be implemented following the steps of the planning and conducting described below [13,14]. 3 of 38 Sensors 2022, 22, 2160 2.1. Planning 2.1.1. Planning the Research Questions 2.1.2. Elaborating the PICOC Once the research questions are made, the next step is to perform the PICOC method proposed by Petticrew and Roberts [12], which assists in the article analysis process. The de- scription about each topic is presented below: • Population (P): Who? • Intervention (I): What and How? • Comparison (C): What to compare? • Outcome (O): The ﬁnal objectives, what does the search obtain or improve? • Context (C): What are the circumstances? 2.1.5. Creating the Search String and Choosing the Sources 2.1.5. Creating the Search String and Choosing the Sources After all the steps before it is possible to select the databases and create the search string easily. The sources chosen to search for the papers were ACM Digital Library, IEEE, Scopus, Springer Link and Web of Science because they are important repositories for research about technology. The search string is also called a query and is an equation that represents all the main terms of the search. This string needs to be put on each database chosen to search for papers related to the theme, but depending on the website the search string varies and needs some speciﬁc characters. p The search string created for this work was: The search string created for this work was: (“robotics competitions” OR “robotic competition”) AND (“benchmark” OR “chal- lenges” OR “challenge” OR “evaluation” OR “performance” OR “robotics application” OR “technologies” OR “technology” OR “validation”) • ACM Digital Library: on the website we used the advanced search and the term “All” for each term in order to ﬁnd it in any place of the paper. The query used in this data base was: [[All: “robotics competitions”] OR [All: “robotic competition”]] AND [[All: “perfor- mance”] OR [All: “challenges”] OR [All: “challenge”] OR [All: “robotics application”] OR [All: “technologies”] OR [All: “technology”] OR [All: “validation”] OR [All: “evaluation”] OR [All: “benchmark”]] • ACM Digital Library: on the website we used the advanced search and the term “All” for each term in order to ﬁnd it in any place of the paper. The query used in this data base was: “evaluation”] OR [All: “benchmark”]] • IEEE: we used the same main equation shown at the beginning and added some terms like “Abstract”, “Author Keywords” and “Title” in the same place as “All”. This way we searched for the terms only in these topics of the paper; y p p p • Scopus: on the website we used the same main equation in the advanced search tab and just added the term “TITLE-ABS-KEY” in the query, indicating that the search for the words is done only on the title, abstract and keywords of the paper. 2.1.3. Selecting the Keywords and Synonyms 2.1.3. Selecting the Keywords and Synonyms The keywords and synonyms will help to obtain the search string, which will be discussed in the next section and it is related to each PICOC item. According to the theme of this work, the keywords and their synonyms chosen are presented in Table 1. Table 1. Keywords and synonyms chosen for the review. Table 1. Keywords and synonyms chosen for the review. Keywords Synonyms Related to Benchmark Outcome Challenges Challenge Outcome Evaluation Outcome Performance Outcome Robotics application Outcome Robotics competitions Robotic competition Population Technologies Technology Outcome Validation Outcome Table 1. Keywords and synonyms chosen for the review. Keywords Synonyms Related to Benchmark Outcome Challenges Challenge Outcome Evaluation Outcome Performance Outcome Robotics application Outcome Robotics competitions Robotic competition Population Technologies Technology Outcome Validation Outcome Sensors 2022, 22, 2160 4 of 38 2.1.4. Inclusion and Exclusion Criteria 2.1.4. Inclusion and Exclusion Criteria The inclusion and exclusion criteria help to deﬁne the relevant papers for the study and which might answer the research questions. A paper which presents all the inclusion topics can be relevant, but if it includes one or more exclusion topics this paper must be excluded. Inclusion criteria: • The work is written in English; The work was published after 2001; • The work must have information about one or more robotics competitions • The work must have included the “robotic competition” term. • The work must have included the “robotic competition” term. Exclusion criteria: • The paper is not accessible; • The work is not written in English; • The work was published before 2001; • Work does not involve a robotic competition context; p • Works that include the term “robotic competition” but does not answer any re- search question. • Works that include the term “robotic competition” but does not answer any re- search question. These criteria were chosen based on the fact that the ﬁrst robotics competitions started to gain space in the 1990s, and even if there were some competitions that have already been created before, we chose to start some years later in order to ensure that concrete research and results could be collected [4]. 2.1.5. Creating the Search String and Choosing the Sources The modiﬁed query was: y p p p • Scopus: on the website we used the same main equation in the advanced search tab and just added the term “TITLE-ABS-KEY” in the query, indicating that the search for the words is done only on the title, abstract and keywords of the paper. The modiﬁed query was: TITLE-ABS-KEY ((“robotic competition” OR “robotics competitions”)) AND TITLE- ABS-KEY ((“performance” OR “challenges” OR “challenge” OR “robotics application” y TITLE-ABS-KEY ((“robotic competition” OR “robotics competitions”)) AND TITLE- ABS-KEY ((“performance” OR “challenges” OR “challenge” OR “robotics application” 5 of 38 Sensors 2022, 22, 2160 OR “technologies” OR “technology” OR “validation” OR “evaluation” OR “bench- mark”)) OR “technologies” OR “technology” OR “validation” OR “evaluation” OR “bench- mark”)) • Springer Link: exactly the same query cited at the beginning was used on the website simple search; • Springer Link: exactly the same query cited at the beginning was used on the website simple search; • Web of Science: the query was put in the search tab of the website and we added some terms at the beginning of the equation like “TI”, “AB” and “AK” indicating a speciﬁc search as explained before. • Web of Science: the query was put in the search tab of the website and we added some terms at the beginning of the equation like “TI”, “AB” and “AK” indicating a speciﬁc search as explained before. TI = ((“robotics competitions” OR “robotic competition”) AND (“benchmark” OR “challenges” OR “challenge” OR “evaluation” OR “performance” OR “robotics appli- cation” OR “technologies” OR “technology” OR “validation”)) OR AB = ((“robotics competitions” OR “robotic competition”) AND (“benchmark” OR “challenges” OR “challenge” OR “evaluation” OR “performance” OR “robotics application” OR “tech- nologies” OR “technology” OR “validation”)) OR AK = ((“robotics competitions” OR “robotic competition”) AND (“benchmark” OR “challenges” OR “challenge” OR “evaluation” OR “performance” OR “robotics application” OR “technologies” OR “technology” OR “validation”)). 2.1.6. Quality Assessment Checklist New questions are deﬁned in this phase in order to verify the quality of a paper when it is read completely and before putting it in the ﬁnal review. These questions can be more speciﬁc and each one has a weight, the quality questions elaborated for this work are presented below. • QQ1: Is the paper based on research and not on expert opinion? • QQ2: Is there a clear objective of the research? QQ3: Are the work results discussed well? • QQ4: Is the work based on one or more robotics competitions? • QQ5: If the work is based on one or more robotics competitions, is the competition described well? • QQ5: If the work is based on one or more robotics competitions, is the competition described well? • QQ6: Does the work describe the challenges and activities of the robotics competition? g p • QQ7: Does the work present the new technologies applied to robotics competitions? • QQ8: Does the work discuss the robotics competitions’ contribution to education or industry? y • QQ9: Does the information or data obtained by the work answer at least one of the research questions? The answer value can be three values: 1.0 (if it answers the question fully), 0.5 (if it answers the question partially) or 0 (if it does not answer the question). Each paper can be evaluated with a maximum score of 9.0 and the cutoff score selected was 6.0 based on the most important questions of the list that needs to be answered fully, these were questions 4, 5, 6, 7, 8 and 9. These questions are more important than the three ﬁrst because they are focused on the topics that we want to discover and are based on the research questions. Therefore, all the papers that exceed the score of 6.0 are included in the ﬁnal review. • DQ7: Which robotics competitions contributes positively to education? • DQ7: Which robotics competitions contributes positively to education? 2.2. Conducting After the planning stage is elaborated, the next step is to perform the conducting, which was done by following the PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analysis) method that describes the phases of the conducting process [11]. The process is illustrated and exempliﬁed in Figure 1. • Identiﬁcation: the papers found in each source using the query are saved and then the duplicate studies are removed. • Identiﬁcation: the papers found in each source using the query are saved and then the duplicate studies are removed. • Screening: just the title, abstract and keywords are read applying the inclusion and exclusion criteria, the papers that are not approved by the criteria are removed too. p p pp y • Eligibility: for the remaining articles, we applied the quality questions, so the papers need to be read fully in order to obtain the answers for those questions and a good score. The papers that do not have a score above the limit must be deleted. • Eligibility: for the remaining articles, we applied the quality questions, so the papers need to be read fully in order to obtain the answers for those questions and a good score. The papers that do not have a score above the limit must be deleted. p p • Included: the papers with a high score are classiﬁed for the ﬁnal review and we performed the data extraction using the data extraction form questions [11]. p p • Included: the papers with a high score are classiﬁed for the ﬁnal review and we performed the data extraction using the data extraction form questions [11]. Figure 1. Conducting process. Source: Author. The tool used to perform this systematic mapping literature review was the Parsi- fal [16]. It is good to organize the steps, plan the review, import the papers, answer the questions and at the end generate a report about the review. Figure 1. Conducting process. Source: Author. Figure 1. Conducting process. Source: Author. The tool used to perform this systematic mapping literature review was the Parsi- fal [16]. It is good to organize the steps, plan the review, import the papers, answer the questions and at the end generate a report about the review. 2.1.7. Data Extraction Form Once we already have all the relevant paper for the research, the last step is to apply the data extraction form, in this phase a new set of questions is created to extract all the important information of the final articles in order to assist to answer the research questions set at the beginning of the mapping. The data extraction questions selected for this work are: • DQ1: What are the main robotics competitions taking place currently and their goals? • DQ2: Where do the main robotics competitions take place? • DQ3: Which ages does the robotics competitions cover? • DQ4: What are the main challenges and activities at the robotics competitions? • DQ6: Which application areas can robotics competitions contribu 6 of 38 Sensors 2022, 22, 2160 3. Results Results of conducting the review process. Source: Author. Figure 2. Results of conducting the review process. Source: Author. For question QQ9, all of the answers were yes, because all the papers answer at least one of the research questions before. The ﬁrst two quality questions obtained good answers because the papers were well elaborated, including clear objectives and based on good research. Most of the QQ7 answers were no, because few articles really described the technologies used in the robotics competitions. For questions QQ3, QQ5, QQ6 and QQ8 the answer distribution was varied since some papers do not have a good results discussion, do not describe in detail the robotics competition which it is based on and do not present a competition application area. Table 2. Results of quality assessment. Table 2. Results of quality assessment. 3. Results This section presents the results obtained in the conducting process. In the identiﬁca- tion stage we searched the databases using the query string and 673 papers were found in total, 63 from ACM Digital Library, 28 from IEEE, 300 from Scopus, 222 from Springer Link and 60 from Web of Science. There were 104 reports duplicates, removing them left 569. In the screening stage we performed the inclusion and exclusion criteria and of the remained articles, 242 passed to the next phase and 327 were excluded because they did not include one or more inclusion and exclusion criteria. In the eligibility stage, it is necessary to read all the articles fully in order to apply the quality assessment, then after this process, 168 papers were removed, leaving 74 papers in the included stage plus 1 which was a recommendation and which contained a mobile robotic competition that was not found in the chosen data sources, but was relevant for the research, totalling 75 papers. The articles classiﬁed for this last stage are those that will be used in the ﬁnal review and which we will perform the data extraction on. All the conducting processes carried out are illustrated in the ﬂowchart in Figure 2. Table 2 shows all 75 papers that went through the quality assessment and were classiﬁed for the data extraction stage, the respective answers to the quality questions and the ﬁnal scores, which were above 6.0. It is possible to notice that for question QQ4 most of the answers were yes, including just two partial answers, indicating that most of the articles were based on any robotic competition. 7 of 38 Sensors 2022, 22, 2160 Figure 2. Results of conducting the review process. Source: Author. For question QQ9, all of the answers were yes, because all the papers answer at least one of the research questions before. The ﬁrst two quality questions obtained good answers because the papers were well elaborated, including clear objectives and based on good research. Most of the QQ7 answers were no, because few articles really described the technologies used in the robotics competitions. For questions QQ3, QQ5, QQ6 and QQ8 the answer distribution was varied since some papers do not have a good results discussion, do not describe in detail the robotics competition which it is based on and do not present a competition application area. Figure 2. 3. Results Reference QQ1 QQ2 QQ3 QQ4 QQ5 QQ6 QQ7 QQ8 QQ9 Final Score [1] Yes Yes Yes Yes Yes Yes Yes Partially Yes 8.5 [2] Yes No Partially Yes Yes Yes No Yes Yes 6.5 [3] Yes Yes Yes Partially Partially Partially No Yes Yes 6.5 [4] Yes Yes Yes Yes Yes Yes No Yes Yes 8.0 [5] Yes Yes Yes Yes Partially Partially Partially Partially Yes 7.0 [6] Partially Yes Yes Yes Yes Yes Yes No Yes 7.5 [7] Yes Yes Yes Yes Partially No Partially Partially Yes 6.5 [8] Yes Yes Yes Yes Yes Yes Yes Yes Yes 9.0 [9] Yes Yes Yes Yes Yes Yes Partially Yes Yes 8.5 [10] Partially Yes Partially Yes Yes Yes No Yes Yes 7.0 [17] Partially Yes Yes Yes Partially Yes No Yes Yes 7.0 [18] Yes Yes No Yes Yes Yes No Yes Yes 7.0 [19] Yes Yes Partially Yes Yes Yes No No Yes 6.5 [20] Yes Yes Yes Yes Yes Yes No Yes Yes 8.0 [21] Yes Yes Yes Yes Yes Yes No Yes Yes 8.0 [22] Yes Yes Partially Yes Yes Yes Yes No Yes 7.5 [23] Yes Yes Yes Yes Yes Yes No Yes Yes 8.0 [24] Yes Yes Partially Yes Partially Partially No Yes Yes 6.5 [25] Yes Yes Partially Yes Yes Yes No No Yes 6.5 [26] Yes Yes Partially Yes Yes Partially Yes Partially Yes 7.5 [27] Yes Yes Yes Yes Yes Yes Partially No Yes 7.5 [28] Yes Yes Partially Yes Yes Yes Partially Yes Yes 8.0 [29] Yes Yes Partially Yes Yes Yes No Yes Yes 7.5 [30] Yes Yes Yes Yes Yes Partially Partially No Yes 7.0 Sensors 2022, 22, 2160 8 of 38 Table 2. Cont. 3. Results Reference QQ1 QQ2 QQ3 QQ4 QQ5 QQ6 QQ7 QQ8 QQ9 Final Score [31] Yes Yes Yes Yes Yes Yes Yes Partially Yes 8.5 [32] Yes Yes Yes Yes Partially Partially Yes Partially Yes 7.5 [33] Partially Yes Partially Yes Yes Yes Partially Yes Yes 7.5 [34] Yes Yes Yes Yes Yes Yes Yes No Yes 8.0 [35] Yes Yes Yes Yes Yes Yes No Yes Yes 8.0 [36] Yes Partially No Yes Yes Yes Partially Partially Yes 6.5 [37] Yes Partially Yes Yes Yes Partially No Partially Yes 6.5 [38] Yes Yes Partially Yes Yes Yes Partially Yes Yes 8.0 [39] Yes Yes Yes Partially Yes Yes No Partially Yes 7.0 [40] Yes Yes Partially Yes Yes Yes No No Yes 6.5 [41] Partially Yes Partially Yes Yes Yes Partially No Yes 6.5 [42] Yes Yes Partially Yes Yes Yes Partially Partially Yes 7.5 [43] Yes Yes Partially Yes Partially Yes Partially Partially Yes 8.0 [44] Yes Partially Partially Yes Yes Yes No Partially Yes 6.5 [45] Yes Yes Yes Yes Yes Yes No No Yes 7.0 [46] Yes Yes Partially Yes Yes Partially Partially Yes Yes 7.5 [47] Yes Yes Yes Yes Yes Yes No Yes Yes 8.0 [48] Yes Yes No Yes Yes Yes Partially Partially Yes 7.0 [49] Yes Yes Yes Yes Partially Partially No Partially Yes 6.5 [50] Yes Yes Yes Yes Yes Yes No Yes Yes 8.0 [51] Partially Yes Partially Yes Yes Partially No Yes Yes 6.5 [52] Yes Yes Yes Yes Yes Yes No Partially Yes 7.5 [53] Yes Yes Yes Yes Partially Yes No No Yes 6.5 [54] Yes Yes Partially Yes Partially Partially Partially Partially Yes 6.5 [55] Yes Yes Yes Yes Yes Partially No No Yes 6.5 [56] Yes Yes Partially Yes Yes Yes Yes Yes Yes 8.5 [57] Yes Yes Yes Yes Yes Yes No No Yes 7.0 [58] Yes Yes Yes Yes Partially Yes No Yes Yes 7.5 [59] Partially Yes Yes Yes Partially Partially No Yes Yes 6.5 [60] Yes Yes Yes Yes Partially No No Yes Yes 6.5 [61] Yes Yes Partially Yes Yes Yes No Yes Yes 7.5 [62] Yes Yes Partially Yes Partially Partially Partially Yes Yes 7.0 [63] Yes Yes Partially Yes Partially Yes Partially No Yes 6.5 [64] Yes Partially Yes Yes Partially Yes No Yes Yes 7.0 [65] Yes Yes Partially Yes Partially No Partially Yes Yes 6.5 [66] Yes Yes Yes Yes Partially Partially No Yes Yes 7.0 [67] Yes Partially Partially Yes Yes Yes No Yes Yes 7.0 [68] Yes Yes Yes Yes Partially Partially Yes No Yes 7.0 [69] Yes Yes Partially Yes Partially Yes No Yes Yes 7.0 [70] Yes Yes Yes Yes Partially Yes No Partially Yes 7.0 [71] Yes Yes Yes Yes Yes Yes Partially Partially Yes 8.0 [72] Yes Yes Partially Yes Partially Partially No Yes Yes 6.5 [73] Yes Partially Partially Yes Yes Yes No Yes Yes 7.0 [74] Partially Yes No Yes Yes Yes No Yes Yes 6.5 [75] Yes Yes No Yes Partially Yes Yes No Yes 6.5 [76] Yes Yes Yes Yes Yes Yes Partially No Yes 7.5 [77] Yes Yes Yes Yes Yes Yes Yes No Yes 8.0 [78] Yes Yes Yes Yes Partially Yes No No Yes 6.5 [79] Partially Yes No Yes Yes Yes No Yes Yes 6.5 [80] Yes Yes Partially Yes Yes Yes No No Yes 6.5 [81] Yes Yes Partially Yes Yes Yes Partially Yes Yes 8.0 Figure 3 represents the number of papers selected per database on the left side and the number of papers classiﬁed per source and year on the right side. 3. Results The selected articles are those obtained at the beginning of the review and the classiﬁed articles are those that Table 2. Cont. Figure 3 represents the number of papers selected per database on the left side and the number of papers classiﬁed per source and year on the right side. The selected articles are those obtained at the beginning of the review and the classiﬁed articles are those that Sensors 2022, 22, 2160 9 of 38 remain at the end of the review, after applying the quality assessment and which will perform the data extraction. remain at the end of the review, after applying the quality assessment and which will perform the data extraction. Figure 3. Selected and classiﬁed papers per source and year. Source: Author Figure 3. Selected and classiﬁed papers per source and year. Source: Author Figure 3. Selected and classiﬁed papers per source and year. Source: Author It is possible to notice that Scopus and Springer Link were the databases in which a large number of articles were found and classiﬁed. The database which was found the lowest number of articles was IEEE but there were few classiﬁed articles were from ACM Digital Library. It is possible to see that most of the articles classiﬁed for the included stage were papers from 2020, 2019, 2016 and 2015, which represents recent data and results that have been added over years of competitions. 4. Discussion After applying the data extraction phase in all the 75 papers we obtained enough information about all the robotics competitions found. Table 3 lists all the 67 robotics competitions found in total and the respective references. It is possible to notice that robotics competitions found in most of the papers were RoboCup and First Robotics Competition, which is famous competitions focused more on education. The DARPA Robotics Challenge was also cited in most articles, which includes autonomous vehicles and complex tasks [3,35,49,53,81]. p Figure 4 illustrates Table 3 and the numbers next to the competition name in the ﬁgure represent the number of articles in which the competition was cited. The spaces where the name is not speciﬁed are competitions cited in only one article. It is possible to see that the competitions most cited by the papers are RoboCup, FIRST and DARPA. The reason is probably that RoboCup is the biggest competition and one of the most famous, including many leagues in several domains, even football games with robots [49,81]. The FIRST is one of the oldest robotics competitions with a greater focus on education, attracting young students to careers in engineering and technology, besides that uses a well-known tool, LEGO kits [9,52]. The DARPA is a robotic competition that is more professional and industry-focused, focused on innovative solutions for problems and it has Sensors 2022, 22, 2160 10 of 38 prize money. The ﬁrst editions were focused on autonomous vehicles and then the other editions included humanoid robots [3,35,53]. prize money. The ﬁrst editions were focused on autonomous vehicles and then the other editions included humanoid robots [3,35,53]. Table 3. All robotics competitions found and their respective references. Table 3. All robotics competitions found and their respective references. 4. Discussion Robotics Competitions References Where It Was Found Robot World Cup Initiative—RoboCup [3,4,6,8,10,17,24,26,28,29,32,35–39,43,44,47– 49,51,59,65–67,76,79,81] World Robot Summit (WRS) [59,75,80] Agile Robotics for Industrial Applications Competition (ARIAC) [77] Mohamed Bin Zayed International Robotics Competition (MBZIRC) [1] e-Yantra Robotics Competition (eYRC) [34,45,69,71,78] Micromouse [3,60,70] Robot@Factory competition/Robot@Factory Lite competition [5,40,41,46,63,67] MicroFactory [41,46] RoboParty [70] Portuguese Robotics Open [70] Bot Olympics [70] Fireﬁgher Robot [70] Robot Competitions Kick Innovation in Cognitive Systems and Robotics (RoCKIn) Competition [36,37,44,48,59,67] Student Autonomous Underwater Vehicles Challenge-Europe (SAUC-E) [2,39,53,62,67,73] An Outdoor Robotics Challenge for Land, Sea and Air (EURATHLON) [2,33,39,44,53,54,62,67,73] The European Robotics League (ERL)/ERL Emergency [53,54,59,66,67,74] DARPA Robotics Challenge [3,4,8,27,28,32,33,35,36,39,44,53,59,62,65,66] FIRA HuroCup [24,65] IEEE Humanoid application challenge [65] Amazon Picking Challenge (APC) [59] European Robotics Challenge (EuRoC) [59] International Aerial Robotics Competition (IARC) [28,49,56] IROS Robotic Grasping and Manipulation Competition [59] Balam Robot Competition [61] National Robotics Competition in Guatemala [61,72] Freescale Cup [61] FIRST Lego League (FLL)/FIRST Robotics Competition/FIRST Technology Challenge [3,4,8–10,18,22,29,32,39,42,43,50,52,55,58,72] 4-H Robotics [50] Mississippi BEST (MSBEST) Robotics [50] VEX Robotics Competition [32,39,64] World Robot Olympiad (WRO) [4,8,10,29,43,55] Brazilian Robotics Olympiad [42] European Land Robot Trial (ELROB) competition [27,28,33,39,44,62] SpaceBot Cup—a German robotics competition [44] BotBall [4,8,10,29] Australian Multi Autonomous Ground-Robotic International Challenge (MAGIC) [27,37] Defense Advance Research Projects Agency Robotics Challenge [37] National Instruments Autonomous Robotics Competition (NIARC) [32] RoboWaiter [25,31] Trinity College Fire-Fighting Home Robot Contest (TCFFHRC) [25] British MoD Grand Challenge [27] Robotic Day Like Follower Competition [68] EUROBOT [20,30] Cybertech [20,21] CiberMouse@RTSS08 [23] Indoor Aerial Robot Competition [19] AAAI Mobile Robot Competition and Exhibition [66] “Schüler bauen Roboter” program [57] Urban Search and Rescue (USAR) [33,39] Humanitarian Robotics and Automation Technology Challenge(HRATC) [35] Robotics Competitions References Where It Was Found Robot World Cup Initiative—RoboCup [3,4,6,8,10,17,24,26,28,29,32,35–39,43,4 49,51,59,65–67,76,79,81] World Robot Summit (WRS) [59,75,80] Agile Robotics for Industrial Applications Competition (ARIAC) [77] Mohamed Bin Zayed International Robotics Competition (MBZIRC) [1] e-Yantra Robotics Competition (eYRC) [34,45,69,71,78] Micromouse [3,60,70] Robot@Factory competition/Robot@Factory Lite competition [5,40,41,46,63,67] MicroFactory [41,46] RoboParty [70] Portuguese Robotics Open [70] Bot Olympics [70] Fireﬁgher Robot [70] Robot Competitions Kick Innovation in Cognitive Systems and Robotics (RoCKIn) Competition [36,37,44,48,59,67] Student Autonomous Underwater Vehicles Challenge-Europe (SAUC-E) [2,39,53,62,67,73] An Outdoor Robotics Challenge for Land, Sea and Air (EURATHLON) [2,33,39,44,53,54,62,67,73] The European Robotics League (ERL)/ERL Emergency [53,54,59,66,67,74] DARPA Robotics Challenge [3,4,8,27,28,32,33,35,36,39,44,53,59,62,6 FIRA HuroCup [24,65] IEEE Humanoid application challenge [65] Amazon Picking Challenge (APC) [59] European Robotics Challenge (EuRoC) [59] International Aerial Robotics Competition (IARC) [28,49,56] IROS Robotic Grasping and Manipulation Competition [59] Balam Robot Competition [61] National Robotics Competition in Guatemala [61,72] Freescale Cup [61] FIRST Lego League (FLL)/FIRST Robotics Competition/FIRST Technology Challenge [3,4,8–10,18,22,29,32,39,42,43,50,52,55, 4-H Robotics [50] Mississippi BEST (MSBEST) Robotics [50] VEX Robotics Competition [32,39,64] World Robot Olympiad (WRO) [4,8,10,29,43,55] Brazilian Robotics Olympiad [42] European Land Robot Trial (ELROB) competition [27,28,33,39,44,62] SpaceBot Cup—a German robotics competition [44] BotBall [4,8,10,29] Australian Multi Autonomous Ground-Robotic International Challenge (MAGIC) [27,37] Defense Advance Research Projects Agency Robotics Challenge [37] National Instruments Autonomous Robotics Competition (NIARC) [32] RoboWaiter [25,31] Trinity College Fire-Fighting Home Robot Contest (TCFFHRC) [25] British MoD Grand Challenge [27] Robotic Day Like Follower Competition [68] EUROBOT [20,30] Cybertech [20,21] CiberMouse@RTSS08 [23] Indoor Aerial Robot Competition [19] AAAI Mobile Robot Competition and Exhibition [66] “Schüler bauen Roboter” program [57] Urban Search and Rescue (USAR) [33,39] Humanitarian Robotics and Automation Technology Challenge(HRATC) [35] References Where It Was Found [3,4,6,8,10,17,24,26,28,29,32,35–39,43,44,47– 49,51,59,65–67,76,79,81] [59,75,80] [77] [1] [34,45,69,71,78] [3,60,70] [5,40,41,46,63,67] [41,46] [70] [70] [70] [70] [36,37,44,48,59,67] [2,39,53,62,67,73] [2,33,39,44,53,54,62,67,73] [53,54,59,66,67,74] [3,4,8,27,28,32,33,35,36,39,44,53,59,62,65,66] [24,65] [65] [59] [59] [28,49,56] [59] [61] [61,72] [61] [3,4,8–10,18,22,29,32,39,42,43,50,52,55,58,72] References Where It Was Found 11 of 38 Sensors 2022, 22, 2160 Table 3. 4. Discussion Cont. Robotics Competitions References Where It Was Found Robomagellan [7] RoboParty [3] Latin American IEEE Robotics Competition [17] Robosub [73] MATE ROV [73] Microtransat Challenge [73] SailBot [73] Underwater robot competition [73] RoboBoat [73] World Robotics Sailing Championship [73] SeaPerch [73] Students Autonomous Underwater Vehicle (SAVe) [73] Oceanology International (OI) China [73] Maritime RobotX Challenge [73] Shell Ocean Discovery XPRIZE [73] Virtual RobotX [73] METRICS [73] Figure 4. Robotics competitions per number of references in which they were found. Source: Author. References Where It Was Found Robotics Competitions Robotics Competitions References Where It Was Found Robomagellan [7] RoboParty [3] Latin American IEEE Robotics Competition [17] Robosub [73] MATE ROV [73] Microtransat Challenge [73] SailBot [73] Underwater robot competition [73] RoboBoat [73] World Robotics Sailing Championship [73] SeaPerch [73] Students Autonomous Underwater Vehicle (SAVe) [73] Oceanology International (OI) China [73] Maritime RobotX Challenge [73] Shell Ocean Discovery XPRIZE [73] Virtual RobotX [73] METRICS [73] Figure 4. Robotics competitions per number of references in which they were found. Source: Author. Figure 4. Robotics competitions per number of references in which they were found. Source: Author. Figure 4. Robotics competitions per number of references in which they were found. Source: Author Table 4 presents the relevant information about each robotics competition extracted from the last review stage and based on the data extraction questions presented in Sec- tion 2.1.7, each topic (second column) associated with each competition (ﬁrst column) is related to the questions. It is noted that not all competitions listed in Table 3 are described in Table 4, because there are some competitions that were only cited in the papers, with- out detailed information. Therefore, in the table below are only the robotics competitions in which it was possible to obtain data through the selected papers, in total there were 38 competitions. Table 5 presents all the mobile robotics competitions linked to the research concepts in which they are involved in each related paper. The proposal of this table is to provide the key researches associated with the competition’s challenges and the papers. Sensors 2022, 22, 2160 12 of 38 Table 4. Description of robotics competitions taken from the data extraction phase. Robotics Competitions Agile Robotics for Industrial Applications Competition (ARIAC) Description It is an annual competition organized by the NIST (National Institute of Standards and Technology) since 2017. The main goal is to test the agility of industrial robot systems and to enable industrial robots on shop ﬂoors to be more productive, autonomous and to require less time from shop ﬂoor workers [77]. Where it takes place This topic was not found in the papers. Target public Researches to practitioners. Challenges and activities Participants needs to implement a robot control system for a robot to overcome agility challenges in a simulated environment. Researches to practitioners. Gazebo (open source robotics simulation environment) and ROS—Robot Operating System. irst land competition: 2013 in Berchtesgaden, Germany. Second sea competition: 2014 in La Spezia, Italy. The ﬁnal euRathlon Grand Challenge (air, land and sea): 2015 in Piombino, Italy. Robotics Competitions an annual competition organized by the NIST (National Institute of Standards and Technology) since 201 The main goal is to test the agility of industrial robot systems and to enable industrial robots on shop ﬂoors to be more productive, autonomous and to require less time from shop ﬂoor workers [77]. Robotics Competitions The robot needs to realize kitting tasks, building the kits by picking up all the required items, which can be found on shelves, on the conveyor belt or in bins. Technologies applied Gazebo (open source robotics simulation environment) and ROS—Robot Operating System. Application area This kind of competition can contribute to the development of robotics applications for the manufacturing industry, realizing repetitive tasks, decreasing the human errors and allowing robots to work in areas inaccessible for humans. An Outdoor Robotics Challenge for Land, Sea and Air (EURATHLON) Description It is a competition funded by European Union and coordinated by the University of the West of England. The main aim of euRathlon was to propose teams from academia and industry real world challenges testing the intelligence and autonomy of outdoor robots in scenarios inspired by the 2011 Fukushima accident [2,33,39,53,54,62,73]. Where it takes place First land competition: 2013 in Berchtesgaden, Germany. Second sea competition: 2014 in La Spezia, Italy. The ﬁnal euRathlon Grand Challenge (air, land and sea): 2015 in Piombino, Italy. Target public Teams from academy, company and industry. Challenges and activities Challenges for autonomous robots of different domains (air, sea and land) in scenarios inspired by the 2011 Fukushima accident. In 2013 Eurathlon coordinates a robotic competition based on land and on the next year based on sea. The third year is the Grand Challenge, where the robots of three domains (land, sea and air) needs to cooperate in order to achieve objectives in a scenario set up to simulate a nuclear power plant ravaged by a tsunami. The scenario was based on the Fukushima disaster. The Grand Challenge is composed of three missions: localizing two missing workers in the disaster area, surveying the area of disaster to identify dangerous leaks and ﬁnally closing valves inside the building and underwater to stem the leaks. Three days of the competition are to practice and the grand challenge is the last two days. Technologies applied This topic is not discussed in the articles. Application area This kind of competition can contribute to increase the state of the art on the air, land and sea autonomous robots to help in natural disasters. Table 4. Description of robotics competitions taken from the data extraction phase. p p Robotics Competitions It is an annual competition organized by the NIST (National Institute of Standards and Technology) since 2017. Robotics Competitions The main goal is to test the agility of industrial robot systems and to enable industrial robots on shop ﬂoors to be more productive, autonomous and to require less time from shop ﬂoor workers [77]. This topic was not found in the papers. Researches to practitioners. Participants needs to implement a robot control system for a robot to overcome agility challenges in a simulated environment. The robot needs to realize kitting tasks, building the kits by picking up all the required items, which can be found on shelves, on the conveyor belt or in bins. Gazebo (open source robotics simulation environment) and ROS—Robot Operating System. This kind of competition can contribute to the development of robotics applications for the manufacturing industry, realizing repetitive tasks, decreasing the human errors and allowing robots to work in areas inaccessible for humans. It is a competition funded by European Union and coordinated by the University of the West of England. The main aim of euRathlon was to propose teams from academia and industry real world challenges testing the intelligence and autonomy of outdoor robots in scenarios inspired by the 2011 Fukushima accident [2,33,39,53,54,62,73]. First land competition: 2013 in Berchtesgaden, Germany. Second sea competition: 2014 in La Spezia, Italy. The ﬁnal euRathlon Grand Challenge (air, land and sea): 2015 in Piombino, Italy. Teams from academy, company and industry. Challenges for autonomous robots of different domains (air, sea and land) in scenarios inspired by the 2011 Fukushima accident. In 2013 Eurathlon coordinates a robotic competition based on land and on the next year based on sea. The third year is the Grand Challenge, where the robots of three domains (land, sea and air) needs to cooperate in order to achieve objectives in a scenario set up to simulate a nuclear power plant ravaged by a tsunami. The scenario was based on the Fukushima disaster. The Grand Challenge is composed of three missions: localizing two missing workers in the disaster area, surveying the area of disaster to identify dangerous leaks and ﬁnally closing valves inside the building and underwater to stem the leaks. Three days of the competition are to practice and the grand challenge is the last two days. This topic is not discussed in the articles. This kind of competition can contribute to increase the state of the art on the air, land and sea autonomous robots to help in natural disasters. Teams from academy, company and industry. Challenges and activities Challenges for autonomous robots of different domains (air, sea and land) in scenarios inspired by the 2011 Fukushima accident. In 2013 Eurathlon coordinates a robotic competition based on land and on the next year based on sea. The third year is the Grand Challenge, where the robots of three domains (land, sea and air) needs to cooperate in order to achieve objectives in a scenario set up to simulate a nuclear power plant ravaged by a tsunami. The scenario was based on the Fukushima disaster. The Grand Challenge is composed of three missions: localizing two missing workers in the disaster area, surveying the area of disaster to identify dangerous leaks and ﬁnally closing valves inside the building and underwater to stem the leaks. Three days of the competition are to practice and the grand challenge is the last two days. This topic is not discussed in the articles. This kind of competition can contribute to increase the state of the art on the air, land and sea autonomous robots to help in natural disasters. Sensors 2022, 22, 2160 13 of 38 Table 4. Cont. Robotics Competitions Balam Robot Competition (BRC) Description It is a local robotics competition in Guatemala started in 2015. The main objective is to show that technology is not complicated and mathematics or science are not boring for students [61]. Where it takes place Outreach Department of Universidad Galileo. Target public Students. Challenges and activities They prepare six weeks having workshops of four hours per week. The main challenge of BRC 1.0 was to build a sumorobot, teams had to compete by rounds against other sumobots and those who remained inside the tatami where who remain as ﬁnalists. After various rounds a winner was determined. Technologies applied 3D printing. Application area This competition can contribute to education. Brazilian Robotics Olympiad (BRO) Description It was started in 2007, created by a team of several university professors with the mission of promoting robotics among brazilian students with or without previous knowledge of robotics, fostering their interest to engage in science, technology and engineering studies and carrers. The olympiad is fully free for participants, being annually organized by volunteers from several brazilian universities [42]. Where it takes place Brazil. Target public Students. Challenges and activities The activities are divided in two modalities, practical and theoretical. Students. lenges and ctivities The activities are divided in two modalities, practical and theoretical. The theoretical exams are designed to give the knowledge and contextualization about robotics, six levels of written tests are prepared by the organizers and based on the age of students. This model allows students to realize that what they are learning at school can be applied to solve real world problems. The practical exams are based on RoboCup Junior—Rescue mission. There is a simulated disaster environment where teams of four participants must build a robot fully autonomous to rescue victims. The robot must follow a safe path, avoid debris, overcome gaps, go over a mountain, identify victims and rescue them, taking them to a safe place. The best teams are selected by the Brazilian RoboCup Committee to represent Brazil in the RoboCup Junior international competition. Teams from academy, company and industry. The theoretical exams are designed to give the knowledge and contextualization about robotics, six levels of written tests are prepared by the organizers and based on the age of students. This model allows students to realize that what they are learning at school can be applied to solve real world problems. The practical exams are based on RoboCup Junior—Rescue mission. There is a simulated disaster environment where teams of four participants must build a robot fully autonomous to rescue victims. The robot must follow a safe path, avoid debris, overcome gaps, go over a mountain, identify victims and rescue them, taking them to a safe place. The best teams are selected by the Brazilian RoboCup Committee to represent Brazil in the RoboCup Junior international competition. Technologies applied Arduino kits. Application area Robotics competition has been an exciting and motivational tool for helping students to learn how to solve real problems in a practical way. It has been a good contribution for education. Table 4. Cont. Robotics Competitions ption It is a local robotics competition in Guatemala started in 2015. The main objective is to show that technology is not complicated and mathematics or science are not boring for students [61]. al robotics competition in Guatemala started in 2015. The main objective is to show that technology is not complicated and mathematics or science are not boring for students [61]. This competition can contribute to education. cription It was started in 2007, created by a team of several university professors with the mission of promoting robotics among brazilian students with or without previous knowledge of robotics, fostering their interest to engage in science, technology and engineering studies and carrers. The olympiad is fully free for participants, being annually organized by volunteers from several brazilian universities [42]. team of several university professors with the mission of promoting robotics among brazilian students edge of robotics, fostering their interest to engage in science, technology and engineering studies and ree for participants, being annually organized by volunteers from several brazilian universities [42]. Arduino kits. Robotics competition has been an exciting and motivational tool for helping students to learn how to solve real problems in a practical way. It has been a good contribution for education. Sensors 2022, 22, 2160 14 of 38 Table 4. Cont. Robotics Competitions CiberMouse @RTSS08 Description It is a robotics simulation competition [23]. Where it takes place The competition took place remotely. Target public This topic was not found in the papers. Challenges and activities The simulation system creates a virtual arena with obstacles, a starting grid, a target area and the bodies of the robots. The bodies are composed of a circular base and are equipped with sensor, actuator and command buttons. The participants must create a software which controls the movements of a team composed of ﬁve virtual robots. Technologies applied Robot simulators. Application area This topic was not found in the papers, but it can be concluded that this kind of competition can contribute to the development of virtual solutions, also for education and dissemination of technology areas through students. Cybertech Description It is a robotic competition organized anually by the Universidad Politécnica de Madrid (UPM) started in 2001 [21]. Where it takes place Madrid, Spain. Target public Undergraduate students from universities all around the world. Challenges and activities The students have to design and build a robot that participates in different events. The events include: Maze event (robot have to get out of a maze in a minimum time), line-following event (robot must follow a black line over a white back-ground), solar cars event (participants have to build an autonomous device that should be able to move inside a circuit being propelled just by solar energy), simulated robots event (participants have to develop a computer program to control a virtual robot that moves in a simulated maze) and bullﬁghting event (each team has to build a bullﬁghter robot that ﬁghts in the arena against a bull robot provided by the organization) Technologies applied This topic was not discussed in the papers. Application area This competition can contribute to the ﬁeld of education, increasing the motivation of them towards engineering domains. DARPA Robotics Competition Description It is an industrial competition focused on autonomous vehicles, which has prize money [3,4,27,33,35,39,53,59,62,66]. Where it takes place This topic was not found in the papers. Target public This topic was not found in the papers. This topic was not found in the papers. tem creates a virtual arena with obstacles, a starting grid, a target area and the bodies of the robots. sed of a circular base and are equipped with sensor, actuator and command buttons. The participants eate a software which controls the movements of a team composed of ﬁve virtual robots. in the papers, but it can be concluded that this kind of competition can contribute to the development o solutions, also for education and dissemination of technology areas through students. mpetition organized anually by the Universidad Politécnica de Madrid (UPM) started in 2001 [21]. Madrid, Spain. This topic was not discussed in the papers. This topic was not discussed in the papers. Arduino kits. Challenges and activities Includes several manipulation tasks. First editions have the objective of promote autonomous driving of road vehicles and then in the others editions promote humanoid robots able to execute complex tasks and in the last editions the focus was to promote the development of adaptive vehicles for military purposes. Started as a competition for autonomous cars and recently a simulated challenge focusing on humanoid robotics using Gazebo. Technologies applied Gazebo. Application area Robotic competitions are important in the learning process of youngsters and it is becoming more and more usual in schools and universities last few years. The competitions can contribute to several areas, like industry, society, search, but one of the most are beneﬁted has been education. Table 4. Cont. This topic was not found in the papers. Application area Robotic competitions are important in the learning process of youngsters and it is becoming more and more usual in schools and universities last few years. The competitions can contribute to several areas, like industry, society, search, but one of the most are beneﬁted has been education. 15 of 38 Sensors 2022, 22, 2160 Table 4. Cont. Robotics Competitions e-Yantra Robotics Competition (eYRC) Description It is an annual competition organized by e-Yantra and hosted at IIT Bombay. The objective is to teach robotics concepts to the college students using a Project Based Learning (PBL). The competition is totally online [34,45,69,71,78]. Where it takes place Indian Institute of Technology Bombay—India. Target public College students of Indian Institute of Technology Bombay. Challenges and activities The competition is divided in different stages. Firstly, there is a preliminary test where participants answer questions related to aptitude programming and electronics knowledge. In Stage 2 the participants combine the software and hardware parts to ﬁnd the best solution it also involves hardware testing, video and code submission. Each stage is subdivided into small tasks. In 2018 it was introduced a them called “Thirsty Crow”, which aims to teach “Marker Based Augmented Reality”, for the ﬁrst time. The teams need to build a robot (called Crow) capable of autonomously following the line and pick up the magnetic pebbles and drops them at the water pitcher marker. They also have to design and construct a 3D model of pebbles, water pitcher and Crow in Blender. They also have to write a python script related to the augmented reality part. Technologies applied Marker based augmented reality using open source python libraries such as OpenCV and OpenGL; 3D modeling using blender software; ros; machine learning; image processing; microcontroller programming. Application area This kind of competition can contribute to education, increasing the students’ interest in STEAM areas and robotics. EUROBOT competition Description It is an international amateur robotics contest, organized by the Eurobot Association and founded in May 2004, but the contest was introduced already in 1998 [20,30]. Where it takes place Annually somewhere in Europe. Target public Young engineering students. Challenges and activities During a match, two opponents robots are competing on the table for 90 seconds, each robot is performing tasks deﬁned in the rules. The robots must be autonomous and a robot should not collide with other opponent, if this happens the team is disqualiﬁed. This topic was not found in the papers. The winner is the robot that collect more points. In the Eurobot 2010 edition the robot must collect fruits and vegetables, represented by balls and cylinders. In the Eurobot 2011 edition two mobile robots must play a “chess up”, the game is played on a playing table of the usual Eurobot size. Technologies applied 3D printing. Application area The main application area is education. Robotics Competitions It is an annual competition organized by e-Yantra and hosted at IIT Bombay. The objective is to teach robotics concepts to the college students using a Project Based Learning (PBL). The competition is totally online [34,45,69,71,78]. Indian Institute of Technology Bombay—India. Indian Institute of Technology Bombay—India. The main application area is education. College students of Indian Institute of Technology Bombay. The competition is divided in different stages. Firstly, there is a preliminary test where participants answer questions related to aptitude, programming and electronics knowledge. In Stage 2 the participants combine the software and hardware parts to ﬁnd the best solution, it also involves hardware testing, video and code submission. Each stage is subdivided into small tasks. In 2018 it was introduced a theme called “Thirsty Crow”, which aims to teach “Marker Based Augmented Reality”, for the ﬁrst time. The teams need to build a robot (called Crow) capable of autonomously following the line and pick up the magnetic pebbles and drops them at the water pitcher marker. They also have to design and construct a 3D model of pebbles, water pitcher and Crow in Blender. They also have to write a python script related to the augmented reality part. ased augmented reality using open source python libraries such as OpenCV and OpenGL; 3D modeling using blender software; ros; machine learning; image processing; microcontroller programming. d of competition can contribute to education, increasing the students’ interest in STEAM areas and robo The main application area is education. Sensors 2022, 22, 2160 16 of 38 16 of 38 Table 4. Cont. Robotics Competitions European Land Robot Trial (ELROB) Description It was founded in 2006 by the European Robotics Group and organized by the Fraunhofer Institute for Communication, Information Processing and Ergonomics [27,33,39]. Where it takes place Annually at changing locations throughout Europe. Target public This topic was not found in the papers. Challenges and activities The ELROB alternates between military and civilian and deﬁnes a variety of scenarios instead of only one single mission. These tasks include: security missions, convoying or reconnaissance by day and night. The team can choose between the alternative scenarios. The scenarios also include detection of objects and transportation, which can be carried out with a single vehicle or a convoy with at least two vehicles. Technologies applied 2D and 3D laser scanner, 3D Lidar sensor, cameras, GPS and inertial sensors. Application area Provides an opportunity to exchange ideas, create solutions as well as a venue to evaluate and encourage state of the art research This competition can be helpful for the members of teams because they are forced to work together in a determined time, contributing to education ﬁeld. This topic is not discussed in the papers. mpetition has a duration of 9 days and the robots has to perform tasks related to land, air and sea domains which emulate world situations inspired by the 2011 Fukushima accident. The missions include: Mission A: Search for missing workers. Mission B: Reconnaissance and environmental survey. Mission C: Pipe inspection and stemming the leak. Robots have to work in a catastrophic scenario. From a starting point, the vehicle had to submerge, pass through the gate and it was then required to perform different tasks without resurfacing. The tasks include inspecting and mapping the area and the objects of interest, identifying mission targets, such as the leaking pipe and the missing worker. This topic was not found in the papers. activities The ELROB alternates between military and civilian and deﬁnes a variety of scenarios instead of only one single mission. These tasks include: security missions, convoying or reconnaissance by day and night. The team can choose between the alternative scenarios. The scenarios also include detection of objects and transportation, which can be carried out with a single vehicle or a convoy with at least two vehicles. 2D and 3D laser scanner, 3D Lidar sensor, cameras, GPS and inertial sensors. Provides an opportunity to exchange ideas, create solutions as well as a venue to evaluate and encourage state of the art research. This competition can be helpful for the members of teams because they are forced to work together in a determined time, contributing to education ﬁeld. tion It is a multidomain robotic competition funded by the European Union Horizon 2020 Programme, which is focused on two indoor robotics competitions (ERL Industrial and ERL Service Robots) and one outdoor robotic competition (ERL Emergency Robots). The 2017 ERL Emergency competitions require ﬂying, land and marine robots acting together to survey the disaster [53,54,66,74]. College students of Indian Institute of Technology Bombay. European Robotics League (ERL)/ ERL Emergency Description It is a multidomain robotic competition funded by the European Union Horizon 2020 Programme, which is focused on two indoo robotics competitions (ERL Industrial and ERL Service Robots) and one outdoor robotic competition (ERL Emergency Robots). The 2017 ERL Emergency competitions require ﬂying, land and marine robots acting together to survey the disaster [53,54,66,74]. Where it takes place Many countries over Europe. Target public This topic is not discussed in the papers. Challenges and activities The competition has a duration of 9 days and the robots has to perform tasks related to land, air and sea domains which emulate real-world situations inspired by the 2011 Fukushima accident. The missions include: Mission A: Search for missing workers. Mission B: Reconnaissance and environmental survey. Mission C: Pipe inspection and stemming the leak. Robots have to work in a catastrophic scenario. From a starting point, the vehicle had to submerge, pass through the gate and it was then required to perform different tasks without resurfacing. The tasks include inspecting and mapping the area and the objects of interest, identifying mission targets, such as the leaking pipe and the missing worker. Technologies applied Cloud resource and 4G connection. Application area This kind of competition can contribute to increase the state of the art on the air, land and sea autonomous robots to help in natural disasters. Table 4. Cont. It was founded in 2006 by the European Robotics Group and organized by the Fraunhofer Institute for Communication, Information Processing and Ergonomics [27,33,39]. Annually at changing locations throughout Europe. Cloud resource and 4G connection. This kind of competition can contribute to increase the state of the art on the air, land and sea autonomous robots to help in natural disasters. Sensors 2022, 22, 2160 17 of 38 Table 4. Cont. Robotics Competitions FIRA HuroCup Description It is a multi-event robot athletic competition intended to encourage breath in humanoid performance [24,65]. Where it takes place First edition in Seoul, Korea. Target public This topic was not found in the papers. Challenges and activities HuroCup is part of the FIRA international robotic competition and consists of robot dash, penalty kicks, lift and carry, basketball, weightlifting, climbing wall and obstacle run, the robot with the best score over all events is the winner. Technologies applied This topic was not found in the papers. Application area This topic was not found in the papers. FIRST Robotics Competition/ FIRST Lego League (FLL) Description It is an international competition which began in 1998 as a joint effort between the FIRST (For Inspiration and Recognition of Science and Technology) Organization and the LEGO Group to introduce robotics to students. The competition has a duration of six weeks. The Lego League is designed for young ages [3,4,9,18,22,29,50,52,58]. Where it takes place In different countries around the world. Target public High school and university students, engineers, technicians, business, leaders and concerned citizens. Lego League: students from 9 to 14 years old. Challenges and activities Teams design and build tele-operated mobile robots to achieve a variety of tasks. In the Lego League they have to use LEGO kits to work on an authentic scientiﬁc-themed challenge, the themes include climate change, senior solutions, food safety, medicine, moving across a ﬁeld, climbing ramps, hanging from bars and placing objects in goals. Each year, there is a new theme. The tasks ﬁrs allow students to connect what they learn about robotics to what they could do in the face of real-world challenges and second, authentic tasks and plausible scenarios are structured to motivate students to overcome potential challenges in learning robotics. Technologies applied LEGO Mindstorms, LabView software, sensors. Application area This competition can connect students with professionals, enable them to solve real-world problems and develop 21st century skills. Robotic competitions have been a good tool for education because it aids universities to teach a variety of multidisciplinary engineering topics including design, programming and mechatronics. In different countries around the world. High school and university students, engineers, technicians, business, leaders and concerned citizens. Lego League: students from 9 to 14 years old. eams design and build tele-operated mobile robots to achieve a variety of tasks. In the Lego League they have to use LEGO kits to work on an authentic scientiﬁc-themed challenge, the themes include climate change, senior solutions, food safety, medicine, ving across a ﬁeld, climbing ramps, hanging from bars and placing objects in goals. Each year, there is a new theme. The tasks ﬁrst allow students to connect what they learn about robotics to what they could do in the face of real-world challenges and second, authentic tasks and plausible scenarios are structured to motivate students to overcome potential challenges in learning robotics. LEGO Mindstorms, LabView software, sensors. This topic was not found in the papers. This topic was not found in the papers. LEGO Mindstorms, LabView software, sensors. manitarian demining international robotics competition, which the goal is to push boundaries of what technology can accomplish in this ﬁeld. The ﬁrst edition happened in 2014 [35]. Cloud resource and 4G connection. Humanitarian Robotics and Automation Technology Challenge (HRATC) Description It is a humanitarian demining international robotics competition, which the goal is to push boundaries of what technology can accomplish in this ﬁeld. The ﬁrst edition happened in 2014 [35]. Where it takes place The entire competition is performed remotely. Target public This topic was not found in the papers. Challenges and activities The competition is divided in three stages: The simulation stage: teams must focus on their ideas and to develop their algorithms. The teams have to focus on the actual problem, humanitarian demining. The ﬁeld trials stage: in this stage each team will be able to run the software developed on the simulator on the actual robot, each team has 3 ﬁeld trials, throughout 3 weeks. Competition day: each team is given two runs on a mineﬁeld using surrogate mines and false positives. Technologies applied Gazebo simulator, TeamBots simulator, ROS and USARSim simulador. Application area The main contribution of this competition is to increase the state of the art in the area of humanitarian demining. Table 4. Cont. Robotics Competitions It is a multi-event robot athletic competition intended to encourage breath in humanoid performance [24,65]. First edition in Seoul, Korea. This topic was not found in the papers. HuroCup is part of the FIRA international robotic competition and consists of robot dash, penalty kicks, lift and carry, basketball, weightlifting, climbing wall and obstacle run, the robot with the best score over all events is the winner. d This topic was not found in the papers. This topic was not found in the papers. It is an international competition which began in 1998 as a joint effort between the FIRST (For Inspiration and Recognition of Science and Technology) Organization and the LEGO Group to introduce robotics to students. The competition has a duration of six weeks. The Lego League is designed for young ages [3,4,9,18,22,29,50,52,58]. In different countries around the world. High school and university students, engineers, technicians, business, leaders and concerned citizens. Lego League: students from 9 to 14 years old. Teams design and build tele-operated mobile robots to achieve a variety of tasks. In the Lego League they have to use LEGO kits to work on an authentic scientiﬁc-themed challenge, the themes include climate change, senior solutions, food safety, medicine, moving across a ﬁeld, climbing ramps, hanging from bars and placing objects in goals. Cloud resource and 4G connection. Each year, there is a new theme. The tasks ﬁrst allow students to connect what they learn about robotics to what they could do in the face of real-world challenges and second, authentic tasks and plausible scenarios are structured to motivate students to overcome potential challenges in learning robotics. d LEGO Mindstorms, LabView software, sensors. This competition can connect students with professionals, enable them to solve real-world problems and develop 21st century skills. Robotic competitions have been a good tool for education because it aids universities to teach a variety of multidisciplinary engineering topics including design, programming and mechatronics. It is a humanitarian demining international robotics competition, which the goal is to push boundaries of what technology can accomplish in this ﬁeld. The ﬁrst edition happened in 2014 [35]. The entire competition is performed remotely. This topic was not found in the papers. The competition is divided in three stages: The simulation stage: teams must focus on their ideas and to develop their algorithms. The teams have to focus on the actual problem, humanitarian demining. The ﬁeld trials stage: in this stage each team will be able to run the software developed on the simulator on the actual robot, each team has 3 ﬁeld trials, throughout 3 weeks. Competition day: each team is given two runs on a mineﬁeld using surrogate mines and false positives. d Gazebo simulator, TeamBots simulator, ROS and USARSim simulador. The main contribution of this competition is to increase the state of the art in the area of humanitarian demining. Robotics Competitions Robotics Competitions It is a multi-event robot athletic competition intended to encourage breath in humanoid performance [24,65]. First edition in Seoul, Korea. This topic was not found in the papers. Gazebo simulator, TeamBots simulator, ROS and USARSim simulador. Sensors 2022, 22, 2160 18 of 38 Table 4. Cont. Robotics Competitions IEEE Humanoid application challenge Description The 2019 theme was robot magic [65]. Where it takes place This topic was not found in the papers. Target public This topic was not found in the papers. Challenges and activities In the robot magic theme a humanoid robot can take on any role in a magic show. Technologies applied OpenCV (image processing) and PocketSphinx (speech recognition). Application area Provides opportunity for improving work in robotics and a large range of areas of artiﬁcial intelligence (vision, speech understanding, interacting with humans). Indoor Aerial Robot Competition Description It was inaugurated in May 2005 with the objective to identify best design practices and gain insight on technical challenges facing the development of unmanned air vehicles [19]. Where it takes place Swarthmore College. Target public This topic was not found in the paper. Challenges and activities The tasks are based on line-following and teleoperation. The teams have to implement a line-following algorithm in real time which is invariant to changing lighting conditions. The points are based on how far the robots are able to travel. Technologies applied This topic was not found in the paper. Application area It has been a means of discovering the best practices to solve real world problems. International Aerial Robotics Competition (IARC) Description It is an international competition focused on aerial robots [49,56]. Where it takes place This topic is not found in the papers. Target public This topic is not found in the papers. Challenges and activities In this competition the agent (aerial robot) is required to contact targets (ground vehicles) sequentially and drive them to a certain boundary to earn score. The agent robot needs to be fully autonomous and the game has a duration of 10 min. In the IARC mission 7 called “Shepherd mission”, there is a drone, 10 ground mobile robots and 4 mobile obstacles. First, the drone should be able to avoid collision with four mobile obstacles. Second, there are two ways to change the moving direction of each ground mobile robot. The ﬁnal target of winning the competition is to drive at least 4 out of the 10 ground mobile robots across the green edge of the square arena within 10 min. Technologies applied This topic is not found in the papers. This topic is not found in the papers. This topic is not found in the papers. This topic was not found in the papers. Application area This topic is not found in the papers. Sensors 2022, 22, 2160 19 of 38 Table 4. Cont. Robotics Competitions Latin American IEEE Robotics Competition Description It is an annually competition organized by the Department of Electrical Engineering of the Universidad de Chile and by the IEEE region 9 [17]. Where it takes place The ﬁrst was held in Santiago—Chile. Target public Engineering students. Challenges and activities The ﬁrst competition (“beginners”) was aimed for students to work in robotics and was based on Lego MindStorms building blocks, the proposed challenge is to design and programming a robot that cross a simulated mineﬁeld. The second competition (“advanced”) is designed for experienced students’ groups and consists of crossing a soccer ﬁeld with obstacles using any kind of legged robots, the robots could be designed by participants, or could be bought or even adapted. Technologies applied LEGO MindStorms. Application area The main contribution is for the area of education. MicroFactory Description It is a robotic competition designed to be low-cost and easily implementable in a small space and it is based on the Portuguese competition called Robot@Factory [41,46]. Where it takes place This topic was not found in the papers. Target public High school students and university undergraduate students. Challenges and activities The challenges are similar to the Robot@factory challenges but the ground area and complexity is reduced and the scenario material were simpliﬁed. In MicroFactory there are just 3 rounds. Technologies applied 3D printing, Arduino, odometry and sensors. Application area The main contribution of this competition is for education. Micromouse Description It is one of the most popular competitions inside the context of mobile robots started in 1970s, being the ﬁrst competition promoted by the IEEE. It is organized at the University of Trás-os-Montes e Alto Douro [3,60,70]. Where it takes place Editions are held worldwide. Target public Students, researches and the general public. Challenges and activities A small autonomous mobile robot put in an unknown labyrinth must be able to map it, look for the best possible route between the starting point and the goal and travel it in the shortest time. The challenge is not solving the maze but how fast the robot can do it. Technologies applied Scanning and path planning algorithms,fFloodﬁll procedure, HIL simulator, self-localization using odometry and distance sensors. This topic was not found in the papers. Application area The Micromouse competition is an important tool for the education, increasing the young students’ interest in STEAM but to introduce other people to the ﬁeld of robotics. Table 4. Cont. Robotics Competitions Sensors 2022, 22, 2160 20 of 38 Table 4. Cont. Robotics Competitions Mississippi BEST (MSBEST) robotics Description It is a competition which has a mission to inspire students to pursue careers in STEM areas through robotic design and competition. [50]. Where it takes place Mississipi—USA Target public Middle and high school students. Challenges and activities The challenge has a duration of six weeks. The participants are supplied with kits of material and they have to put those material together to build a robot, participants have to do a search about the competition theme for that particular year, realize a brainstorm with the ideas on how to design the robot to perform tasks related to the theme. All the students are required to submit their notebook, team demographics and consent forms. Technologies applied This topic is not discussed in the papers. Application area The main application area is education. Mohamed Bin Zayed International Robotics Competition (MBZIRC) Description It is an international robotics competition [1]. Where it takes place This topic was not found in the papers. Target public This topic was not found in the papers. Challenges and activities The Challenge 1 of the MBZIRC competition consists of aerial drone interception scenario. First, there are ﬁxed balloons randomly around the arena and the autonomous aerial system must automatically detect, get close and blow up. Second, another autonomou aerial system should capture a ball that is suspended from another drone that ﬂies at high speed on a variety trajectory. All these tasks must be performed autonomously. Technologies applied Time-of-ﬂight cameras, machine learning, computer vision and Kalman ﬁlters. Application area This competition can contribute to increase the state of the art in autonomous vehicles and drones, which has been attracting a lot of attention, for example, for urban air mobility (UAM). National Instruments Autonomous Robotics Competition (NIARC) Description It is a competition started in 2012 and its focus on fully autonomous robots to complete a given theme challenge. These themes have included search and rescue, mining and agriculture [32]. Where it takes place Universities across Australia and New Zealand. Target public Students. This topic was not found in the papers. n area This competition can contribute to increase the state of the art in autonomous vehicles and drones, which has been attracting a lot of attention, for example, for urban air mobility (UAM). ion It is a competition started in 2012 and its focus on fully autonomous robots to complete a given theme challenge. These themes have included search and rescue, mining and agriculture [32]. es place Universities across Australia and New Zealand. ublic Students. s and es In NIARC 2012 the theme was search and rescue, where teams have to develop a robot to navigate a grid based maze environment. The objective is the robot navigate in unknown maze and differentiate the victims. NIARC’s 2013 theme was the mining industry, where the objective was the robot navigate to the mining area through the unknown entrances and differentiate the desired gold cubes and undesired grey rubble cubes. NIARC’s 2014 theme was the agriculture industry and the objective is the robot navigate accurately to known but unmarked seeding areas to plant seeds. applied Real time control, FPGA, LabView. n area Studies have shown the beneﬁts of using robotics competitions to generate interest and motivation in studying engineering for high school students and general public. Besides that it can also contribute to develop the ability of the teams to work together in multidisciplinary work. It is a competition which has a mission to inspire students to pursue careers in STEM areas through robotic design and competition. [50]. This topic was not found in the papers. Time-of-ﬂight cameras, machine learning, computer vision and Kalman ﬁlters. This topic was not found in the papers. Challenges and activities In NIARC 2012 the theme was search and rescue, where teams have to develop a robot to navigate a grid based maze environment The objective is the robot navigate in unknown maze and differentiate the victims. NIARC’s 2013 theme was the mining industry, where the objective was the robot navigate to the mining area through the unknown entrances and differentiate the desired gold cubes and undesired grey rubble cubes. NIARC’s 2014 theme was the agriculture industry and the objective is the robot navigate accurately to known but unmarked seeding areas to plant seeds. Technologies applied Real time control, FPGA, LabView. Application area Studies have shown the beneﬁts of using robotics competitions to generate interest and motivation in studying engineering for high school students and general public. Besides that it can also contribute to develop the ability of the teams to work together in multidisciplinary work. Table 4. Cont. Robotics Competitions ion It is a competition which has a mission to inspire students to pursue careers in STEM areas through robotic design and competition. [50]. es place Mississipi—USA ublic Middle and high school students. s and es The challenge has a duration of six weeks. The participants are supplied with kits of material and they have to put those material together to build a robot, participants have to do a search about the competition theme for that particular year, realize a brainstorm with the ideas on how to design the robot to perform tasks related to the theme. All the students are required to submit their notebook, team demographics and consent forms. applied This topic is not discussed in the papers. n area The main application area is education. ion It is an international robotics competition [1]. es place This topic was not found in the papers. ublic This topic was not found in the papers. s and es The Challenge 1 of the MBZIRC competition consists of aerial drone interception scenario. First, there are ﬁxed balloons randomly around the arena and the autonomous aerial system must automatically detect, get close and blow up. Second, another autonomous aerial system should capture a ball that is suspended from another drone that ﬂies at high speed on a variety trajectory. All these tasks must be performed autonomously. applied Time-of-ﬂight cameras, machine learning, computer vision and Kalman ﬁlters. Real time control, FPGA, LabView. 21 of 38 Sensors 2022, 22, 2160 Table 4. Cont. Robotics Competitions RoboCup Description It is an international competition which runs by the RoboCup Federation and the goal is: “By the year 2050, a team of fully-autonomous humanoid robot soccer players shall win a soccer game, complying with the ofﬁcial FIFA rules, against the winner of the most recent World Cup of Human Soccer” [4,6,8,10,17,26,28,29,36,43,47–49,51,59,76,79,81]. Where it takes place The ﬁrst edition took place in Nagoya, Japan and now the competition take place in many countries all over the world annually. Target public Since senior participants like researchers and university students to hobbyists, high school, primary and secondary students. Challenges and activities There are different leagues: Junior: for young students. There are three ages categories and three leagues being them soccer, rescue and dance. This league keeps the same activities over the years to help students improving their solutions. Soccer game: teams with autonomous robots compete each other in a soccer game; Search and rescue: robots that can assist ﬁrst responders in mitigatin a disaster such as an earth-quake or an accident in an industrial environment; Home: service robots to realize household activities Work: deﬁnes nine challenges inspired by industrial mobile manipulation and transport tasks; Logistics League: groups of three robots have to plan, execute and optimize the material ﬂow and deliver products according to dynamic orders in a simpliﬁed factory; Humanoid Challenge: humanoid robots compete in three events: walking, penalty kicks and a free demonstration; Simulation 2D and 3D: two teams of eleven software agents compete against each other on a simulated soccer pitch; Small size: semi-autonomous soccer robots (diameter of 18 cm and height of up to 15 cm); Middle size: slow driving robots (that drive up to 4m/s) on small soccer ﬁelds enclosed by walls; Standard Platform: soccer game in which all teams compete with identical robot Technologies applied Sensor, actuators, AI solutions, machine learning, multi-agent coordination, ROS, SLAM, image processing, wireless standard communication interfaces, LEGO Mindstorms, object recognition, speech recognition and gesture recognition. Application area How the competition includes several leagues the application area can be more than one, increase the state of the art in the leagues areas, attract more students to STEM concepts contributing to education through RoboCup Junior and also increase the development of solutions for industry, daily life and natural disasters like RoboCup Home, Logistics League and Search and Rescue. Real time control, FPGA, LabView. Robomagellan Description It is an outdoor navigation competition hosted by RoboGames [7]. Where it takes place This topic is not discussed in the papers. Target public This topic is not discussed in the papers. Challenges and activities The competition requires the robot moves in an unconstrained and unstructured real-world outdoor environment with different obstacles. Technologies applied ROS, FSM and extended Kalman ﬁlter (EKF). Application area The robotic competition contributes with the advances in the ﬁeld of robotics. Robotics Competitions It is an international competition which runs by the RoboCup Federation and the goal is: “By the year 2050, a team of fully-autonomous humanoid robot soccer players shall win a soccer game, complying with the ofﬁcial FIFA rules, against the winner of the most recent World Cup of Human Soccer” [4,6,8,10,17,26,28,29,36,43,47–49,51,59,76,79,81]. The ﬁrst edition took place in Nagoya, Japan and now the titi t k l i t i ll th ld The ﬁrst edition took place in Nagoya, Japan and now the i i k l i i ll h ld p g y , J p competition take place in many countries all over the world annually. ke researchers and university students to hobbyists, high school, primary and secondary students. 22 of 38 Sensors 2022, 22, 2160 Table 4. Cont. Robotics Competitions Roboparty Description It is an educational robotic event with a duration of three non-stop days [3,70]. Where it takes place Universidade do Minho, Guimarães, Portugal Target public School-age children. Challenges and activities The students learn by experience how to build the Bot’n Roll robotic platform (mechanics, soldering electronics components and assembling the parts). Then, three challenges are executed to test their robots and the developed algorithms. Technologies applied This topic is not discussed in the papers. Application area The main application is education. Robosub Description It is the ﬁrst AUV competition and the ﬁrst edition was in 1997. Currently it is the most popular competition in the AUV world and every year the competition has a different theme [73]. Where it takes place USA. Target public Students (high school and university) Challenges and activities The AUV mission consists of passing a gate, touching a buoy, dropping and retrieving objects and launching a plastic marker inside a target hole. Technologies applied This topic was not found in the papers. Application area The main application area is education. RoboWaiter Description The ﬁrst robot competition in the area of assistive robotics, it is conducted in conjunction with the annual international Trinity College Fire-Fighting Home Robot Contest (TCFFHRC) in 2009. The vision of the competition is: bringing people with disabilities as clients of RoboWaiter design and Integrating the RoboWaiter project in a robotics course [25,31]. Where it takes place Hartford, Connecticut Target public Traditional participants are students, hobbyists and engineers. Challenges and activities Each robot has three runs and must navigate autonomously from its home position to a scale-model refrigerator, pick up a plate of food from a shelf, navigate to the table where a person with mobility impairment is sitting, places the plate on it and return to home position. Robots must avoid collisions with obstacles (sink, chair and elderly person). Technologies applied This topic was not found in the paper. Application area Development of solutions based on assistive robots to help people with disabilities realizes activities more easily. Other application area can be the education once the competition encourages students to STEAM areas. Table 4. Cont. Sensors 2022, 22, 2160 23 of 38 Table 4. Cont. ke researchers and university students to hobbyists, high school, primary and secondary students. Robotics Competitions Robot Competitions Kick Innovation in Cognitive Systems and Robotics (RoCKIn) Competition Description It aims to provide tools for benchmarking to the robotics community by designing and setting up competitions that increase scientiﬁc and technological knowledge. It is inspired by the RoboCup [44,48,59]. Where it takes place The ﬁrst was held in Toulouse, France in 2014. The ﬁnal was held in Lisbon, Portugal in 2015. Target public This topic was not found in the papers. Challenges and activities RoCKIn@Work: there is a medium sized factory which produces small to medium sized lots of mechanical parts and assembled mechanical products, the robots must try to optimize its production process to meet the increasing demands of their customers. RoCKIn@Home: the robots must assist a person and supporter quality life, it is based in an apartment with all common household items like windows, doors, furniture and decorations. Technologies applied This topic was not found in the papers. Application area Robotics competitions have been a good way to promote comparison of different algorithms and systems, allowing for the replication of their results. Robotics competitions also contributes for promoting education and research to push the ﬁeld forward. Robotic Day Line Follower Competition Description It is an annual competition which has been occurring during the last 15 years and it is growing every year. This competition used topics that can be used as benchmark, comparing different performances [68]. Where it takes place Prague, Czech Republic. Target public This topic is not discussed in the article. Challenges and activities The participants’ robots must to run in a way and follow a black line. They need to pass obstacles and the robot that complete the route in the shortest time qualify for the ﬁnale. In the ﬁnal round the races are held on a knock-out. Technologies applied Time of ﬂight distance sensor and computer vision. Application area In this competition context it is possible to apply the activities in multidisciplinary approach contributing to education. It can also have an importance in research and development, because the outcomes can be applied to solve real world problems, for example, in manufacturing and service robots. Robot@Factory Robot@Factory Lite Description It is an annually competition started in 2011 recently included in Robotica, the main Portuguese Robotics Competition. Robot@Factory Lite is a simpliﬁed version. The goal is to stimulate students and researchers to develop solutions to the challenges proposed by them [5,40,41,46,63,67]. ke researchers and university students to hobbyists, high school, primary and secondary students. Where it takes place Portugal. Target public Secondary school and universities students. Challenges and activities The competition deal with the problem of the transportation of materials inside a factory. The main idea is an AGV organize the materials in warehouses with processing machines. There are four warehouses with two machines, the robot must deliver the parts in their correct locations. The AGV must be fully autonomous. The competition is divided in three days, each day has a round. Technologies applied SimTwo simulator, which is provided by the competition and hardware in the loop (HIL), a software where the competitors insert their microcontroller in the loop of the simulation. Application area The main application area is education. Table 4. Cont. Robotics Competitions It aims to provide tools for benchmarking to the robotics community by designing and setting up competitions that increase scientiﬁc and technological knowledge. It is inspired by the RoboCup [44,48,59]. ace The ﬁrst was held in Toulouse, France in 2014. The ﬁnal was held in Lisbon, Portugal in 2015. This topic was not found in the papers. vities RoCKIn@Work: there is a medium sized factory which produces small to medium sized lots of mechanical parts and assembled mechanical products, the robots must try to optimize its production process to meet the increasing demands of their customers. RoCKIn@Home: the robots must assist a person and supporter quality life, it is based in an apartment with all common household items like windows, doors, furniture and decorations. ied This topic was not found in the papers. a Robotics competitions have been a good way to promote comparison of different algorithms and systems, allowing for the replication of their results. Robotics competitions also contributes for promoting education and research to push the ﬁeld forward. It is an annual competition which has been occurring during the last 15 years and it is growing every year. This competition used topics that can be used as benchmark, comparing different performances [68]. ace Prague, Czech Republic. This topic is not discussed in the article. vities The participants’ robots must to run in a way and follow a black line. They need to pass obstacles and the robot that complete the route in the shortest time qualify for the ﬁnale. In the ﬁnal round the races are held on a knock-out. ied Time of ﬂight distance sensor and computer vision. ke researchers and university students to hobbyists, high school, primary and secondary students. a In this competition context it is possible to apply the activities in multidisciplinary approach contributing to education. It can also have an importance in research and development, because the outcomes can be applied to solve real world problems, for example, in manufacturing and service robots. It is an annually competition started in 2011 recently included in Robotica, the main Portuguese Robotics Competition. Robot@Factory Lite is a simpliﬁed version. The goal is to stimulate students and researchers to develop solutions to the challenges proposed by them [5,40,41,46,63,67]. ace Portugal. Secondary school and universities students. vities The competition deal with the problem of the transportation of materials inside a factory. The main idea is an AGV organize the materials in warehouses with processing machines. There are four warehouses with two machines, the robot must deliver the parts in their correct locations. The AGV must be fully autonomous. The competition is divided in three days, each day has a round. ied SimTwo simulator, which is provided by the competition and hardware in the loop (HIL), a software where the competitors insert their microcontroller in the loop of the simulation. a The main application area is education. Robotics Competitions Robotics Competitions The ﬁrst was held in Toulouse, France in 2014. The ﬁnal was held in Lisbon, Portugal in 2015. nd activities RoCKIn@Work: there is a medium sized factory which produces small to medium sized lots of mechanical parts and assembled mechanical products, the robots must try to optimize its production process to meet the increasing demands of their customers. RoCKIn@Home: the robots must assist a person and supporter quality life, it is based in an apartment with all common household items like windows, doors, furniture and decorations. This topic is not discussed in the article. pants’ robots must to run in a way and follow a black line. They need to pass obstacles and the robot the route in the shortest time qualify for the ﬁnale. In the ﬁnal round the races are held on a knock-out. an annually competition started in 2011 recently included in Robotica, the main Portuguese Robotics petition. Robot@Factory Lite is a simpliﬁed version. The goal is to stimulate students and researchers to develop solutions to the challenges proposed by them [5,40,41,46,63,67]. 24 of 38 Sensors 2022, 22, 2160 Table 4. Cont. Robotics Competitions “Schüler bauen Roboter” program Description “Schüler bauen Roboter” is a German project that brings together schools and universities [57]. Where it takes place Technical University of Munich, Germany. Target public Target group is 14 to 18 years old high school students. Challenges and activities In the ﬁrst school year the students can build a robot that solve a given task and at the end of the year, the different groups can compete against each other. Usually the competition starts in September, when the school year begin. Technologies applied This topic was not found in the paper. Application area The main application area is education, once the competition was created to take place inside a university to help them to encourage students in the STEM areas and get skills in programming, electronics, robotics, etc. SICK robot day Description It is a bi-annual competition hosted by SICK AG, a producer of sensor systems [38]. Where it takes place Waldkirch, Germany. Target public This topic was not found in the papers. Challenges and activities The robots must navigate autonomously and avoid obstacles and collision with other robots. The goal is to deliver as many objects as possible, where each correctly delivered object was awarded one point and each erroneous delivery one penalty point. With a limit time of 10 min, each robot had to alternately collect labelled objects at ﬁlling stations and transport them to delivery stations based on the object label. Technologies applied This topic was not found in the papers. Application area This topic was not found in the papers. SpaceBot Cup Description It is a German robotics competition started in 2013. The second edition occurred in 2015. The main of the competition is to accomplish (conclude, ﬁnish) this activity as autonomous as possible by means of unmanned vehicles. The focus is mobile manipulation for planetary exploration [44]. This topic was not found in the papers. tion It is a German robotics competition started in 2013. The second edition occurred in 2015. The main of the competition is to accomplish (conclude, ﬁnish) this activity as autonomous as possible by means of unmanned vehicles. The focus is mobile manipulation for planetary exploration [44]. This topic was not found in the paper. This topic is not discussed in the article. Where it takes place Germany. Target public Universities, research institutes and subject matter experts (SMEs). Challenges and activities SpaceBot has only one scenario which involves typical exploration tasks carried out on a planetary surface after landing on a planet. The robots have to locate and identify objects in a complex terrain. The target objects needs to be conveyed (transmitted) to a base station. The robots needs to be autonomous, communication was only allowed through a shaped network connection that imposed restrictions on the ports used. The tasks to be accomplished were: explore and map the terrain, ﬁnd artiﬁcial objects, collect the two objects and bring them to a third and ﬁnally return to landing site. Technologies applied This topic was not found in the paper. Application area This topic was not found in the paper. Table 4. Cont. Universities, research institutes and subject matter experts (SMEs). 25 of 38 Sensors 2022, 22, 2160 Table 4. Cont. Robotics Competitions Student Autonomous Underwater Vehicle Challenge- Europe (SAUC-E) Description It is the ﬁrst underwater robotics competition in Europe. SAUC-E started in 2006 in the UK and then has been hosted by CMRE since 2010, the main goals are: advance the state of art of AUV, promote creative environment among researches, get closer contact between the university teams and companies invited to participate [2,53,62,73]. Where it takes place In many countries around Europe. Target public University students. Challenges and activities The typical tasks includes passing through the gate, mapping and inspecting an underwater pipeline structure, localizing on the seaﬂoor a pinger that emit an acoustic wave and localizing underwater buoys and objects. The task must be done totally autonomous by the robot. Technologies applied This topic was not found in the papers. Application area The main application area is education. VEX Robotics Competition Description It is a competition which aim to engages participants from elementary through university students in learning about STEM concepts. This competition was launched in 2005 and today it is one of the largest extracurricular robotics program in the world [32,39,64]. Where it takes place The place is not discussed in the articles. Target public Middle school, high school and university students. Challenges and activities This topic was not found in the papers. Technologies applied This topic was not found in the papers. Application area The main application area is education. World Robot Olympiad (WRO) Description It was founded in 2004 and the initial mission is: to bring together young people all over the world to develop their creativity, design and problem solving skills through challenging and educational robot competitions and activities [4,55]. Where it takes place This topic is not discussed in the papers. Target public Students. Challenges and activities There are three categories: regular category (the robots complete tasks and it is open for 13–15 years), open category (build a robot model) and football category (teams build two robots who compete against another team’s robots in a robot football match). The theme of the 2018 edition was “Precision Farming” requires the students to design a robot which is able to plant different coloured seedlings in the corresponding farm areas. Technologies applied LEGO Mindstorms. Application area The main application area is education. 26 of 38 Sensors 2022, 22, 2160 26 of 38 Table 4. Cont. Universities, research institutes and subject matter experts (SMEs). Robotics Competitions World Robot Summit (WRS) Description It is an international competition started in October 2018, organized by the Japanese government to accelerate research and development of robots in the areas of daily life, society and industry in order to promote a world where humans and robots successfully live and work together [59,75]. Where it takes place Tokyo, Japan Target public This topic was not found in the papers. Challenges and activities The leagues: rescue, service and assembly. Service: the tidy up here task consists of moving objects from incorrect positions to the right positions. There are four rooms, children, dining, kitchen and a living room. There are two types of objects, 45 known units and 10 unknown units. Assembly: aims to develop robots to allow the assembly of complex systems with varied products. There some tasks like task board, kitting, assembly and surprise assembly. Technologies applied Simultaneous localization and mapping (vSLAM), vision system (TensorFlow). Application area The competition can contribute to development of novel solutions, providing a benchmark for robot assistance, not only for disabled people but also for elderly people and healthy people, by assisting daily housekeeping tasks. Can also contribute to the manufacture industry developing solution to assembly products. ption It is an international competition started in October 2018, organized by the Japanese government to accelerate research and development of robots in the areas of daily life, society and industry in order to promote a world where humans and robots successfully live and work together [59,75]. s an international competition started in October 2018, organized by the Japanese government to accelera research and development of robots in the areas of daily life, society and industry in order to promote a world where humans and robots successfully live and work together [59,75]. This topic was not found in the papers. vities The leagues: rescue, service and assembly. Service: the tidy up here task consists of moving objects from incorrect positions to the right positions. There are four rooms, children, dining, kitchen and a living room. There are two types of objects, 45 known units and 10 unknown units. Assembly: aims to develop robots to allow the assembly of complex systems with varied products. There some tasks like task board, kitting, assembly and surprise assembly. Simultaneous localization and mapping (vSLAM), vision system (TensorFlow). Application area The competition can contribute to development of novel solutions, providing a benchmark for robot assistance, not only for disabled people but also for elderly people and healthy people, by assisting daily housekeeping tasks. Can also contribute to the manufacture industry developing solution to assembly products. Sensors 2022, 22, 2160 27 of 38 Table 5. Research topics associated with the mobile robotics competitions and related papers. Robotics Competitions Simulation Control Localization and/or Mapping Obstacle Avoidance Prototyping Computer Vision Navigation Artiﬁcial Intelligence ARIAC [77] [77] [77] [77] [77] [77] EURATHLON [2,33,39,53,54,62, 73] [2,33,39,53,54,62, 73] [2,33,39,53,54,62, 73] [2,33,39,53,54,62, 73] [2,33,39,53,54,62, 73] BRC [61] [61] BRO [42] [42] [42] [42] [42] [42] Cibermouse [23] [23] [23] [23] Cybertech [21] [21] [21] [21] DARPA [3,4,27,33,39,53,59, 62,66] [3,27,33,35,39,53, 59,62,66] [3,27,33,35,39,53, 59,62,66] [3,27,33,35,39,53, 59,62,66] [3,27,33,35,39,53, 59,62,66] [3,27,33,35,39,53, 59,62,66] eYRC [34,45,69,71,78] [34,45,69,71,78] [34,45,69,71,78] [34,45,69,71,78] EUROBOT [20,30] [20,30] [20,30] [20,30] [30] [20,30] ELROB [27,33,39] [27,33,39] [27,33,39] ERL [53,54,66,74] [53,54,66,74] [53,54,66,74] [53,54,66] [53,54,66,74] FIRA HuroCup [24,65] [24] [65] [65] FIRST [3,4,9,18,22,29,50, 52,58] [3,4,9,18,29,50,52, 58] [3,4,9,18,29,50,52, 58] HRATC [35] [35] IEEE Humanoid application challenge [65] [65] [65] Indoor Aerial Robot Competition [19] [19] IARC [56] [49] Latin American IEEE [17] [17] [17] Microfactory [41] [41,46] [41,46] Table 5. Research topics associated with the mobile robotics competitions and related papers. Sensors 2022, 22, 2160 28 of 38 Table 5. Cont. Table 5. Cont. This topic was not found in the papers. Some competitions found have sports as a theme and often have soccer games, like RoboCup@Soccer, FIRA HuroCup and WRO football category [4,24,81]. Besides that, there are competitions that include magic and dance like IEEE Humanoid Application Chal- lenge [65], in which the robot needs to perform a magic show and RoboCup@Junior Dance Challenge [10,29] which the robot needs to perform a dance and its focus is more on educa- tion. There also are competitions totally online for example the CiberMouse@RTSS08 [23]. p y p Many of the robotics competitions found have a focus on search and rescue based on natural disaster scenarios, including outdoor, indoor, terrain and even underwater environmental. Some examples are RoboCup Search and Rescue League, NIARC 2012 edition, EURATHLON, ERL and ERL Emergency, being the last three focused on the 2011 Fukushima accident. The NIARC is a competition that changes the theme every year, in the 2013 edition the theme was the mining industry and in 2014 was the agriculture industry. The EUROBOT competition follows the same way, changing its theme every year, in the 2010 edition the theme was collecting fruits and vegetables and the 2011 edition was a chess game [20,32,53,54,66,81]. There are also robotics competitions that are important tools to push the state of the art in a speciﬁc ﬁeld, for example in the area of drones like MBZIRC and IARC [1,56]. HRATC contributes in the area of humanitarian demining and DARPA in autonomous vehicles [35]. SAUC-E, which can also cited, was the ﬁrst underwater robotic competition in Europe and promotes advances in the state of the art of Autonomous Underwater Vehicle (AUV) [73]. p RQ2: Where do the mobile robotics competitions take place currently and who is their target public? Most of the international robotics competitions take place in many countries all over the world because it has an international scope [3,6,43,58]. Some educational competitions take place in universities or in a speciﬁc region, for example the “Schüler bauen Roboter” program that takes place in the University of Munich, Germany or Cybertech that take place in Universidad Politécnica de Madrid (UPM), Spain [21,57]. The target public of the competitions can vary a lot, but we can conclude that for the most part, it is students. This topic was not found in the papers. botics etitions Simulation Control Localization and/or Mapping Obstacle Avoidance Prototyping Computer Vision Navigation Artiﬁcial Intelligence mouse [3,60] [3,60] [3,60] [3,70] BEST [50] [50] ZIRC [1] [1] [1] [1] ARC [32] [32] [32] oCup [4,6,8,17,29,43,47, 48] [4,6,8,10,17,26,28, 29,36,43,47,48,59] [4,6,8,17,26,28,29, 36,43,47,48] [4,6,8,17,28,29,43, 47,48] [4,6,8,10,17,29,36, 43,47,48] [4,6,8,17,26,28,29, 43,47,48] [4,6,8,17,29,43,47, 48] [4,6,8,17,26,28,29, 43,47– 49,51,76,79,81] magellan [7] [7] [7] [7] oparty [3,70] [3,70] osub [73] [73] [73] Waiter [25,31] [25,31] [25,31] [25,31] [25,31] [25,31] CKIn [44,48,59] [44,48,59] [44,48,59] tic Day [68] [68] [68] [68] @ Factory [5,40,41,46,63,67] [5,40,41,46,63,67] [5,40,41,46,63,67] [5,41,46,63,67] [41,46,63,67] [5,40] er bauen oter” [57] [57] obot day [38] [38] [38] Bot Cup [44] [44] [44] [44] [44] UC-E [2,53,62,73] [2,53,62,73] [2,53,62,73] [2,53,62,73] EX [32,39,64] [32,39,64] RO [4,55] [4,55] WRS [59,75] [59,75] [59,75] [59,75] Sensors 2022, 22, 2160 29 of 38 29 of 38 At the end of the review and after all the information is extracted, the research questions proposed in Section 2.1.1, in the beginning of the review, are ﬁnally answered. As Table 4 shows, there are a lot of robotic competitions around the world, since there are big and international competitions with several challenges and also simple competitions that are often done in a speciﬁc region or school. The following discussion is related to answering the research questions. g q RQ1: What type of mobile robotics competitions exist in the last few decades and with what aim? There are robotics competitions focused on education, that is, willing to teach robotics concepts to students, attracting them to STEM areas and to encourage them to enter in technologies careers. Most of the competitions found were focused on education like Robosub, Latin American IEEE Robotics Competition, RoboParty, Microfactory, Cy- bertech, EUROBOT, NIARC, Brazilian Robotics Olympiad, WRO, MSBEST, VEX, BRC, FIRST, RoboCup@Junior, Robot@Factory, eYRC and the “Schüler bauen Roboter” pro- gram [3,4,8–10,17,20–22,29,30,32,41–43,45,46,50,52,55,57,61,63,64,67,70,71,73,78]. A theme very commonly found in the robotics competitions was industry, like RoboCup Work and RoboCup Logistics League, WRS Assembly task, ARIAC, RoCKIn@Work and SICK robot day [37,38,48,59,75–77]. Robot@Factory and MicroFactory have a focus on edu- cation, but their themes are focused on industry and logistics too [41,46,63,67]. Other types of competitions found were those facing the domestic ﬁeld, like RoboCup@Home, WRS Service Challenge, RoCKIn@Home and RoboWaiter, which their challenges usually are to perform house tasks in order to help elderly people or those with disabilities [6,25,28,37,75]. RQ3: What type of robotics challenges are addressed by the mobile robotics com- petitions? p The challenges addressed by the educational robotics competitions usually includes tasks that the participants have to build a robot to perform some activity, in order to allow them to apply the knowledge learned in classes in real-world problems and put it into practice. Sometimes there are several stages, like eYRC for example, in which the students ﬁrstly answer questions, realize a preliminary test or learn concepts about programming and electronics and then, they use tools provided by the competition to ﬁnd the best solution for a problem, involving hardware and software approaches [21,29,42,45,50,57,71,78]. This kind of competition usually elaborates simple tasks like dance and games and with a focus on students working in teams and developing soft skills [3,10,29]. However, there also are competitions focused on education which includes industry challenges too, for example the Robot@Factory and MicroFactory [41,46,63,67]. y g p y y In the robotics competitions focused on industry, the challenges can vary between transport products and logistics tasks, in which the robots must navigate, avoid obstacles and deliver products in correct positions, perform assembly tasks and kitting processes, build kits by picking up items, organize materials in warehouses, among others [37,38,41,46,48,59,63,67,75–77]. There also are challenges addressed to evaluate the robot speed, like activities in a maze (Micromouse) or delivering as many objects as possible (SICK robot day), in these kind of tasks the robots needs to be as fast as possible [3,38,60,70]. y p Home challenges are usually focused on the robot helping in household activities inside an environment that simulates a house including rooms. The tasks found in this kind of competition were organizing objects from an incorrect place to a correct place, assisting people in opening or closing windows and doors and kitchen activities like opening the refrigerator, picking up a plate of food from a shelf and putting it in a table [6,25,28,37,75]. The challenges included in search and rescue competitions can be exploration tasks, climbing ramps, walking on dirty terrain with poor visibility, looking for missing people, pipe inspection and stemming the leak, reconnaissance and environmental survey and usually, the scenarios simulate a natural disaster and it can vary between underwater, buildings, small places, indoor or outdoor environments [19,20,32,53,54,66,81]. g p In soccer games, the challenges usually include teams of autonomous robots competing against each other. This topic was not found in the papers. The competitions focusing on education always include students and some other big, famous and international competitions have a larger target public including young students and senior participants, like engineers and business [3,6,43,58]. 30 of 38 Sensors 2022, 22, 2160 30 of 38 RQ4: What type of technologies are used in the mobile robotics competitions? y g The main technologies used by the robotics competitions found in the papers were methods like AI solutions, machine learning, computer vision, simultaneous localization and mapping (SLAM), image processing, speech recognition, object recognition, gesture recognition, extended Kalman ﬁlters (EKF), real-time control and 3D printing. It was also found that there were a lot of software and robot simulators like ROS, HIL, SimTwo, Gazebo, TeamBots, USARSim, OpenCV, TensorFlow, LabView and also components like sensors, actuators, cameras, microcontrollers and LEGO Mindstorms kits [1,6,8,19,22,24,35, 40,63,70,77,79]. RQ3: What type of robotics challenges are addressed by the mobile robotics com- petitions? FIRA Hurocup also includes other sports activities for the robots for example basketball, climbing wall, penalty kicks, lift and carry, weightlifting and obstacle run [4,24,81]. ] RQ5: What is the ﬁnal application area of the mobile robotics competitions? In general, the mobile robotics competitions can contribute with advances in many ﬁelds, like industry, daily life, search and rescue in disaster scenarios, but one of the most beneﬁted has been education. Most of the competitions have been contributing to education recently, increasing the students’ interest in STEM concepts, introducing other people to the ﬁeld of robotics, connecting students with professionals, enabling them to solve real world problems, encouraging them to join engineering careers and developing 21st-century skills. It is becoming more and more usual in schools and universities last few years because it aids them to teach a variety of multidisciplinary engineer- ing topics including design, programming and mechatronics. Besides that it can also Sensors 2022, 22, 2160 31 of 38 31 of 38 contribute to develop the ability of the teams to work together in a multidisciplinary work [3,4,8–10,17,20–22,29,30,32,41–43,45,46,50,52,55,57,61,63,64,67,70,71,73,78]. contribute to develop the ability of the teams to work together in a multidisciplinary work [3,4,8–10,17,20–22,29,30,32,41–43,45,46,50,52,55,57,61,63,64,67,70,71,73,78]. Another application area very common in robotics competitions is industry, contributing to the development of new solutions related to manufacturing and logistics, assisting people in repetitive tasks and reducing human errors. Besides that, the competitions have been a good way to promote comparison of different algorithms and systems and a means of discovering the best practices to solve real-world prob- lems [37,38,41,46,48,59,63,67,75–77]. The competitions focused on home environments contributes to the development of solutions based on assistive robots, providing a benchmark for robot assistance, not only for disabled people but also for elderly people and healthy people, by assisting daily housekeeping tasks [6,25,28,37,75]. Search and rescue area can be beneﬁted by the robotics competitions focused on this theme because encourages the development of solutions that allow autonomous robots to work and help in areas inaccessible for humans or in natural disasters areas [20,32,53,54,66,81]. There are some competitions that assist to push the state of the art in domains that are growing like air, land and sea, providing an opportunity to exchange ideas, create solutions as well as a venue to evaluate and encourage state of art research. Autonomous vehicles and drones are ﬁelds that have been attracting a lot of attention, mainly through robotics competitions [4,53,54,65,66]. p RQ6: How have these competitions been contributing positively to education? p RQ6: How have these competitions been contributing positively to education? As we can see in Table 4 a large part of the competitions has a ﬁnal application area in education. Associated with the last extracted form question DQ7 (Which robotics competi- tions contributes positively to education?) we listed most of the robotics competitions with a focus on education. • RoboCup Junior; • RoboCup Junior; • e-Yantra Robotics Competition (eYRC); • Micromouse; • Robot@Factory/Robot@Factory Lite; • Balam Robot Competition (BRC); • FIRST Robotics Competition/FIRST Lego League (FLL); • VEX Robotics Competition; • Student Autonomous Underwater Vehicle Challenge-Europe (SAUC-E); • Mississippi BEST (MSBEST) robotics; • World Robot Olympiad (WRO); • Brazilian Robotics Olympiad; • National Instruments Autonomous Robotics Competition (NIARC); • EUROBOT competition; • Cybertech; • “Schüler bauen Roboter” program; • MicroFactory; • Roboparty; • Latin American IEEE Robotics Competition; • Robosub. • e-Yantra Robotics Competition (eYRC); • e-Yantra Robotics Competition (eYRC); • Micromouse; • Robot@Factory/Robot@Factory Lite; • Balam Robot Competition (BRC); p • FIRST Robotics Competition/FIRST Lego League (FLL); • VEX Robotics Competition; p • Student Autonomous Underwater Vehicle Challenge-Europe (SAUC-E); Mi i i i BEST (MSBEST) b ti Student Autonomous Underwater Vehicle Challenge-Europe (SAUC-E); Mississippi BEST (MSBEST) robotics; World Robot Olympiad (WRO); Brazilian Robotics Olympiad; • National Instruments Autonomous Robotics Competition (NIARC); • Latin American IEEE Robotics Competition Most of them have as objective the dissemination of technology and STEM concepts through young students, encouraging them to pursue career in these fields, develop- ing skills in programming, electronics, robotics, working in team, increasing the moti- vation towards engineering besides that assist universities to teach multidisciplinary do- mains [3,4,8–10,17,20–22,29,30,32,41–43,45,46,50,52,55,57,61,63,64,67,70,71,73,78]. The SAUC- E, for example, has one of the objectives like getting closer contact between the university teams and companies invited to participate [53]. p p p The target public involves young students of primary and secondary schools, high school students and university and engineering students. A famous robotic competition Sensors 2022, 22, 2160 32 of 38 32 of 38 called FIRST Lego League accept students from 9 to 14 years old, a lower age level than e-Yantra Robotics Competition for example, which includes college students of the Indian Institute of Technology Bombay [4,58,78]. called FIRST Lego League accept students from 9 to 14 years old, a lower age level than e-Yantra Robotics Competition for example, which includes college students of the Indian Institute of Technology Bombay [4,58,78]. p RQ6: How have these competitions been contributing positively to education? Education: include tasks in which the participants have to build a robot to perform some activity or sometimes just program the robot to realize a speciﬁc task, for example follow a line, dance, games, etc. Industry: the challenges vary between transport products and logistics tasks, in which the robot must navigate, avoid obstacles, pick up items, etc. Domestic: usually the robots have to help people with household tasks inside an environment that simulates a house. Some tasks include organizing items, opening the refrigerator, picking up a plate of food, etc. Search and rescue: tasks like climbing ramps, walking on dirty terrain with poor visibility, looking for missing people, pipe inspection, etc. Besides that this kind of competition can include underwater ﬁelds, buildings, small places, indoor and outdoor environments. Soccer: the soccer challenges usually includes teams of autonomous robots to compete against each other. RQ4: What type of technologies are used in mobile robotics competitions? Machine Learning, computer vision, localization and mapping (SLAM), speech, object and gesture recognition, real-time control, 3D printing, ROS, HIL, SimTwo, Gazebo, OpenCV, TensorFlow, LEGO Mindstorms, etc. Table 6. Summary of the research questions answers. Table 6. Summary of the research questions answers. Table 6. Summary of the research questions answers. Research Questions Answers Educational: attracting students to STEM areas encouraging them to enter in technologies careers. Industry: manufacturing and logistics. Domestic: assist people in household activities, especially those with disabilities. Sports: amusement for young students with focus on education. Search and rescue: creation of technologies to assist in natural disaster. State of the art: push the state of the art in a speciﬁc ﬁeld like autonomous vehicles, drones and underwater robots. Most take place in many countries all over the world. Some educational competitions usually take place inside a university or school in a speciﬁc region. The target public vary a lot, from young students until professionals and engineers. Education: include tasks in which the participants have to build a robot to perform some activity or sometimes just program the robot to realize a speciﬁc task, for example follow a line, dance, games, etc. Industry: the challenges vary between transport products and logistics tasks, in which the robot must navigate, avoid obstacles, pick up items, etc. Domestic: usually the robots have to help people with household tasks inside an environment that simulates a house. p RQ6: How have these competitions been contributing positively to education? Some competitions take place directly at universities like NIARC, which occurs in universities across Australia and New Zealand, Cybertech, which occur in Univer- sidad Politécnica de Madrid, Balam Robot Competition (BRC) in Universidad Galileo and also “Schüler bauen Roboter” program in Technical University of Munich [21,32,57,61]. The other robotics competitions can take place in any other place or more than one like SAUC-E and EUROBOT competition, for example, which occur in many countries around Europe [20,30,62,73] or even RoboCup and FIRST which are competitions that take place all over the world [3,6,43,58]. FIRST Robotics Competition is one of the oldest and the most famous competition focused on education which began in 1998 with the FIRST Organization and the LEGO Group [52]. The most recent VEX robotics world championship run by the Robotics Education and Competition (REC) Foundation in April of 2018, became the “largest robot competition” in the world according to Guinness World Records [64]. Table 6 summarizes the research questions answers. On the left side there are the six research questions that this work aims to answer and on the right side are presented the main topics of each answer which were already discussed above but with fewer details, in order to provide better insights. Table 6. Summary of the research questions answers. Research Questions Answers RQ1: What type of mobile robotics competitions exist in the last few decades and with what aim? Educational: attracting students to STEM areas encouraging them to enter in technologies careers. Industry: manufacturing and logistics. Domestic: assist people in household activities, especially those with disabilities. Sports: amusement for young students with focus on education. Search and rescue: creation of technologies to assist in natural disaster. State of the art: push the state of the art in a speciﬁc ﬁeld like autonomous vehicles, drones and underwater robots. RQ2: Where do the mobile robotics competitions take place currently and who is their target public? Most take place in many countries all over the world. Some educational competitions usually take place inside a university or school in a speciﬁc region. The target public vary a lot, from young students until professionals and engineers. RQ3: What type of robotics challenges are addressed by the mobile robotics competitions? 5. Conclusions In this paper, a systematic mapping literature review was developed and it was found a large number of articles cited or/and described many mobile robotics competitions that took place over the last few decades. It was possible to analyze in detail most of the competitions and to conclude that these competitions are growing and becoming more common in several domains with diverse objectives, mainly in education. It was observed that the number of competitions since 2001 is gradually increasing each year. Among the most cited robotics competitions in the articles are RoboCup, FIRST and DARPA. RoboCup is the biggest robotic competition with more than 10 leagues, covering several areas in a single competition but with different challenges. FIRST is one of the oldest robotics competitions, very famous too, and has a focus on education and each year a new theme is chosen. DARPA is a competition that is more professional and has prize money and is focused on autonomous vehicles. It is possible to conclude that education is the area most beneﬁted by the mobile robotics competitions. The number of competitions focused on contributing to education is growing because they have been provided how powerful they could be in attracting students for technological areas and positive results have been observed. The robotics competitions focused on education usually have objectives focused on encouraging young students to pursue careers in STEM areas, develop skills, teach how to work in team, assist teachers and universities in multidisciplinary domains and expose students to real problem solving and practical application of their knowledge. Therefore, the robotics competitions have been a good contribution tool not only for education but for different areas, helping people, engineers, researchers, business and students to solve real problems through the use of robots and creating innovative solutions, showing that the robots can be assist us to construct a better quality of life for people and consequently a world better for all. q y As future work, we intend to perform a systematic literature review, which is more speciﬁc than a mapping, of the mobile robotics competitions all over the world and the research questions could be more focused on education, industry or benchmarking. Author Contributions: Data curation, L.B.; Formal analysis, L.B., J.G., M.Á.C. and P.C.; Investigation, L.B., J.G., M.Á.C. and P.C.; Methodology, J.G., M.Á.C. and P.C.; Supervision, J.G., M.Á.C. and P.C.; Validation, J.G., M.Á.C. p RQ6: How have these competitions been contributing positively to education? Some tasks include organizing items, opening the refrigerator, picking up a plate of food, etc. Search and rescue: tasks like climbing ramps, walking on dirty terrain with poor visibility, looking for missing people, pipe inspection, etc. Besides that this kind of competition can include underwater ﬁelds, buildings, small places, indoor and outdoor environments. Soccer: the soccer challenges usually includes teams of autonomous robots to compete against each other. Machine Learning, computer vision, localization and mapping (SLAM), speech, object and gesture recognition, real-time control, 3D printing, ROS, HIL, SimTwo, Gazebo, OpenCV, TensorFlow, LEGO Mindstorms, etc. 33 of 38 Sensors 2022, 22, 2160 Table 6. Cont. Research Questions Answers RQ5: What is the ﬁnal application area of the mobile robotics competitions? Education, industry, daily life, household tasks, search and rescue in disaster scenarios and amusement. RQ6: How have these competitions been contributing positively to education? Dissemination of technology and STEM concepts through young students, encouraging them to pursue a career in these ﬁelds, developing skills in programming, electronics, robotics, working in a team, increasing the motivation and besides that assisting universities to teach multidisciplinary domains. Table 6. Cont. Answers Dissemination of technology and STEM concepts through young students, encouraging them to pursue a career in these ﬁelds, developing skills in programming, electronics, robotics, working in a team, increasing the motivation and besides that assisting universities to teach multidisciplinary domains. 5. Conclusions and P.C.; Writing—original draft, L.B.; Writing—review & editing, L.B., J.G., M.Á.C. and P.C. All the authors have collaborated in the same way. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The dataset containing information about the mobile robotics competi- tions and related URLs to each paper in which the competition is cited, can also be accessed in Zenodo repository through the link: https://doi.org/10.5281/zenodo.6337324 (accessed on 8 March 2022). Conﬂicts of Interest: The authors declare no conﬂict of interest. 34 of 38 34 of 38 Sensors 2022, 22, 2160 Abbreviations The following abbreviations are used in this manuscript: PRISMA Preferred Reporting Items for Systematic Reviews and Meta-Analyses 6 STEM Science, Technology, Engineering and Mathematics STEAM Science, Technology, Engineering, Arts and Mathematics AGV Automated Guided Vehicle AUV Autonomous Underwater Vehicle FIRST For Inspiration and Recognition of Science and Technology SLR Systematic Literature Review PICOC Population, Intervention, Comparison, Outcomes, Context RQ Research Question QQ Quality Question DQ Data Question WRS World Robot Summit ARIAC Agile Robotics for Industrial Applications Competition MBZIRC Mohamed Bin Zayed International Robotics Competition eYRC e-Yantra Robotics Competition RoCKIn Robot Competitions Kick Innovation in Cognitive Systems and Robotics SAUC-E Student Autonomous Underwater Vehicles Challenge-Europe ERL European Robotics League APC Amazon Picking Challenge EuRoC European Robotics Challenge IARC International Aerial Robotics Competition FLL FIRST Lego League MSBEST Mississippi BEST WRO World Robot Olympiad BRC Balam Robot Competition ELROB European Land Robot Trial MAGIC Australian Multi Autonomous Ground-Robotic International Challenge NIARC National Instruments Autonomous Robotics Competition TCFFHRC Trinity College Fire-Fighting Home Robot Contest USAR Urban Search and Rescue HRATC Humanitarian Robotics and Automation Technology Challenge SAVe Students Autonomous Underwater Vehicle OI Oceanology International ROS Robot Operating System SLAM Simultaneous Localization and Mapping AI Artiﬁcial Intelligence NIST National Institute of Standards and Technology vSLAM Visual Simultaneous Localization and Mapping UAM Urban Air Mobility PBL Project Based Learning HIL Hardware in the Loop FSM Finite State Machine EKF Extended Kalman Filter FPGA Field-programmable Gate Array GPS Global Positioning System UPM Universidad Politécnica de Madrid USARSim Uniﬁed System for Automation and Robot Simulation USA United States of America Abbreviations Sensors 2022, 22, 2160 35 of 38 35 of 38 References 1. 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https://openalex.org/W2147960301	https://pure.eur.nl/ws/files/46768373/fulltext.pdf	English	null	Gene-expression of metastasized versus non-metastasized primary head and neck squamous cell carcinomas: A pathway-based analysis	BMC cancer	2,008	cc-by	8,142	Open Acc Research article Gene-expression of metastasized versus non-metastasized primary head and neck squamous cell carcinomas: A pathway-based analysis Erik F Hensen1,2, Maria J De Herdt2, Jelle J Goeman3, Jan Oosting2, Vincent THBM Smit2, Cees J Cornelisse2 and Robert J Baatenburg de Jong4 Open Access Address: 1Department of Otolaryngology and Head and Neck Surgery, Leiden University, Medical Centre, Leiden, The Netherlands, 2Department of Pathology, Leiden University Medical Centre, Leiden, The Netherlands, 3Department of Medical Statistics, Leiden University Medical Centre, Leiden, The Netherlands and 4Department of Otolaryngology and Head and Neck Surgery, Erasmus Medical Centre Rotterdam, The Netherlands Email: Erik F Hensen - e.f.hensen@lumc.nl; Maria J De Herdt - m.deherdt@erasmusmc.nl; Jelle J Goeman - j.j.goeman@lumc.nl; Jan Oosting - j.oosting@lumc.nl; Vincent THBM Smit - v.t.h.b.m.smit@lumc.nl; Cees J Cornelisse - c.j.cornelisse@lumc.nl; Robert J Baatenburg de Jong - r.j.baatenburgdejong@erasmusmc.nl * Corresponding author Received: 18 October 2007 Accepted: 10 June 2008 Received: 18 October 2007 Accepted: 10 June 2008 BMC Cancer 2008, 8:168 doi:10.1186/1471-2407-8-168 This article is available from: http://www.biomedcentral.com This article is available from: http://www.biomedcentral.com/1471-2407/8/168 © 2008 Hensen et al; licensee BioMed Central Ltd. © 2008 Hensen 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. BioMed Central BioMed Central BioMed Central Background way. First, pathways and functional gene clusters that are involved in the process of metastasis in carcinoma were defined using pathways described in literature and the publicly available Kyoto Encyclopedia of Genes and Genomes (KEGG) and Biocarta pathway databases, with a focus on pathways involved in survival, proliferation, apoptosis, cell adhesion, extra cellular matrix signaling and remodeling, hypoxia and angiogenesis [11-13]. Using this supervised analysis strategy, we found considerable concordance between the datasets for pathways involved in survival, proliferation, apoptosis, cell adhesion, extra cellular matrix signaling and remodeling, hypoxia and angiogenesis. Gene sets that were validated by the inde- pendent validation dataset were matrix metalloprotein- ases (MMPs) and pathways involved in MMP regulation, the uPA system and pathways involved in uPA regulation, and HIF1α regulated invasion and angiogenesis. This approach to microarray analysis generates an outcome with readily interpretable biological meaning. Further- more, by concentrating on groups of genes with a known biological relation rather than individual genes, compara- bility of microarray studies performed on different micro- array platforms is improved [14]. g Head and neck squamous cell carcinoma is a relatively common malignancy, associated with severe disease- and treatment-related morbidity. One of the most predictive factors of poor clinical outcome is the presence of regional lymph node metastasis, and nodal status of the neck plays a decisive role in the choice of treatment [1,2]. The com- plex process of metastasis in HNSCC is still incompletely understood at a molecular level; however, multiple marker studies have been performed in order to identify markers that predict the presence of metastasis. Recently, high-throughput gene expression studies have been able to identify a metastatic gene expression signature in pri- mary HNSCC tumors, and Roepman et al. were able to predict the presence of lymph node metastasis based on gene expression of the primary tumor [3]. Analyses in this study were performed in a 'data-driven' way, by means of computational statistics without prior implementation of existing knowledge about functionally related genes and pathways. This technique is very useful in the search for new biomarkers, new subgroups, and differences in their gene expression profiles. Although the authors were able to identify a number of genes that are known to be involved in metastatic disease within this gene set, the interpretation of statistical differences in a meaningful molecular biological context is not self-evident. Background It has been demonstrated that classification gene sets are pro- foundly influenced by the microarray methodology, such as the microarray technique, microarray platform, and preprocessing methods [4-8]. Furthermore, it has been shown that classifying gene sets are highly dependent on the chosen analysis strategy [9,10]. This is illustrated by the fact that the authors were able to generate several dif- ferent classifying gene sets that were all able to predict nodal metastasis with reasonable accuracy [10]. The dependence of classifying gene sets on statistical methods, as well as technical methods such as choice of microarray platform, hampers comparability of results from different microarray studies and raises questions about the biolog- ical relevance of the classifying genes. It is therefore neces- sary that differences in gene expression are validated in an independent dataset. Here, we present an independent gene expression validation study of metastasized versus non-metastasized HNSCC. Differences in gene expression between metastasized and non-metastasized HNSCC were determined in the publicly available dataset gener- ated by Roepman et al., and subsequently validated in an independent gene expression dataset of 11 metastasized and 11 non-metastasized HNSCC tumors of three ana- tomical localizations (the oral cavity, the oropharynx and the larynx). In addition to the validation of individual dif- ferentially expressed genes, we performed a supervised, pathway-based analysis. Gene expression was evaluated within predefined subgroups of genes with a known bio- logical context, i.e. genes within a metastasis related path- Methods Reference dataset h bl l l The publicly available HNSCC gene expression dataset published by Roepman and co-workers was used as the reference dataset [3,15]. All 104 samples (49 non-metas- tasized and 55 metastasized primary head and neck carci- noma's) analyzed in this study were included the reference dataset (Table 1). For sample selection, cRNA preparation, microarray hybridization and normalization methods in this study see elsewhere [3]. http://www.biomedcentral.com/1471-2407/8/168 BMC Cancer 2008, 8:168 Page 2 of 10 (page number not for citation purposes) Abstract Background: Regional lymph node metastasis is an important prognostic factor in head and neck squamous cell carcinoma (HNSCC) and plays a decisive role in the choice of treatment. Here, we present an independent gene expression validation study of metastasized versus non-metastasized HNSCC. Methods: We used a dataset recently published by Roepman et al. as reference dataset and an independent gene expression dataset of 11 metastasized and 11 non-metastasized HNSCC tumors as validation dataset. Reference and validation studies were performed on different microarray platforms with different probe sets and probe content. In addition to a supervised gene-based analysis, a supervised pathway-based analysis was performed, evaluating differences in gene expression for predefined tumorigenesis- and metastasis related gene sets. Results: The gene-based analysis showed 26 significant differentially expressed genes in the reference dataset, 21 of which were present on the microarray platform used in the validation study. 7 of these genes appeared to be significantly expressed in the validation dataset, but failed to pass the correction for multiple testing. The pathway-based analysis revealed 23 significant differentially expressed gene sets, 7 of which were statistically validated. These gene sets are involved in extracellular matrix remodeling (MMPs, MMP regulating pathways and the uPA system), hypoxia and angiogenesis (HIF1α regulated angiogenic factors and HIF1α regulated invasion). Conclusion: Pathways that are differentially expressed between metastasized and non- metastasized HNSCC are involved in the processes of extracellular matrix remodeling, hypoxia and angiogenesis. A supervised pathway-based analysis enhances the understanding of the biological context of the results, the comparability of results across different microarray studies, and reduces multiple testing problems by focusing on a limited number of pathways of interest instead of analyzing the large number of probes available on the microarray. Page 1 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1471-2407/8/168 http://www.biomedcentral.com/1471-2407/8/168 http://www.biomedcentral.com/1471-2407/8/168 BMC Cancer 2008, 8:168 Table 1: Characteristics of tumors included in the reference and validation dataset Characteristic Reference set Validation set Sex (n – %) Male 60 (58%) 14 (64%) Female 44 (42%) 8 (36%) Location of primary tumor (n – %) oral cavity 87 (84%) 7 (32%) oropharynx 17 (16%) 7 (32%) larynx 0 (0%) 8 (36%) Primary tumor size (n – %) Maximal diameter (cm.) < 2.5 33 (32%) 0 (0%) 2.5 – 5.0 49 (47%) 13 (59%) > 5.0 22 (21%) 9 (41%) Nodal metastasis (n – %) no (N0) 49 (47%) 11 (50%) yes (N+) 55 (53%) 11 (50%) Table 1: Characteristics of tumors included in the reference and validation dataset Total RNA was precipitated with glycogen and isopropa- nol, washed with ethanol and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA). Total RNA yield and purity were measured using spectrophotometric analysis and in addition RNA quality was checked on a 1% agarose gel. Double-stranded cDNA synthesis was performed using the Superscript Choice system (Life Technologies, Rockville, MD); incorporating the T7 RNA polymerase promoter in the first round. The resulting ds cDNA was purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA), and applied as a template for in vitro tran- scription using the RNA Transcript Labeling Kit (Enzo Diagnostics, Inc., Farmingdale, NY), incorporating bioti- nylated ribonucleotides required for the staining proce- dures after hybridization. The resulting cRNA was purified as described above and cRNA quantity and quality were measured using spectrophotometric analysis and the 2100 Bioanalyzer (Agilent, Palo Alto, CA). 15 μg frag- mented, biotinylated cRNA was hybridized to the Affyme- trix HG-Focus Target arrays at 45°C for 16 h. After hybridization the arrays were washed, stained and scanned, as described in the Affymetrix users' manual. laryngeal HNSCC) from patients with lymph node metas- tasis at the time of surgery or during the follow-up period (Table 1). In all cases fresh frozen tissue of the primary tumor containing a tumor percentage of 50% or more, paraffin material, an unequivocal pathological classifica- tion of the resection material and clinical data were avail- able. Patients who had undergone radiotherapy before surgical excision of the tumor or had a previous malig- nancy in the same region were excluded. http://www.biomedcentral.com/1471-2407/8/168 All specimens were handled according to the ethical guidelines, as described in the Code for Proper Secondary Use of Human Tissue in the Netherlands of the Dutch Federation of Medical Scientific Societies (FEDERA). The data dis- cussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO), and are accessible through GEO Series accession number GSE9349 [16]. laryngeal HNSCC) from patients with lymph node metas- tasis at the time of surgery or during the follow-up period (Table 1). In all cases fresh frozen tissue of the primary tumor containing a tumor percentage of 50% or more, paraffin material, an unequivocal pathological classifica- tion of the resection material and clinical data were avail- able. Patients who had undergone radiotherapy before surgical excision of the tumor or had a previous malig- nancy in the same region were excluded. All specimens were handled according to the ethical guidelines, as described in the Code for Proper Secondary Use of Human Tissue in the Netherlands of the Dutch Federation of Medical Scientific Societies (FEDERA). The data dis- cussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO), and are accessible through GEO Series accession number GSE9349 [16]. Statistical analysis Normalization and expression analysis Normalization and expression analysis In the validation study, acquisition and quantification of array images was performed using the MAS software pack- age (Affymetrix). All arrays were normalized with gcrma normalization and custom probe definitions based on EntrezGene identifiers using the R statistical software package available on Bioconductor [17-20]. Validation dataset Sample selection p An independent validation dataset was constructed using fresh frozen tumor tissue of 24 patients with an HNSCC originating from the oral cavity, the oropharynx, or the larynx. Primary tumors were removed between 1990 and 2000. Specimens were selected from the frozen tissue bank of the Leiden University Medical Centre (LUMC), the Netherlands. Neck dissection specimens of all cases were available for histological evaluation of lymph nodes. Based on histology obtained from surgical specimens as well as clinical and radiological data collected during a follow-up period of four years or more, the group was divided into a non-metastasized and a metastasized sub- group. The non-metastasized group consisted of 12 tumors (4 from the oral cavity, 4 oropharyngeal and 4 laryngeal HNSCC) from patients without indication of metastasis at the time of surgery, nor during the follow-up period. The metastasized group also consisted of 12 tumors (4 from the oral cavity, 4 oropharyngeal and 4 Page 2 of 10 (page number not for citation purposes) Page 2 of 10 (page number not for citation purposes) Supervised analysis p y The R package 'Linear Models for Microarray Data' (LIMMA) was used for the assessment of differential expression between N0 and N+ HNSCC subgroups in the reference dataset [21]. Multiple testing correction was per- formed using False Discovery Rate (FDR) analysis [22]. The reference dataset was used as the hypothesis generat- ing dataset, and genes with a FDR adjusted p-value < 0.1 were included in the gene set that was subsequently vali- dated using the independent validation dataset. Genes were considered to be validated by the validation dataset if an FDR adjusted p-value < 0.05 was reached. High-density oligonucleotide arrays Data from the reference dataset were obtained with the Human Array-Ready Oligo set, version 2.0 (Qiagen) printed on Corning UltraGAPS slides, containing over 21,000 genes. These data were compared to the data from the validation set, obtained with Affymetrix HG-Focus Target arrays (Affymetrix, Inc., Santa Clara, CA) as micro- array platform, containing over 190,000 unique oligonu- cleotides, representing more than 8,500 of the best characterized human genes. In the study by Roepman et al. it was reported that longer sample storage time had an adverse effect on the predic- tive value of classifying genes, and that the classifying gene set based on samples stored after 1998 had a higher accu- racy [3]. In order to evaluate whether storage time had an effect on the gene-based analysis in this study, we per- formed an additional LIMMA analysis on samples stored after 1998 in the reference dataset and subsequently vali- dated the outcome using samples stored after 1998 in the validation dataset. Total RNA isolation, probe preparation and hybridization to arrays Total RNA isolation, probe preparation and hybridization to arrays were performed according to the Affymetrix pro- tocols. In short, total RNA was extracted from fresh frozen tumor sections using Trizol (Invitrogen, San Diego, CA). Page 3 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1471-2407/8/168 BMC Cancer 2008, 8:168 Pathway selection and analysis chip used in creating the validation dataset. Seven of these 21 genes appeared to be significantly expressed in the val- idation dataset (raw p value < 0.05). However, all failed to pass the correction for multiple testing (FDR adjusted p- value < 0.05). Supervised pathway-based analysis p p y y Analysis of the selected pathways related to survival, pro- liferation, apoptosis, cell adhesion, extra cellular matrix signaling and remodeling, hypoxia and angiogenesis, revealed significant differential expression for 23 gene sets in the reference dataset. These gene sets mainly encode extracellular matrix components (i.e. collagens), and pathways involved in extracellular matrix remodeling (MMPs, the uPA system, uPA regulating pathways, and HIF1α regulated invasion), hypoxia (upregulation of HIF1α target genes) and hypoxia induced angiogenesis (HIF1α regulated angiogenic factors). Of these 23 gene sets, 7 gene sets were validated by analysis of the inde- pendent expression dataset when corrected for multiple testing (Table 3 and 4). These gene sets with significant differential expression between N0 and N+ tumors in both datasets are involved in extracellular matrix remode- ling (MMPs, MMP regulating pathways and the uPA sys- tem), hypoxia and angiogenesis (HIF1α regulated angiogenic factors and HIF1α regulated invasion) (Table 4 and 5, Figure 1). No significant differential expression between metastasized and non-metastasized HNSCC was shown for 215 gene sets in both datasets, including almost all survival, proliferation and apoptosis related gene sets (Table 3). Discussion It has been demonstrated that the outcome of microarray studies is profoundly influenced by the chosen analysis strategy and highly dependent on technical aspects such as sample preparation methods and choice of microarray platform [4-10]. This raises questions about the biological validity of the outcome of individual studies, and the val- idation of microarray studies is therefore essential. Here we present the results of an independent validation anal- ysis of differences in gene expression between metasta- sized and non-metastasized HNSCC. The reference study Unsupervised analysis of the validation dataset p y f 2 out of 24 hybridizations were of insufficient quality, leaving 22 samples in the analysis (11 metastasized and 11 non-metastasized samples) (Table 1). No differences were observed in total RNA and cRNA yield or quality between samples with different storage time. After nor- malization of the validation dataset, there was a great sim- ilarity in gene-expression profiles irrespective of their N- stage or location in the oral cavity, oropharynx, or larynx. Complete linkage two-way hierarchical clustering revealed no well-defined grouping of samples according to their N-stage or location. No grouping was found according to sample storage time. We also performed this clustering analysis separately for each subgroup (accord- ing to localization or N-stage), but found no stable clus- ters (data not shown). 2 out of 24 hybridizations were of insufficient quality, leaving 22 samples in the analysis (11 metastasized and 11 non-metastasized samples) (Table 1). No differences were observed in total RNA and cRNA yield or quality between samples with different storage time. After nor- malization of the validation dataset, there was a great sim- ilarity in gene-expression profiles irrespective of their N- stage or location in the oral cavity, oropharynx, or larynx. Supervised analysis These genes are lethal giant larvae protein 2 of Drosophila (LLGL2), fibroblast activation protein alpha (FAP), urokinase plasminogen activator (PLAU), laminin beta 1 (LAMB1), musculin (MSC), and collagen type V alpha 1 and 3 subunits (COL5A1 and COL5A3) (Table 2). Next, in analogy with the study by Roepman et al., we performed LIMMA analysis on the samples stored after 1998 only (66 samples in the reference dataset and 8 in the validation dataset) [3]. This revealed 352 differen- tially expressed well-defined genes in the reference dataset when corrected for multiple testing. However, none of these genes could be validated (FDR adjusted p-value < 0.05) in the validation dataset (data not shown). y y In order to acquire metastatic potential, a primary tumor cell must complete a series of sequential steps, including progressive growth at the primary tumor site, vascularisa- tion of the primary tumor, invasion of the surrounding stroma, detachment from other tumor cells, embolization of tumor cells in blood vessels or lymph vessels, survival within these vessels, extravasation and proliferation at the metastatic site [23]. Each of these steps is regulated by transient or permanent changes in DNA, RNA or proteins. We have defined pathways and gene sets involved in each of these steps using the publicly available pathway data- bases KEGG and Biocarta, and by researching literature on metastasis in (head and neck) carcinoma [11-13]. We cat- egorized these pathways in the following subgroups: sur- vival, proliferation, differentiation, apoptosis, cell adhesion, extra cellular matrix signaling and remodeling, hypoxia and angiogenesis. In all, 241 gene sets were thus created. In order to determine if gene expression was sig- nificantly different between metastasized and non-metas- tasized HNSCC in the reference dataset, we performed the global test designed by J.J. Goeman (available as the R package 'globaltest' at Bioconductor) on each predefined pathway [19,20,24]. The reference dataset was used as the hypothesis generating dataset, and all pathways with a FDR adjusted p-value < 0.1 were accepted for validation in the independent validation dataset. Pathways were con- sidered to be validated by the validation dataset if an FDR adjusted p-value < 0.05 was reached. Page 4 of 10 (page number not for citation purposes) Supervised gene-based analysis P-values for genes that were not represented on the Affymetrix HG U95Av2 microarray used for the validation dataset are missing. Differentially expressed genes are considered to be validated by the validation dataset if the FDR adjusted p-value is smaller than 0.05. Although the first 7 genes (LLGL2, FAP, PLAU, LAMB1, MSC, COL5A1 and COL5A3) score a raw p-value smaller than 0.05, none of them pass the correction for multiple testing. and validation study were performed in different centers by different investigators, using different microarray plat- forms with different probe content. In this study, we con- centrated on the validation and biological interpretation of the differences in gene expression between N0 and N+ HNSCC subgroups, and did not attempt to validate classi- fying gene sets that predict N-status in the reference study or other HNSCC microarray studies, because the data- driven way in which these classifying gene sets are created makes them too dependent on the microarray platform, the microarray technique, the preprocessing methods and the laboratory used to create them [4-8]. Furthermore, gaining insight into the process of metastasis on the basis of these classifying gene sets is troublesome: although some probes within the classifier encode a gene with a known role in tumorigenesis or metastasis, many others have unrelated or unknown functions [3]. The fact that multiple classifying gene sets can be constructed on basis of the reference study data casts further doubts on their biological validity [10]. In this study, a gene-based analy- Table 3: Number of pathways with significant differential expression between N0 and N+ HNSCC in the reference and validation datasets Pathways significant in validation dataset Pathways not significant in validation dataset Pathways significant in reference dataset 7 16 Pathways not significant in reference dataset 3 215 In all, 241 different tumorigenesis and metastasis related pathways were tested. 23 significant differentially expressed pathways were found in the reference dataset, and 10 in the validation dataset, 7 of which overlapped. 215 pathways showed no significant differential gene expression in both datasets. ays with significant differential expression between N0 and N+ HNSCC in the reference and validation 7 3 Pathways significant in reference dataset Pathways not significant in reference dataset Pathways significant in reference dataset Pathways not significant in reference dataset In all, 241 different tumorigenesis and metastasis related pathways were tested. Supervised gene-based analysis LIMMA analysis revealed 31 significant differentially expressed oligonucleotide transcripts within the reference dataset, when corrected for multiple testing (FDR adjusted p-value < 0.1). Twenty-six of these 31 transcripts repre- sented well-defined genes. Twenty-one of these 26 well- defined genes were also represented on the HG U95 Av2 Page 4 of 10 (page number not for citation purposes) Page 4 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1471-2407/8/168 BMC Cancer 2008, 8:168 Table 2: Differentially expressed genes between N0 and N+ HNSCC as validated by the independent expression dataset Gene GenBank ID Function P-Value P-Value (FDR adjusted) LLGL2 NM_001031803 cell division and migration 0.011 0.116 FAP NM_004460 ecm remodelling 0.013 0.116 PLAU NM_002658 ecm remodelling 0.017 0.116 LAMB1 NM_002291 basement membrane component 0.022 0.116 MSC NM_005098 transcription factor 0.030 0.126 COL5A1 NM_000093 ecm component 0.039 0.134 COL5A3 NM_015719 ecm component 0.045 0.134 COL4A1 NM_001845 ecm component 0.077 0.201 LAMA4 NM_002290 basement membrane component 0.103 0.240 CYB5R3 NM_000398 Metabolism 0.237 0.488 CSTA NM_005213 cell envelope 0.255 0.488 PPL NM_002705 cell envelope 0.283 0.496 NTHL1 NM_002528 DNA damage repair 0.454 0.682 TNFAIP3 NM_006290 inhibit TNF-mediated apoptosis. 0.477 0.682 TTYH1 NM_020659 chloride anion channel 0.487 0.682 PALM2-AKAP2 NM_147150 Unknown 0.617 0.809 SCRG1 NM_007281 response to prion-associated infection 0.727 0.866 ADAM12 NM_021641 ecm remodelling 0.742 0.866 FADS1 NM_013402 Metabolism 0.869 0.888 SOX4 NM_003107 apoptosis and survival 0.876 0.888 IVL NM_005547 cell envelope 0.888 0.888 FN1 NM_212482 cell adhesion and migration - - PELO NM_181501 cell cycle control - - FBXL19 NM_019085 F-box protein - - DMKN NM_033317 Unknown - - EPPK1 NM_031308 cell adhesion - - ecm = extra cellular matrix. P-value = raw p-value of the LIMMA analysis. P-value (FDR adjusted) = p-value of the LIMMA analysis, corrected for multiple testing. P-values for genes that were not represented on the Affymetrix HG U95Av2 microarray used for the validation dataset are missing. Differentially expressed genes are considered to be validated by the validation dataset if the FDR adjusted p-value is smaller than 0.05. Although the first 7 genes (LLGL2, FAP, PLAU, LAMB1, MSC, COL5A1 and COL5A3) score a raw p-value smaller than 0.05, none of them pass the correction for multiple testing. ntially expressed genes between N0 and N+ HNSCC as validated by the independent expression dataset ecm = extra cellular matrix. P-value = raw p-value of the LIMMA analysis. P-value (FDR adjusted) = p-value of the LIMMA analysis, corrected for multiple testing. Supervised gene-based analysis 23 significant differentially expressed pathways were found in the reference dataset, and 10 in the validation dataset, 7 of which overlapped. 215 pathways showed no significant differential gene expression in both datasets. Page 5 of 10 (page number not for citation purposes) Page 5 of 10 (page number not for citation purposes) BMC Cancer 2008, 8:168 http://www.biomedcentral.com/1471-2407/8/168 ween non-metastasized and metastasized primary HNSCC ssion between non-metastasized and metastasized Heatmap of pathways with validated differential gene expression between non-metastasized and metastasized primary HNSCC Figure 1 Heatmap of pathways with validated differential gene expression between non-metastasized and metastasized primary HNSCC. Each row in the figure denotes a gene and each column denotes a sample. Panel on the left: validated dif- ferentially expressed pathways in the reference study. Panel on the right: validated differentially expressed pathways in the val- idation study. Relative gene-expression is shown for each gene in the pathway, and for each sample in the N0 and N1 HNSCC subgroups, red indicating overexpression and green indicating underexpression. White brackets indicate genes not repre- sented on the respective microarray platforms. Heatmap of pathways with validated differential gene expression between non metastasized and metastasized primary HNSCC Figure 1 Heatmap of pathways with validated differential gene expression between non-metastasized and metastasized primary HNSCC. Each row in the figure denotes a gene and each column denotes a sample. Panel on the left: validated dif- ferentially expressed pathways in the reference study. Panel on the right: validated differentially expressed pathways in the val- idation study. Relative gene-expression is shown for each gene in the pathway, and for each sample in the N0 and N1 HNSCC subgroups, red indicating overexpression and green indicating underexpression. White brackets indicate genes not repre- sented on the respective microarray platforms. Supervised gene-based analysis Page 6 of 10 (page number not for citation purposes) Page 6 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1471-2407/8/168 BMC Cancer 2008, 8:168 BMC Cancer 2008, 8:168 Table 4: Validated pathways with significant differential expression between N0 and N+ HNSCC Pathways Genes in pathway Probes in reference dataset (n) Probes in validation dataset (n) p-value p-value (FDR adjusted) MAPK 1/3 regulated MMPs 16 17 14 0.003 0.028 JNK/MAPK regulated MMPs 25 28 19 0.003 0.028 MAPK 1/3 regulated invasion 35 38 30 0.004 0.028 MMPs 9 8 8 0.005 0.028 HIF1α regulated invasion 14 11 11 0.011 0.048 HIF1α regulated angiogenesis 26 21 19 0.013 0.048 uPA system 3 3 2 0.015 0.048 Genes in pathway = number of genes theoretically involved in the pathway. Probes in reference dataset = number of probes representing pathway genes in the reference dataset. Probes in validation dataset = number of probes representing pathway genes in the validation dataset. P-value = raw p-value of the global test. P-value (FDR adjusted) = p-value of the global test, corrected for multiple testing using the false discovery rate. Differentially expressed pathways are considered to be validated by the validation dataset if the FDR adjusted p-value is smaller than 0.05. Table 4: Validated pathways with significant differential expression between N0 and N+ HNSCC ys with significant differential expression between N0 and N+ HNSCC able 4: Validated pathways with significant differential expression between N0 and N+ HNSCC t differential expression between N0 and N+ HNSCC Genes in pathway = number of genes theoretically involved in the pathway. Probes in reference dataset = number of probes representing pathway genes in the reference dataset. Probes in validation dataset = number of probes representing pathway genes in the validation dataset. P-value = raw p-value of the global test. P-value (FDR adjusted) = p-value of the global test, corrected for multiple testing using the false discovery rate. Differentially expressed pathways are considered to be validated by the validation dataset if the FDR adjusted p-value is smaller than 0.05. Genes in pathway = number of genes theoretically involved in the pathway. Probes in reference dataset = number of probes representing pathway genes in the reference dataset. Probes in validation dataset = number of probes representing pathway genes in the validation dataset. P-value = raw p-value of the global test. Page 7 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1471-2407/8/168 PLAU in particular seems to correlate well with unfavourable outcome, and in this study PLAU correlated well with metastatic HNSCC in both the reference and validation datasets. Hypoxia is a common feature in solid tumors and their metastasis, and can lead to tumor progression in a variety of ways. It induces HIF1α a transcription factor that regulates angio sis revealed 7 genes that appeared to be significantly expressed in the validation dataset (raw p value < 0.05) (Table 2). All of these 7 genes are known to be involved in processes of tumorigenesis or metastasis. LLGL2 belongs to a group of genes that act as tumor suppressor genes. Loss of function is associated with disruption of cell polar- ity and tissue architecture, uncontrolled proliferation and growth of neoplastic lesions [25]. FAP is a cell-surface pro- tease expressed in reactive stromal fibroblasts of epithelial cancers, and is associated with invasion and metastasis in gastric, colorectal and cervical carcinoma [26-28]. PLAU encodes a serine protease involved in degradation of the extracellular matrix. Its plays a well-known role in inva- sion and metastasis of carcinoma, and is a prognostic fac- tor for metastasis and outcome [29,30]. LAMB1 encodes the β1 subunit of members from the laminin family, extracellular matrix glycoproteins that are the major non- collagenous constituent of basement membranes. Lam- inins containing the β1 subunit (i.e. laminin 8 and 10) have been implicated in the metastasis related processes of angiogenesis, invasion, and migration [31,32]. The pro- tein encoded by MSC is a transcriptional repressor that attenuates E2A-mediated gene activation. MSC overex- pression is associated with loss of differentiation in mul- tiple tissues and is associated with B-cell lymphoma, but no association with epithelial cancer has been described to date [33,34]. COL5A1 and COL5A3 encode alpha chains of collagen type 5. Upregulation is associated with metastatic potential in carcinoma [3,35]. However, when corrected for multiple testing, none of these genes could be statistically validated. Roepman et al. have reported an adverse effect of long- term storage of tissue samples on its predictive accuracy. No explanation was found for this phenomenon, but it did not seem to be attributable to differences in total RNA and cRNA yield or quality [3]. We have evaluated the effect of storage time on the gene-based validation analy- sis in this study. http://www.biomedcentral.com/1471-2407/8/168 BMC Cancer 2008, 8:168 ing groups of functionally related genes, we were able to study the same biological processes in both reference and validation datasets, even though not all genes involved in these processes were present, and the number and nature of the represented genes varied in the respective datasets. In this way, 7 metastasis-related pathways and function- ally related gene sets that differentiate between metasta- sized and non-metastasized HNSCC were statistically validated (Table 4 and Figure 1). These validated path- ways are metalloproteinases and regulatory pathways of metalloproteinases, HIF1α induced invasion- and angio- genesis related target genes and the urokinase plasmino- gen activator system, key pathways involved in invasion, extra cellular matrix remodelling, detachment and angio- genesis, essential steps in the progression to metastatic disease (Tables 4 and 5). Metalloproteinases play a com- plex role in tumor progression and metastasis. Not only do they facilitate invasion by degrading components of the extracellular matrix, there is also evidence that they are involved in angiogenesis. MMPs that induce metastasis are not only produced by the tumor cells but also by stro- mal cells and leucocytes, especially along the invasive front of the tumor [36]. The second messenger signalling pathways that lead to expression of MMPs are not fully understood, but there is evidence that MAPK pathways are involved [37]. Three different regulatory MAPK pathways of MMPs have been identified, and in this study two of them show significant differential expression between metastasized and non-metastasized HNSCC: the MAPK1/ 3 (ERK1/2) and JNK/MAPK pathways. The MAPK1/3 pathway, which is activated by a variety of mitogenic and growth factors, induces FOS and JUN phosphorylation and expression. The JNK/MAPK pathway, which is induced by various inflammatory cytokines, increases transcriptional activity and protein stability of JUN. FOS and JUN are leucine zipper proteins that can dimerize forming the AP-1 transcription factor complexes. JUN, FOS and AP-1 complexes seem to regulate expression of multiple MMPs [37]. The urokinase plasminogen activa- tor system (uPA) mediates invasion and metastasis by cat- alysing extracellular matrix dissolution, and there is evidence that the uPA system plays a role in cell prolifera- tion, migration and modulation of cell adhesion as well. The potential of components of this system as prognosti- cators in cancer has been evaluated most extensively in breast cancer, but also in HNSCC [38]. Supervised gene-based analysis P-value (FDR adjusted) = p-value of the global test, corrected for multiple testing using the false discovery rate. Differentially expressed pathways are considered to be validated by the validation dataset if the FDR adjusted p-value is smaller than 0.05. Table 5: Genes included in the pathways and functional gene sets that are differentially expressed between metastasized and non- metastasized HNSCC uPA system HIF1A regulated angiogenesis HIF1A regulated invasion MMPs in HNSCC MAPK 1/3 regulated invasion JNK/MAPK regulated MMPs MAPK 1/3 regulated MMPs PLAU HGF HIF1A MMP1 SHC1 HRAS SHC1 PLAUR FGF2 MMP2 MMP2 GRB2 RAC1 GRB2 PLG PDGFB MMP13 MMP3 SOS1 PAK1 SOS1 IL8 PLAUR MMP6 HRAS PAK2 HRAS PGF P4HA1 MMP7 RAF1 MAP3K4 RAF1 ANGPT2 CXCL12 MMP9 MAP2K1 MAP3K1 MAP2K1 TEK MMP14 MMP10 MAP2K2 MAP3K12 MAP2K2 MMP2 COL5A1 MMP11 MAPK3 MAP2K7 MAPK3 MMP13 CTGF MMP14 MAPK1 MAP2K4 MAPK1 PLAUR ITGB2 RPS6KA5 MAPK8 FOS CCL2 SERPINE 1 RPS6KA1 MAPK9 MMP1 CXCL12 CXCR 4 MKNK1 MAPK10 MMP3 VEGFA MET MKNK2 JUN MMP7 FLT 1 ETS 1 MYC ATF2 MMP9 IGFBP1 ELK1 SP1 MMP10 SERPINE 1 STAT3 ELK1 MMP13 P4HA1 TERT JUND CXCR 4 SRF CDC42 COL5A1 ELK4 CD44 CTGF ATF4 MMP1 MMP14 FOS MMP3 HIF1A RASA2 MMP7 ENG NF1 MMP9 EDN 1 RASA1 MMP10 LRP 1 RASGRF1 MMP13 CITED 2 RASGRP1 RAPGEF2 PRKCA CD44 MMP1 MMP3 MMP7 MMP9 MMP10 MMP13 Table 5: Genes included in the pathways and functional gene sets that are differentially expressed between metastasized and non- metastasized HNSCC the pathways and functional gene sets that are differentially expressed between metastasized and non- able 5: Genes included in the pathways and functional gene sets that are differentially expressed between m metastasized HNSCC Page 7 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1471-2407/8/168 Page 8 of 10 (page number not for citation purposes) Authors' contributions EFH participated in the design of the study, in the valida- tion microarray experiments and the statistical analysis, and drafted the manuscript. MJDH carried out the valida- tion microarray experiments and helped with the statisti- cal analysis. JJG participated in the design of the study, carried out part of the statistical analysis and helped to draft the manuscript. JO carried out part of the statistical analysis and helped to draft the manuscript. VTHBMS helped with designing the study and drafting the manu- script. CJC participated in the design of the study, its coor- dination and drafting of the manuscript. RJBdJ conceived of the study, participated in its coordination and helped to draft the manuscript. All authors read and approved the final manuscript. http://www.biomedcentral.com/1471-2407/8/168 BMC Cancer 2008, 8:168 involved in angiogenesis and invasion are significantly upregulated in metastasized HNSCC in this study [39-41]. As oligonucleotide microarrays measure mRNA levels, results reflect the gene-expression levels in N0 and N+ HNSCC. Post-transcriptional events such as splicing, translation or activation of the proteins encoded by these genes are not measured. Upregulation of genes in a spe- cific pathway as determined by oligonucleotide microar- rays therefore may not necessarily mean heightened activity of the pathway. However, it is very plausible that the observed differences in gene-expression levels of genes involved in metastasis-related pathways are responsible for the differences in metastatic potential of N0 and N+ HNSCC. sets instead of individual probes in the analysis of micro- array data, the biological context of the results is readily interpretable. Furthermore, a supervised, pathway-based analysis reduces multiple testing problems associated with microarray analysis by focusing on a limited number of pathways instead of analyzing all of the probes availa- ble on the microarray. Most importantly, the comparabil- ity of results from different microarray studies is greatly improved. A supervised, pathway based analysis can reveal biologically relevant similarity between results of different gene-expression studies, even if studies have used different microarray platforms with different probes and probe content. Abbreviations The aim of this study is not only to identify and validate a gene-expression profile that characterizes metastatic dis- ease in head and neck squamous cell carcinoma, but to provide an analysis strategy that incorporates the available insights in the pathways that lead to metastasis. This supervised pathway-based analysis will not reveal new, previously unknown metastasis related biomarkers. It does however increase our understanding of the biologi- cal context of the results. By focusing on pathways and functional gene sets, rather than individual genes, the insight into the biological steps that lead from carcinogen- esis to metastatic disease in HNSCC is enhanced. Further- more, by leaving probes that are not relevant to the biological processes of interest out of the analysis, statisti- cal noise and multiple testing problems associated with microarray analysis are reduced. The most important advantage of this strategy however is the increased compa- rability of data from different microarray studies. Microar- ray analyses based on individual genes are highly dependent on the exact gene content of the microarray used in the study, and thus on the chosen microarray plat- form. In a pathway-based analysis however, gene expres- sion does not have to be measured from every single gene involved in a specific pathway, as long as a representative subset of genes is assessed. These representative subsets of genes involved in a specific pathway may vary between studies. A pathway-based analysis thus can reveal biologi- cally relevant similarity between results of different micro- array studies even though the gene contents of the microarray platforms used do not match exactly. LLGL2: lethal giant larvae homolog 2; FAP: fibroblast acti- vation protein alpha; PLAU: plasminogen activator uroki- nase; LAMB1: laminin beta 1; MSC: musculin; COL5A1: collagen type V alpha 1; COL5A3: collagen type V alpha 3; MMP: matrix metalloproteinase; uPA: urokinase plas- minogen activator system; HIF1α: hypoxia-inducible fac- tor 1; MAPK: mitogen-activated protein kinase; JNK: c-jun N-terminal kinase; FOS: v-fos FBJ murine osteosarcoma viral oncogene homolog; JUN: c-jun oncogene; AP-1: DNA binding/transcription factor AP1. Conclusion h This research was financially supported by a grant from the Dutch Cancer Society (DCS), Amsterdam, the Netherlands. In this gene-expression study, we were able to identify and validate several pathways that are differentially expressed between metastasized and non-metastasized HNSCC. These pathways are involved in the processes of extracel- lular matrix remodeling (MMPs, MMP regulating path- ways and the uPA system), hypoxia and angiogenesis (HIF1α regulated angiogenic factors and HIF1α regulated invasion). By focusing on pathways and functional gene http://www.biomedcentral.com/1471-2407/8/168 Our LIMMA analysis of the most recent tumor samples within the reference dataset identified more differentially expressed genes, an observation that seems to correlate well with the findings of Roepman et al. [3]. However, we do not find an effect of sample storage time on the outcome of our validation analysis as none of these genes are statistically validated by the most recent samples in the validation dataset. The gene-based validation between the reference and val- idation studies was hampered by the use of different microarray platforms with different probes and probe content. In order to overcome this problem, a pathway- based supervised analysis was performed, evaluating dif- ferences in gene expression between metastasized and non-metastasized HNSCC for predefined tumorigenesis- and metastasis related pathways and gene sets. By analyz- Page 8 of 10 (page number not for citation purposes) Page 8 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1471-2407/8/168 Page 9 of 10 (page number not for citation purposes) References 1. Baatenburg de Jong RJ, Hermans J, Molenaar J, Briaire JJ, Le Cessie S: Prediction of survival in patients with head and neck cancer. Head Neck 2001, 23(9):718-724. 2. Mamelle G, Pampurik J, Luboinski B, Lancar R, Lusinchi A, Bosq J: Lymph node prognostic factors in head and neck squamous cell carcinomas. Am J Surg 1994, 168(5):494-498. 3. 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https://openalex.org/W2596216072	https://jurnal.untirta.ac.id/index.php/jft/article/download/1261/1016	Indonesian	null	ANALISIS BANJIR MENGGUNAKAN SOFTWARE HEC-RAS 4.1.0 (Studi Kasus Sub DAS Ciberang HM 0+00 - HM 34+00)	Fondasi: Jurnal Teknik Sipil/Fondasi	2,016	cc-by-sa	5,809	ANALISIS BANJIR MENGGUNAKAN SOFTWARE HEC-RAS 4.1.0 (Studi Kasus Sub-DAS Ciberang HM 0+00 - HM 34+00) Restu Wigati1), Soedarsono2), Tia Mutia3) Jurusan Teknik Sipil, Fakultas Teknik, Universitas Sultan Ageng Tirtayasa Jl. Jenderal Sudirman Km.3 Kota Cilegon – Banten Indonesia 3) 3)timutia1402@yahoo.co.id Jurnal Fondasi, Volume 5 No 2 2016 Jurnal Fondasi, Volume 5 No 2 2016 INTISARI Salah satu fenomena alam yang mengancam keberadaan hidup manusia dibeberapa wilayah di Indonesia setiap masuk musim penghujan yaitu banjir.Setiap tahun Kabupaten Lebak umumnya mengalami banjir terutama di daerah masyarakat yang bermukim di sepanjang bantaran sungai. Hal ini disebabkan berkurangnya kapasitas penampang sungai sehingga dimensi sungai tidak mampu menampung debit yang ada dan menyebabkan Sungai Ciberang meluap. Tujuan dari penelitian ini untuk mengetahui besarnya debit banjir Sungai Ciberang dengan Q50dan mengidentifikasi daerah rawan banjir serta memberikan solusi masalah banjir yang terjadi di Sungai Ciberang. Pada Penelitian ini data yang digunakan adalah data sekunder berupa Curah hujan harian selama 19 tahun dan data dimensi penampang melintang sub-DAS Ciberang. hasil hitungan hujan rencana dengan kala ulang 50 tahun yaitu 105,875 mm, hujan jam-jaman durasi 6 jam dengan durasi puncak pada jam ke 3 yaitu = 57,713 mm sedangkan debit rencana dengan metode HSS SCS didapat nilai debit puncak sebesar 523,174m3/s, adapun dengan menggunakan metode HSS Snyder didapat sebesar 1228,162 m3/s, langkah dilanjutkan menggunakan softwareHEC-RAS 4.1.0 untuk mengetahui kapasitas tampung sungai dengan menggunakan debit Snyder. gg y Setelah dianalisis menggunakan software, sub-DAS Ciberang tidak dapat menampung debit aliran yang terjadi, oleh karenanya perlu adanya perbaikan sungai berupa normalisasi sungai dan peninggian tanggul. C. Lokasi Penelitian C. Lokasi Penelitian Lokasi yang ditinjau dari penelitian ini terletak di alur Sungai Ciberang Kecamatan Rangkasbitung, Kabupaten Lebak Provinsi Banten. Untuk mengetahui lokasi penelitian seperti diperlihatkan pada gambar 1 berikut ini: p p g Gambar 1. Titik Lokasi yang Ditinjau (Sumber: Hasil Analisa Menggunakan Google Map, 2015) Gambar 1. Titik Lokasi yang Ditinjau (Sumber: Hasil Analisa Menggunakan Google Map, 2015) 1. PENDAHULUAN dilakukan pada kondisi sungai yang ada saat ini dengan tujuan untuk mengetahui kapasitas pengaliran maksimum pada masing-masing segmen sungai. Analisis ini dilakukan dengan menggunakan kala ulang 50 tahun. B. Tujuan Penelitian B. Tujuan Penelitian Tujuan dari penelitian ini untuk mengetahui besarnya debit banjir Sungai Ciberang dengan Q50dan mengidentifikasi daerah rawan banjir serta memberikan solusi masalah banjir yang terjadi di Sungai Ciberang. B. Tujuan Penelitian Tujuan dari penelitian ini untuk mengetahui besarnya debit banjir Sungai Ciberang dengan Q50dan mengidentifikasi daerah rawan banjir serta memberikan solusi masalah banjir yang terjadi di Sungai Ciberang. Salah satu fenomena alam yang mengancam keberadaan hidup manusia di beberapa wilayah di Indonesia setiap masuk musim penghujan yaitu banjir. Setiap tahun Kabupaten Lebak umumnya mengalami banjir terutama di daerah masyarakat yang bermukim di sepanjang bantaran sungai. Banjir di Kabupaten Lebak yang terjadi menimbulkan dampak yang merugikan masyarakat yang dapat menimbulkan korban jiwa maupun kerugian harta. Banjir juga dapat merusak bangunan, lahan pertanian, sarana dan prasarana,lingkungan hidup serta tata kehidupan masyarakat sekitar sungai ciberang yang merupakan Sub- DAS Ciujung. Banjir yang terjadi disebabkan oleh beberapa faktor diantaranya curah hujan yang tinggi dan berkurangnya kapasitas penampang sungai sehingga dimensi saluran air sungai tidak cukup untuk menampung debit aliran sungai yang ada dan menyebabkan air Sungai Ciberang meluap dan mengenai pemukiman rumah warga. Kondisi seperti ini tentu saja sangat berbahaya bagi warga yang memiliki rumah di pinggiran sungai karena lama kelamaan air sungai terus mengikis tanah sebagai pijakan rumah mereka. \| Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa A. Latar Belakang Sungai merupakan salah satu sumber daya air yang mempunyai sejumlah potensi yang dapat dimanfaatkan bagi kesejahteraan manusia. Salah satu manfaat sungai sebagai sumber air di antaranya adalah sebagai sumber penghidupan dan kehidupan masyarakat yang tinggal disekitar sungai. Salah satu sungai yang berada di Indonesia yaitu Sungai Ciberang yang berada di Provinsi Banten Kabupaten Lebak dengan panjang 51,537 Km dan luas 342 Km2. Sungai merupakan salah satu sumber daya air yang mempunyai sejumlah potensi yang dapat dimanfaatkan bagi kesejahteraan manusia. Salah satu manfaat sungai sebagai sumber air di antaranya adalah sebagai sumber penghidupan dan kehidupan masyarakat yang tinggal disekitar sungai. Salah satu sungai yang berada di Indonesia yaitu Sungai Ciberang yang berada di Provinsi Banten Kabupaten Lebak dengan panjang 51,537 Km dan luas 342 Km2. ABSTRACT Flood is one of the natural phenomenon that can be threatens the existence of human life inseveral regions in Indonesia especially in rainy season. Lebak regency is generallyexperienced floods every year, especially in the living area along the riverbanks. This is causedthe reduced of the tiver capacity so that the cross section of the river dimensions are cannot to accommodate the existing discharge and cause Ciberang River to overflowed. The purpose of this research is to know about the rate of the discharge flood in Ciberang River by using Q50 and to identify the flood-prone areas and also to give the solutions for any problems about flood in Ciberang River. This research uses the secondary data ina form of the data in daily rainfall for 19 years and the cross- section dimensions of Ciberang sub-watersheds. The result of the count rains plan with a return period of 50 years is 105.875 mm, rain with the 6 hours durations there’s on the peak position on the third hour is 57.713 mm, while the discharge plan with the method of SCS obtained the peak discharge valueis 523.174 m3/s and while using HSS Snyder method, it is obtained the peak discharge value is 1228.162 m3/s, the next step is using HEC-RAS 4.1.0 software to determine the carrying capacity of the river by using discharge flood Snyder. After analyze using the software, the sub-watershed of Ciberang River cannot accommodate the rate flow that occurred, therefore the rivers need an improvements in the form of normalization and raising the levee river. Keywords: Discharge flood, HEC-RAS 4.1.0, HSS Snyder method, HSS SCS method, Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa \| Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa \| 51 Jurnal Fondasi, Volume 5 No 2 2016 2. TINJAUAN PUSTAKA A. Umum Beberapa istilah yang berkaitan dengan sungai menurut Peraturan Pemerintah Republik Indonesia Nomor 38 Tahun 2011 Tentang Sungai diantaranya: 1. Sungai adalah alur atau wadah air alami dan atau buatan berupa jaringan pengaliran air beserta air di dalamnya, mulai dari hulu sampai muara, dengan dibatasi kanan dan kiri oleh garis sempadan. Berdasarkan latar belakang yang ada penulis memandang perlu adanya analisa pengendalian banjir di wilayah Sub DAS Ciberang. Analisis dilakukan dengan menggunakan program pemodelan matematik HEC-RAS 4.1.0.HEC-RAS (Hydrologic Engineering Center’s - River Analysis System). Analisis kapasitas penampang Sungai Ciberang p 2. Banjir adalah peristiwa meluapnya air sungai melebihi palung sungai. \| Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa 52 Jurnal Fondasi, Volume 5 No 2 2016 = (5) 5. Koefisien Kurtosis (Ck) = ∑( ) ( )( )( )( ) (6) Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa \| D. Distribusi Curah Hujan (9) Keterangan : = Besarnya hujan rencana untuk periode ulang T tahun = Nilai tengah sampel = Standar deviasi sampel = Faktor frekuensi (9) 1. Uji Konsistensi Data ( ) 5. Koefisien Kurtosis (Ck) = ∑( ) ( )( )( )( ) (6) (6) B. Curah Hujan = (5) B. Curah Hujan 1. Uji Konsistensi Data Data hujan disebut konsisten jika data yang terukur dan dihitung adalah teliti dan benar serta sesuai dengan fenomena saat hujan itu terjadi. Metode yang digunakan untuk mengoreksi data dengan cara Double Mass Curve (Kurva Massa Ganda) yang menggambarkan grafik hubungan antara curah hujan kumulatif stasiun yang diuji dengan curah hujan kumulatif stasiun disekitarnya. C. Analisa Frekuensi Curah Hujan C. Analisa Frekuensi Curah Hujan Analisa frekuensi curah hujan diperlukan untuk menentukan jenis sebaran (distribusi) yang mempunyai parameter statistik sebagai berikut : 1. Curah hujan rata-rata ( ) = ∑ (2) Keterangan : ∑ = Jumlah Curah Hujan n = Jumlah data 2. Standar Deviasi (s) = ∑( ) (3) 3. Koefisien Skewness (Cs) = ∑( ) ( )( )( ) (4) 4. Koefisien Variasi (Cv) Analisa frekuensi curah hujan diperlukan untuk menentukan jenis sebaran (distribusi) yang mempunyai parameter statistik sebagai berikut : (10) 1. Curah hujan rata-rata ( ) ) = ∑ (2) ∑ = Jumlah Curah Hujan 2. Standar Deviasi (s) D. Distribusi Curah Hujan j Tujuan dari analisa distribusi frekuensi curah hujan adalah untuk memperkirakan besarnya variate-variate masa ulang tertentu.Untuk menganalisis probabilitas banjir biasanya dipakai beberapa macam distribusi yaitu: p y 1. Gumbel p 1. Gumbel = + . (7) gan : (7) (7) Keterangan : 2. Hujan Kawasan (DAS) Curah hujan yang diperlukan untuk penyusunan suatu rancangan pemanfaatan air dan rancangan pengendalian banjir adalah curah hujan rata-rata di seluruh daerah yang bersangkutan. Curah hujan ini disebut curah hujan wilayah atau daerah dan dinyatakan dalam mm. Data untuk perhitungan curah hujan rata-rata maksimum dari beberapa stasiun menggunakan cara aljabar. 2. Hujan Kawasan (DAS) Curah hujan yang diperlukan untuk penyusunan suatu rancangan pemanfaatan air dan rancangan pengendalian banjir adalah curah hujan rata-rata di seluruh daerah yang bersangkutan. Curah hujan ini disebut curah hujan wilayah atau daerah dan dinyatakan dalam mm. Data untuk perhitungan curah hujan rata-rata maksimum dari beberapa stasiun menggunakan cara aljabar. = Besarnya hujan rencana untuk periode ulang T tahun = Nilai tengah sampel = Standar deviasi sampel = Faktor frekuensi 2. Log Pearson Type III = + . (8) Keterangan : (8) g = Besarnya curah hujan rancangan untuk periode ulangpada T tahun = Besarnya curah hujan rancangan untuk periode ulangpada T tahun = ( 1 + 2 … … … … + ) (1) Keterangan : K = faktor frekuensi yang merupakan fungsi dari periode ulang dan tipe distribusi frekuensi. K = faktor frekuensi yang merupakan fungsi dari periode ulang dan tipe distribusi frekuensi. R = Curah hujan daerah (mm) 3. Distribusi Normal = + . (9) Keterangan : 3. Distribusi Normal = + . (9) Keterangan : = Besarnya hujan rencana untuk periode ulang T tahun = Nilai tengah sampel = Standar deviasi sampel = Faktor frekuensi 4. Distribusi Log Normal = + . (10) Keterangan : = Variabel standart, besarnya bergantung pada koefisien Kemiringan E. Uji Kecocokan Distribusi Data Untuk menguatkan perkiraan pemilihan distribusi yang diambil, maka dilakukan pengujian distribusi yaitu: 3. Distribusi Normal = + . (9) Keterangan : = Besarnya hujan rencana untuk periode ulang T tahun = Nilai tengah sampel = Standar deviasi sampel = Faktor frekuensi 4. Distribusi Log Normal = + . (10) Keterangan : = Variabel standart, besarnya bergantung pada koefisien Kemiringan E. Uji Kecocokan Distribusi Data Untuk menguatkan perkiraan pemilihan distribusi yang diambil, maka dilakukan pengujian distribusi yaitu: 3. Distribusi Normal = + . E. Uji Kecocokan Distribusi Data Hidrograf Satuan Sintetis Soil Conservation Servise (HSS SCS) qp = Puncak hidrograf satuan (m3/s) = 0,6 (20) , ( ) tp = Lama waktu kelambatan (lag time) ( ) tp = Lama waktu kelambatan (lag time) time) = 0,01947 , , (21) Tc = Waktu konsentrasi (menit) ) = 0,01947 , , (21) Tc = Waktu konsentrasi (menit) Tp = Waktu naik(time of rise) (jam) \| Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa E. Uji Kecocokan Distribusi Data ( )( 4. Koefisien Variasi (Cv) ( )( 4. Koefisien Variasi (Cv) Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa \| 53 Jurnal Fondasi, Volume 5 No 2 1. Metode Chi-kuadrat = ∑ ( ) (11) Keterangan : X2Cr = Harga chi-kuadrat, Efi = Banyaknya frekuensi yang diharapkan Ofi = Frekuensi yang terbaca pada kelas yang sama n = Jumlah data 2. Metode Smirnov-Kolmogorov = \| ( ) − ′( )\|(12) Keterangan : D=Selisih terbesar antara peluang pengamatan dan peluang teoritis P(Xm) = Peluang pengamatan P’(Xm) =Peluang teoritis dan di t ib i di k i ′ = + 0,25( − ) (17) = Waktu penaikan banjir (time rise to speak) Qp = qp.A (18) = Debit maksimum total (m3/s) 2. Hidrograf Satuan Sintetis Soil Conservation Servise (HSS SCS) = (19) qp = Puncak hidrograf satuan (m3/s) = 0,6 (20) tp = Lama waktu kelambatan (lag time) = 0,01947 , , (21) Tc = Waktu konsentrasi (menit) = + (22) Tp = Waktu naik(time of rise) (jam) ′ = + 0,25( − ) (17) = Waktu penaikan banjir (time rise to speak) Qp = qp.A (18) = Debit maksimum total (m3/s) 2. Hidrograf Satuan Sintetis Soil Conservation Servise (HSS SCS) = (19) qp = Puncak hidrograf satuan (m3/s) = 0,6 (20) tp = Lama waktu kelambatan (lag time) = 0,01947 , , (21) Tc = Waktu konsentrasi (menit) = + (22) Tp = Waktu naik(time of rise) (jam) 1. Metode Chi-kuadrat = ∑ ( ) (11) Keterangan : X2Cr = Harga chi-kuadrat, Efi = Banyaknya frekuensi yang diharapkan Ofi = Frekuensi yang terbaca pada kelas yang sama n = Jumlah data 2. Metode Smirnov-Kolmogorov = \| ( ) − ′( )\|(12) Keterangan : D=Selisih terbesar antara peluang pengamatan dan peluang teoritis P(Xm) = Peluang pengamatan P’(Xm) =Peluang teoritis dan persamaan distribusi yang dipakai. 1. Metode Chi-kuadrat = ∑ ( ) (11) Keterangan : X2Cr = Harga chi-kuadrat, Efi = Banyaknya frekuensi yang diharapkan Ofi = Frekuensi yang terbaca pada kelas yang sama n = Jumlah data 2. Metode Smirnov-Kolmogorov = \| ( ) − ′( )\|(12) Keterangan : D=Selisih terbesar antara peluang pengamatan dan peluang teoritis P(Xm) = Peluang pengamatan P’(Xm) =Peluang teoritis dan persamaan distribusi yang dipakai. (11) Keterangan : 2 2. F. Distribusi Hujan jam-jaman Perencanaan penampang melintang diperlukan untuk mendapatkan penampang yang ideal dan efisien dalam penggunaan lahan serta dapat mengalirkan debit air agar tidak sampai meluap ke daerah yang akan dikeringkan. F. Distribusi Hujan jam-jaman Untuk menghitung hidrograf banjir rancangan dengan hidrograf satuan tertentu perlu diketahui terlebih dahulu distribusi curah hujan jam-jaman dengan interval tertentudengan cara Mononobe (R SNI 03-2415-1991): Gambar 2. Sungai Berpenampang Majemuk(Ganda) (Sumber : Analisis Penulis, 2015) ) = / (13) n : (13) Keterangan : I = Intensitas curah hujan (mm/jam) I = Intensitas curah hujan (mm/jam) j ( T = waktu curah hujan (jam) Gambar 2. Sungai Berpenampang Majemuk(Ganda) (Sumber : Analisis Penulis, 2015) Gambar 2. Sungai Berpenampang Majemuk(Ganda) (Sumber : Analisis Penulis, 2015) R24 = Curah hujan maksimum dalam 24 jam (mm) R24 = Curah hujan maksimum dalam 24 jam (mm) G. Debit Rencana Pemodelan HEC-RAS 4.1.0 Progam HEC RAS merupakan paket program dari ASCE (American Society of Civil Engineers).HEC-RAS dirancang untuk membuat simulasi aliran satu dimensi. Perangkat lunak ini memberikan kemudahan dengan tampilan grafisnya. Pada software HEC-RAS ini, dapat ditelusuri kondisi air sungai dalam pengaruh hidrologi dan hidrolikanya, serta penanganan sungai lebih lanjut sesuai kebutuhan. Pada software HEC-RAS ini, dapat ditelusuri kondisi air sungai dalam pengaruh hidrologi dan hidrolikanya, serta penanganan sungai lebih lanjut sesuai kebutuhan. B. Analisa Hidrologi Melakukan analisa hidrologi untuk mendapatkan nilai debit banjir rencana yang digunakan untuk menentukan penampang yang cukup dan mampu menampung debit banjir dengan menggunakan metode HSS SCS dan Snyder. Secara umum perangkat lunak ini menyediakan fungsi-fungsi sebagai berikut: Secara umum perangkat lunak ini menyediakan fungsi-fungsi sebagai berikut: 1. Manajemen File 2. Input data dan pengeditan 3. Analisa Hidraulika 4. Keluaran (tabel, grafik dan gambar) C. Analisa Hidrolika Menghitung penampang sungai yang mampu menampung debit banjir tersebut dan juga mengetahui keperluan perbaikan sungai. Analisa banjir dan analisa penanggulangan banjir ini dibantu dengan menggunakan software HEC-RAS 4.1.0. Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa \| G. Debit Rencana = (23) = ( + ) (24) = + 2 √ + 1 (25) = (26) = / / (27) = (23) = ( + ) (24) = + 2 √ + 1 (25) = (26) = / / (27) Perhitungan debit rencana sangat diperlukan untuk memperkirakan besarnya debit hujan maksimum yang sangat mungkin pada periode tertentu. Pada penelitian ini menggunakan metode Hidrograf Satuan Sintetis Soil Conservation Servise (HSS SCS) dan Snyder. Keterangan : Snyder. 1. Metode Snyder = ( ) , (14) = Waktu dari titik berat curah = 2,75 (15) = Debit maksimum unit hidrograf (m3/s/km2) = , (16) = Lamanya curah hujan efektif Q = Debit aliran (m3/s) A = Luas penampang basah (m2) V = Kecepatan aliran (m/s) B = Lebar dasar saluran (m) h = kedalaman sungai (m) P = Keliling panampang basah (m) m = Perbandingan kemiringan R = Jari-jari hidrolis (m) I = Kemiringan dasar saluran n = Koefisien kekasaran dinding Manning y 1. Metode Snyder y 1. Metode Snyder . Metode Snyder = ( ) , (14) = Waktu dari titik berat curah = 2,75 (15) = Debit maksimum unit hidrograf (m3/s/km2) = , (16) = Lamanya curah hujan efektif = Debit maksimum unit hidrograf (m3/s/km2) 54 Jurnal Fondasi, Volume 5 No 2 I. Penanganan Sungai Konsep pengelolaan sungai ekohidraulik menekankan pentingnya mengelola aliran sungai secara terintegrasi sehingga bisa memelihara sumber daya abiotik dan biotik atau kehidupan biota air Di Indonesia lebih dikenal konsep One River, One Plan, One Integrated Management. curah hujan, dan gambar penampang melintang sungai, dsb. curah hujan, dan gambar penampang melintang sungai, dsb. 3. Data Observasi Data observasi diperoleh dari penelusuran sungai (walk trought) digunakan untuk mendapatkan gambaran yang jelas dari lokasi penelitian. Data yang peroleh berupa foto kondisi lapangan. 4. Literatur (Pustaka) Data literatur merupakan data formal yang diperoleh dari sumber informasi. Dalam penelitian ini, data literatur diperoleh dari buku naskah (teks book), Peraturan Pemerintah dan SNI, bahan ajar (kuliah) dari dosen serta literatur yang diperoleh dari sumber internet dan juga jurnal yang berkaitan. 4. Literatur (Pustaka) Data literatur merupakan data formal yang diperoleh dari sumber informasi. Dalam penelitian ini, data literatur diperoleh dari buku naskah (teks book), Peraturan Pemerintah dan SNI, bahan ajar (kuliah) dari dosen serta literatur yang diperoleh dari sumber internet dan juga jurnal yang berkaitan. J. 3. METODOLOGI PENELITIAN A. Teknik dan Pengumpulan Data 1. Data Primer Data primer adalah data yang langsung diambil dari objeknya oleh peneliti berupa tanya jawab atau wawancara dengan pihak-pihak yang bersangkutan yang sudah berpengalaman. Dalam penelitian ini wawancara dilakukan dengan pihak Balai Besar Wilayah Sungai Cidanau-Ciujung-Cidurian (BBWS C3) dan masyarakat yang tinggal dibantaran Sungai Ciberang. D. Alur Pikir Penelitian Agar Penelitian ini terstruktur maka alur pikir dalam penelitian Analisis Banjir Sungai Ciberang dengan HEC- RAS 4.1.0 seperti diperlihatkan pada gambar alir dibawah ini: D. Alur Pikir Penelitian Agar Penelitian ini terstruktur maka alur pikir dalam penelitian Analisis Banjir Sungai Ciberang dengan HEC- RAS 4.1.0 seperti diperlihatkan pada gambar alir dibawah ini: 2. Data Sekunder Data sekunder adalah data yang diambil oleh peneliti secara tidak langsung dari objeknya berupa data tertulis. Data sekunder diperoleh dari instansi terkait yaitu BBWS C3. Data sekunder yang diperlukan diantaranya peta DAS, data hidrologi Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa \| 55 Jurnal Fondasi, Volume 5 No 2 Gambar 3. Bagan Alir Penelitian Analisa Banjir Sungai Ciberang (Sumber : Hasil Analisa, 2015) C. Distribusi Frekuensi Curah Hujan Tabel 1.Persyaratan Parameter Statistik Suatu Distribusi Tabel 1.Persyaratan Parameter Statistik Suatu Distribusi (Sumber : Hasil Perhitungan 2016) (Sumber : Hasil Perhitungan 2016) Gambar 3. Bagan Alir Penelitian Analisa Banjir Sungai Ciberang (Sumber : Hasil Analisa, 2015) A. Uji Konsistensi A. Uji Konsistensi Metode yang digunakan untuk menguji konsistensi data adalah dengan cara Double Mass Curve Analysis (Analisa Kurva Massa Ganda) dengan meninjau data lima pos stasiun hujan pengamatan (Sta. Ciboleger, Sta. Ciminyak/Cilaki, Sta. Sampang Peundeuy, Sta. Banjar Irigasi, dan Sta. Pasir Ona). Berdasarkan persyaratan distribusi terpilih pada tabel 1 dapat disimpulkan hampir semua distribusi memenuhi persyaratan dan sementara dipilih distribusi Normal, Log Normal dan Log Pearson III maka untuk lebih meyakinkan metode yang digunakan dilakukan uji metode Chi-Kuadrat dan Smirnov Kolmogorov. Gambar 4.Grafik Konsistensi Data Sta. Pasir Ona (Sumber: Hasil Perhitungan, 2016) B. Hujan Kawasan (DAS) Penentuan hujan kawasan diambil dari data curah hujan selama 19 tahun (1997-2015). Mencari nilai data curah hujan rata-rata menggunakan metode Rata-rata Aljabar. Dari hasil perhitungan dapat disimpulkan bahwa nilai hujan kawasan maksimum selama 19 tahun terakhir sebesar 102.2 mm dan nilai hujan kawasan minimum 54.2 mm. D. Uji Distribusi Probabilitas 1. Metode Chi-Kuadrat ( ) Parameter (terhitung) yang digunakan harus lebih kecil dari nilai (Chi-Kuadrat Kritik) Gambar 4.Grafik Konsistensi Data Sta. Pasir Ona (Sumber: Hasil Perhitungan, 2016) Gambar 4.Grafik Konsistensi Data Sta. Pasir Ona (Sumber: Hasil Perhitungan, 2016) Gambar 4.Grafik Konsistensi Data Tabel 2. Rekapitulasi Nilai dan Sta. Pasir Ona Dari hasil perhitungan menunjukan bahwa ke lima data 5 pos stasiun hujan pengamatan (Sta. Ciboleger, Sta. Ciminyak/Cilaki, Sta. Sampang Peundeuy, Sta. Banjar Irigasi, dan Sta. Pasir Ona) memenuhi syarat kepanggahan yang ditunjukan dengan gambar grafik dari metode Double Mass \| Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa \| Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa 56 Jurnal Fondasi, Volume 5 No 2 Pada analisa hujan rencana kala ulang 50 tahun, didapat hujan rencana X50 = 105.875 mm. Dari hasil perhitungan uji Chi- Kuadrat distribusi Log Normal dan Gumbel merupakan distribusi yang paling diterima karena nilai terhitung yang paling kecil nilainya diantara distribusi yang lain. Selanjutnya akan diadakan satu pengujian lagi diantara sebaran distribusi yang lolos yaitu uji SmirnovKolmogorof. Dari hasil perhitungan uji Chi- Kuadrat distribusi Log Normal dan Gumbel merupakan distribusi yang paling diterima karena nilai terhitung yang paling kecil nilainya diantara distribusi yang lain. Selanjutnya akan diadakan satu pengujian lagi diantara sebaran distribusi yang lolos yaitu uji SmirnovKolmogorof. Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa \| F. Distribusi Hujan Jam-jaman F. Distribusi Hujan Jam-jaman Dari hasil hujan rencana tersebut akan diubah menjadi hujan rencana jam- jaman, dengan menggunakan metode ABM (Alternatif Block Methode). Dari hasil perhitungandidapatkan bahwa pada jam ketiga tinggi hujan jam-jaman mencapai puncaknya yaitu 57,713 mm atau 54,545% dari rencana hujan kala ulang 50 tahun yaitu 105,875 mm, F. Distribusi Hujan Jam-jaman Dari hasil hujan rencana tersebut akan diubah menjadi hujan rencana jam- jaman, dengan menggunakan metode ABM (Alternatif Block Methode). Dari hasil perhitungandidapatkan bahwa pada jam ketiga tinggi hujan jam-jaman mencapai puncaknya yaitu 57,713 mm atau 54,545% dari rencana hujan kala ulang 50 tahun yaitu 105,875 mm, 2. Metode Smirnov Kolmogorof Syarat untuk pengujian ini adalah∆ <∆ kritik, jika “tidak” artinya distribusi probabilitas yang dipilih tidak dapat diterima, demikian sebaliknya. 2. Metode Smirnov Kolmogorof Syarat untuk pengujian ini adalah∆ <∆ kritik, jika “tidak” artinya distribusi probabilitas yang dipilih tidak dapat diterima, demikian sebaliknya. G. Debit Rencana Debit Rencana 1. Metode Snyder Berdasarkan hasil perhitungan, debit maksimum dari metode Hydrograf Satuan Snyder adalah 1228,162 m3/detik. Gambar 5.Hidrograf banjir dengan metode Snyder (Sumber: Hasil Perhitungan, 2016) G. Debit Rencana 1. Metode Snyder G. Debit Rencana 1. Metode Snyder Berdasarkan hasil perhitungan, debit maksimum dari metode Hydrograf Satuan Snyder adalah 1228,162 m3/detik. Gambar 5.Hidrograf banjir dengan metode Snyder (Sumber: Hasil Perhitungan, 2016) 1. Metode Snyder Tabel 3. Rekapitulasi Hasil Perhitungan Uji Smirnov- Kolmogorov Tabel 3. Rekapitulasi Hasil Perhitungan Uji Smirnov- Kolmogorov Tabel 3. Rekapitulasi Hasil Perhitungan Uji Smirnov- Kolmogorov Berdasarkan hasil perhitungan, debit maksimum dari metode Hydrograf Satuan Snyder adalah 1228,162 m3/detik. Berdasarkan hasil perhitungan, debit maksimum dari metode Hydrograf Satuan Snyder adalah 1228,162 m3/detik. (Sumber : Hasil Perhitungan 2016) Dari hasil pengujian SmirnovKolmogorov dipilih distribusi Log Normal.Karena dari ketiga pengujian menunjukan distribusi Log Normal yang sering dipilih maka disimpulkan bahwa untuk wilayah Sub-DAS Ciberang untuk mencari hujan rencana distribusi yang digunakan adalah distribusi Log Normal. Gambar 5.Hidrograf banjir dengan metode Snyder (Sumber: Hasil Perhitungan, 2016) Gambar 5.Hidrograf banjir dengan metode Snyder (Sumber: Hasil Perhitungan, 2016) 2. Metode SCS (Soil Conservation Service) Berdasarkan hasil perhitungan, debit maksimum dari metode Hydrograf Satuan SCS adalah 523,174 m3/detik. Berdasarkan hasil perhitungan, debit maksimum dari metode Hydrograf Satuan SCS adalah 523,174 m3/detik. E. Analisis Hujan Rencana = + Keterangan: Nilai = 1,8803mm Nilai S = 0,07034 mm = + . = 1,8803+ (2,05 x 0,07034) = 2,0248= 10 , = 105,875 E. Analisis Hujan Rencana = + Keterangan: Nilai = 1,8803mm Nilai S = 0,07034 mm = + . = 1,8803+ (2,05 x 0,07034) = 2,0248= 10 , = 105,875 Gambar 6.Hidrograf banjir dengan metode SCS (Sumber: Hasil Perhitungan, 2016) , , Tabel 4.Perhitungan Hujan Rencana dengan Metode Distribusi Log Normal Gambar 6.Hidrograf banjir dengan metode SCS (Sumber: Hasil Perhitungan, 2016) Gambar 6.Hidrograf banjir dengan metode SCS (Sumber: Hasil Perhitungan, 2016) (Sumber : Hasil Perhitungan 2016) (Sumber : Hasil Perhitungan 2016) Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa \| 57 Jurnal Fondasi, Volume 5 No 2 Atas dasar perolehan nilai debit tersebut, maka untuk angka debit yang diinput pada software HEC-RAS 4.1.0 adalah debit terbesar hasil dari perhitungan yaituMetode Snyder sebesar1228,162 m3/detik. Atas dasar perolehan nilai debit tersebut, maka untuk angka debit yang diinput pada software HEC-RAS 4.1.0 adalah debit terbesar hasil dari perhitungan yaituMetode Snyder sebesar1228,162 m3/detik. \| Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa \| 2. Simulasi Aliran Unsteady Flow 6.54 / 0,00006 / = 1.09 m/detik Q =A x V = 815.39 x 1.09 = 889.24 m3/detik b. Bagian II Q = A x V A = [ + ( + 2 )] = [162.85 + (162.85 +2 x 1 x 3.54)] x 3.54 = 589.021 m2 P2.1= . + √ + 1 = 14 + 3.54√1 + 1= 19 m P2.2 = . + √ + 1 = 16 + 3.54√1 + 1= 21 m P= P2.1 + P2.1= 19 + 21= 40 m R = = . = 14.72 m V= = . . 14.72 / 0,00006 / = 1.88m/detik Gambar 13.Dimensi sungai rencana pada penampang melintang HM 11+50 (Sumber: Analisis Pribadi, 2016) Menghitung debit dengan rumus : a. Bagian I Q = A x V A = [ + ( + 2 )] = [103.91 + (103.91+2 x 1 x 7.33] 7.33 = 815.39 m2 P = + 2 √ + 1 = 103.91 + 2 7.33√1 + 1 =124.64 m R = = . . = 6.54m V = I = = . . = 0,00006 m V = . . 6.54 / 0,00006 / = 1.09 m/detik Q =A x V = 815.39 x 1.09 = 889.24 m3/detik b. Bagian II Q = A x V A = [ + ( + 2 )] = [162.85 + (162.85 +2 x 1 x 3.54)] x 3.54 = 589.021 m2 P2.1= . + √ + 1 = 14 + 3.54√1 + 1= 19 m P2.2 = . + √ + 1 = 16 + 3.54√1 + 1= 21 m P= P2.1 + P2.1= 19 + 21= 40 m R = = . = 14.72 m V= = . . 14.72 / 0,00006 / = 1.88m/detik Berikut beberapa tampilan hasil analisis yang didapat dari program HEC- RAS 4.1.0 : RAS 4.1.0 : Gambar 11.Tampilan Cross Section HM 0+00 (Sumber: HEC-RAS 4.1.0, 2016) Gambar 12.Tampilan memanjang Sungai Ciberang (Sumber: HEC-RAS 4.1.0, 2016) Gambar 11.Tampilan Cross Section HM 0+00 (Sumber: HEC-RAS 4.1.0, 2016) Gambar 13.Dimensi sungai rencana pada penampang melintang HM 11+50 (Sumber: Analisis Pribadi, 2016) Gambar 13.Dimensi sungai rencana pada penampang melintang HM 11+50 (Sumber: Analisis Pribadi, 2016) Menghitung debit dengan rumus : a. 2. Simulasi Aliran Unsteady Flow 2. Simulasi Aliran Unsteady Flow Pada simulasi aliran Unsteady Flow data yang dibutuhkan adalah data debit hidrograf satuan sintetis pada beberapa jam, serta data tinggi muka air dengan bantuan interpolasi pada program HEC- RAS 4.1.0. H. Analisis Penampang Sungai Menggunakan HEC-RAS 4.1.0 1. Simulasi Aliran Steady Flow a. Input nilai debit Untuk menginput nilai debit aliran steady flow, klik menubar Edit lalu klik sub menu steady flow data.menginput nilai debit Q50 yang sudah dihitung sebesar 1228,162 m3/detik. Untuk penginputan dataflow Hydrographadalah sebagai berikut. Gambar 9.Tampilan Flow Hidrograph (Sumber: HEC-RAS 4.1.0, 2016) Gambar 7.Penginputan nilai debit (Sumber: HEC-RAS 4.1.0, 2016) Gambar 9.Tampilan Flow Hidrograph (Sumber: HEC-RAS 4.1.0, 2016) Sedangkan untuk menginput stage Hydrograph adalah sebagai berikut. Gambar 7.Penginputan nilai debit (Sumber: HEC-RAS 4.1.0, 2016) Gambar 10.Tampilan Stage Hidrograph (Sumber: HEC-RAS 4.1.0, 2016) b. Input nilai kemiringan Menginput nilai kemiringan yang sudah dihitung pada kolom Downstream, yaitu 0,000107, klik ikon Reach Boundary conditions, kemudian pilih Normal Depthdan klik OK│Apply Data. Gambar 8.Penginputan nilai kemiringan (Sumber: HEC-RAS 4.1.0, 2016) c. Run AnalisysSteady flow Setelah data tersimpan selanjutnya dianalisis oleh program dengan cara klik menubar Run│SteadyFlow Analysis│Compute. Gambar 8.Penginputan nilai kemiringan (Sumber: HEC-RAS 4.1.0, 2016) Gambar 10.Tampilan Stage Hidrograph (Sumber: HEC-RAS 4.1.0, 2016) Gambar 8.Penginputan nilai kemiringan (Sumber: HEC-RAS 4.1.0, 2016) Gambar 8.Penginputan nilai kemiringan (Sumber: HEC-RAS 4.1.0, 2016) Setelah data tersimpan selanjutnya dianalisis oleh program dengan cara klik menubar Run│Steady Flow Analysis│Compute. Sebelumnya mengisi kotak Starting Date, Ending Date, Starting Time dan Ending Time diisi c. Run AnalisysSteady flow Setelah data tersimpan selanjutnya dianalisis oleh program dengan cara klik menubar Run│SteadyFlow Analysis│Compute. \| Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa \| Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa 58 Jurnal Fondasi, Volume 5 No 2 2 Gambar 13.Dimensi sungai rencana pada penampang melintang HM 11+50 (Sumber: Analisis Pribadi, 2016) sesuai waktu ketika terjadi muka air tertinggi. Gambar 13.Dimensi sungai rencana pada penampang melintang HM 11+50 (Sumber: Analisis Pribadi, 2016) Menghitung debit dengan rumus : a. Bagian I Q = A x V A = [ + ( + 2 )] = [103.91 + (103.91+2 x 1 x 7.33] 7.33 = 815.39 m2 P = + 2 √ + 1 = 103.91 + 2 7.33√1 + 1 =124.64 m R = = . . = 6.54m V = I = = . . = 0,00006 m V = . . 2. Simulasi Aliran Unsteady Flow Bagian I Q = A x V A = [ + ( + 2 )] = [103.91 + (103.91+2 x 1 x 7.33 7.33 = 815.39 m2 P = + 2 √ + 1 = 103.91 + 2 7.33√1 + 1 =124.64 m R = = . . = 6.54m V = I = = . . = 0,00006 m V = . . 6.54 / 0,00006 / = 1.09 m/detik Q =A x V = 815.39 x 1.09 = 889.24 m3/detik b. Bagian II Q = A x V A = [ + ( + 2 )] = [162.85 + (162.85 +2 x 1 x 3.54)] x 3.54 = 589.021 m2 P2.1= . + √ + 1 = 14 + 3.54√1 + 1= 19 m P2.2 = . + √ + 1 = 16 + 3.54√1 + 1= 21 m P= P2.1 + P2.1= 19 + 21= 40 m R = = . = 14.72 m V= = . . 14.72 / 0,00006 / = 1.88m/detik Menghitung debit dengan rumus : g g a. Bagian I a. Bagian I Gambar 11.Tampilan Cross Section HM 0+00 (Sumber: HEC-RAS 4.1.0, 2016) Gambar 12.Tampilan memanjang Sungai Ciberang (Sumber: HEC-RAS 4.1.0, 2016) Gambar 12.Tampilan memanjang Sungai Ciberang (Sumber: HEC-RAS 4.1.0, 2016) Gambar 12.Tampilan memanjang Sungai Ciberang (Sumber: HEC-RAS 4.1.0, 2016) Dari output program HEC-RAS 4.1.0 kondisi eksistingSungai Ciberang dengan debit kala ulang 50 tahun tidak dapat menampung debit yang ada, sehingga perlu adanya normalisasi sungai. I. Perencanaan Dimensi Sungai Perencanaan dimensi sungai dilakukan untuk mendapatkan dimensi sungai yang dapat dialiri oleh debit rencana dengan tinggi muka air yang lebih rendah dari pada dimensi eksisting sungai sebelumnya. Dari penelitian ini diambil 6 penampang melintang pada HM 01+00, HM 07+50, HM 11+50, HM 17+50, HM 26+00 dan HM 32+00. Berikut ini adalah perhitungan untuk mendapatkan dimensi sungai yang baru: Berikut ini adalah perhitungan untuk mendapatkan dimensi sungai yang baru: . 1.88m/detik 59 Jurnal Fondasi, Volume 5 No 2 \| Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa Tabel 5.Rekapitulasi data teknis penampang Tabel 5.Rekapitulasi data teknis penampang Tabel 5.Rekapitulasi data teknis penampang sungai (Sumber : Hasil Perhitungan 2016) 3. Setelah dilakukan normalisasi sungai dan peninggian tanggul bahwa Sungai Ciberang mampu menampung debit banjir kala ulang 50 tahun dengan penurunan muka air yang terjadi rata- rata sebesar 10.25 % dan kenaikan debit rata-rata sebesar 10.49%. (Sumber : Hasil Perhitungan 2016) A. Kesimpulan Dari uraian pembahasan dapat disimpulkan sebagai berikut: Dari uraian pembahasan dapat disimpulkan sebagai berikut: 1. Debit banjir Sungai Ciberang dengan kala ulang 50 tahun adalah 1228,162 m3/detik. = 1995.3 m3/detik 1995.3m3/detik >1228,162 m3/detik (AMAN)Qkapasitas > Q50 (OK) Tabel 5.Rekapitulasi data teknis penampang sungai (Sumber : Hasil Perhitungan 2016) 2. Kondisi eksisting SungaiCiberang dengan debit kala ulang 50 tahun tidak dapat menampung debit yang direncanakan. Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa \| B. Saran Beberapa alternatif atau solusi yang dapat dilakukan untuk mengatasi permasalahan banjir yang ada yaitu: Dari tabel diatas dapat disimpulkan bahwa kapasitas debit sungai > dari debit yang digunakanQkapasitas > Q50(OK) aman. 1. Tahap yang dapat segera dikerjakan karena kondisi sungai saat ini, dikhawatirkan bila tidak segera ditangani akan terjadi luapan adalah pekerjaan normalisasi sungai dan peninggian tanggul sungai. Dari Hasil software HEC-RASsetelah normalisasi semua penampang melintang sungai elevasi muka air banjir setelah normalisasi mengalami penurunan rata-rata 10.25%, sehingga dengan diperlebarnya penampang dan dibuatnya tanggul dapat mereduksi tinggi muka air banjir pada kondisi eksisting.Hal ini dapat dilihat dari elevasi maksimum yang masih lebih tinggi dari muka air banjir. Kemudian dari ke 69 penampang sungai 66 diantaranya mengalami kenaikan debit yang konstan dengan rata-rata kenaikan 10.49% dengan kata lain dari luas masing-masing penampang sungai dengan kondisi unsteady mampu menampung debit dengan kala ulang 50 tahun. Sedangkan 3 penampang sungai terakhir bagian hilir mengalami kenaikan debit yang sangat signifikan hal ini disebabkan pada HM 33+00 mengalami penyempitan sungai serta penurunan kemiringan yang tinggi sehingga permukaan air turun secara drastis juga. Debit air yang mengalir pada penampang saluran yang menyempit maka meningkatkan kecepatan aliran. p gg gg g 2. Salah satu upaya non-struktural pengendalian banjir di Sungai Ciberang yaitu dengan melakukan restorasi sungai atau mengembalikan fungsi alami sungai serta menerapkanKonsep Pembangunan Sungai Berwawasan Ekologi Hidraulik (Eko-Hidraulik). Untuk mengefektifkan upaya pengendalian banjir dengan prinsip ekohidraulik ini perlu adanya pengaturan dari sungai yaitu satu sungai, satu perencanaan dan satu menejemen terintegrasi (Pengaturan Secara Terpadu) istilah yang sering dipaparkan yaitu One River, One Plan and One Integrated Managment (ORPIM). Adapun regulasi dan himbauan kepada masyarakat dalam pengendalian banjir adalah: 1. Regulasi : Peraturan Pemerintah Daerah berupa sanksi hukum p 2. Himbauan : p 2. Himbauan : \| Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa \| Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa 60 Jurnal Fondasi, Volume 5 No 2 2016 Jurnal Fondasi, Volume 5 No 2 a. Jangan membuang sampah sembarangan a. Jangan membuang sampah sembarangan b. Menjaga kelestarian lingkungan b. Menjaga kelestarian lingkungan c. Penanaman pohon c. Penanaman pohon d. Jangan tinggalkan air mata kepada anak cucu, tapi wariskan mata air. 6. DAFTAR PUSTAKA Sosrodarsono, Suyono dan Kensaku Takeda. Hidrologi untuk Pengairan, PT. Pradnya Paramita, Jakarta, 2003. Triatmodjo, Bambang, Hidrologi Terapan, Beta Offset, Yogyakarta. 2008. Soemarto (1987), Hidrologi Teknik, Usaha Nasional, Surabaya, 2008. Maryono, Agus. Menangani Banjir, Kekeringan dan Lingkungan, Universitas Gajah Mada, Yogyakarta, 2005. Maryono, Agus. Restorasi Sungai, Universitas Gajah Mada, Yogyakarta, 2007. Peraturan Pemerintah Republik Indonesia Nomor 38 tahun 2011 TentangSungai. R SNI 03-2415-1991, Tata Cara Perhitungan Debit Banjir. Wigati, Restu.Bahan Ajar Hidrologi dan Pengembangan Sumber Daya Air, UNTIRTA, Cilegon, 2013. Cahyono, Agung Tri. Perencanaan Pengendalian Banjir Kali Kemuning, Sampang, ITS, Jawa Timur, 2010. Martin, Fransiskus Higang dan Stefanus Barlian Soeryamassoeka. Normalisasi Sungai Rantauan Sebagai Alternatif Penanggulangan Banjir Di Kecamatan Jelimpo Kabupaten Landak, UNTAN, KalimantanBarat, 2013. Noor, M. Azhari dan Budi Utomo.Studi Kapasitas Sungai Riam Kiwa Menggunakan HEC – RAS 4.1.0., UniversitasLambung Mangkurat, Kalimantan Selatan, 2013. Wahyudin. Analisis Banjir Sungai Ciliwung (Studi Kasus Ruas Sungai Lenteng Agung-Manggarai), UNTIRTA, Cilegon, 2013. Istiarto. Simulasi Aliran 1-Dimensi Dengan Bantuan Program Hidrodinamika HEC-RAS, Jurusan Teknik Sipil FT.UGM, Yogyakarta, 2012. Jurusan Teknik Sipil Universitas Sultan Ageng Tirtayasa \| 61
https://openalex.org/W2122226992	https://europepmc.org/articles/pmc3890588?pdf=render	English	null	Measures and procedures utilized to determine the added value of microprocessor-controlled prosthetic knee joints: a systematic review	BMC musculoskeletal disorders	2,013	cc-by	8,250	Abstract Background: The effectiveness of microprocessor-controlled prosthetic knee joints (MPKs) has been assessed using a variety of outcome measures in a variety of health and health-related domains. However, if the patient is to receive a prosthetic knee joint that enables him to function optimally in daily life, it is vital that the clinician has adequate information about the effects of that particular component on all aspects of persons’ functioning. Especially information concerning activities and participation is of high importance, as this component of functioning closely describes the person’s ability to function with the prosthesis in daily life. The present study aimed to review the outcome measures that have been utilized to assess the effects of microprocessor-controlled prosthetic knee joints (MPK), in comparison with mechanically controlled prosthetic knee joints, and aimed to classify these measures according to the components and categories of functioning defined by the International Classification of Functioning, Disability and Health (ICF). Subsequently, the gaps in the scientific evidence regarding the effectiveness of MPKs were determined. Methods: A systematic literature search in 6 databases (i.e. PubMed, CINAHL, Cochrane Library, Embase, Medline and PsychInfo) identified scientific studies that compared the effects of using MPKs with mechanically controlled prosthetic knee joints on persons’ functioning. The outcome measures that have been utilized in those studies were extracted and categorized according to the ICF framework. Also, a descriptive analysis regarding all studies has been performed. Results: A total of 37 studies and 72 outcome measures have been identified. The majority (67%) of the outcome measures that described the effects of using an MPK on persons’ actual performance with the prosthesis covered the ICF body functions component. Only 31% of the measures on persons’ actual performance investigated how an MPK may affect performance in daily life. Research also typically focused on young, fit and active persons. g p p p the ICF body functions component. Only 31% of the measures on persons’ actual performance investigated how an MPK may affect performance in daily life. Research also typically focused on young, fit and active persons. Conclusions: Scientifically valid evidence regarding the performance of persons with an MPK in everyday life is limited. Future research should specifically focus on activities and participation to increase the understanding of the possible functional added value of MPKs. Conclusions: Scientifically valid evidence regarding the performance of persons with an MPK in everyday life is limited. * Correspondence: p.theeven@adelante-zorggroep.nl 1Adelante, Centre of Expertise in Rehabilitation and Audiology, Hoensbroek, The Netherlands 2Department of Rehabilitation Medicine, Maastricht University, Research School CAPHRI, Maastricht, The Netherlands Full list of author information is available at the end of the article Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Open Access Measures and procedures utilized to determine the added value of microprocessor-controlled prosthetic knee joints: a systematic review trick JR Theeven1,2, Bea Hemmen1, Peter RG Brink3, Rob JEM Smeets2,1 and Henk AM Seelen1,2 Correspondence: p.theeven@adelante-zorggroep.nl 1Adelante, Centre of Expertise in Rehabilitation and Audiology, Hoensbroek, The Netherlands 2Department of Rehabilitation Medicine, Maastricht University, Research School CAPHRI, Maastricht, The Netherlands Full list of author information is available at the end of the article © 2013 Theeven 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. Background Prosthetic componentry has evolved considerably over the years, incorporating advanced materials and techniques into the modern-day prostheses. Presently, a multitude of prosthetic knee joints exists that, based on how these joints are controlled, can generally be divided into two groups, i.e. prosthetic knee joints with exclusively mechan- ical control properties, and microprocessor-controlled prosthetic knee joints (MPKs) [1]. Mechanical control of the knee joint during the swing phase of gait is achieved either by constant friction (spring loaded or elastic exten- sion), hydraulic dampening, or pneumatic dampening. Mechanical control during stance phase is obtained by manual locking, a weight-activated friction brake, a poly- centric axis, or by hydraulic dampening [1]. MPKs on the other hand, have a built-in microcomputer that continu- ously controls the flexion and extension resistance of the prosthetic knee during the stance phase and/or swing phase of each gait cycle. According to its manufacturers, the benefits of using an MPK, in comparison with a mech- anically controlled prosthetic knee, are especially associ- ated with the possibility to walk with varying walking speeds due to the adaptive swing phase control, and to walk with a higher level of safety due to the MPKs’ adap- tive stance phase control. These features may allow per- sons with an amputation to ambulate with a walking pattern that closely resembles natural gait, which in turn may lead to, for instance, a reduction in metabolic cost and cognitive demand. Ever since the MPKs became available, a debate is on- going as to what characteristics an amputee should have in order for an MPK to be a more suitable prosthetic so- lution than a mechanically controlled prosthetic knee joint to function optimally in daily life. Finding an an- swer to this issue is an important step in streamlining the current prescription guidelines and reimbursement policies regarding the MPKs. Various studies with di- verse degrees of quality have been performed that evalu- ated the possible effects on persons’ level of functioning of using the more advanced MPKs in comparison with the mechanically controlled prosthetic knee joints. The effects of using an MPK have been assessed using a var- iety of outcome measures in a variety of health and health-related domains. Abstract Future research should specifically focus on activities and participation to increase the understanding of the possible functional added value of MPKs. Keywords: Rehabilitation, Functioning, Review, Classification, Amputation, Lower extremities, Microprocessor-controlled knee joint © 2013 Theeven 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. Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Page 2 of 12 level of functioning in daily life. Patients’ level of function- ing encompasses multiple health and health-related do- mains that are described in the framework provided by the International Classification of Functioning, Disability and Health (ICF) [2]. The ICF describes health and the health-related domains in two parts, i.e. “functioning and disability” and “contextual factors”. The part that covers functioning and disability is further categorised into the components “body functions”, “body structures” and “ac- tivities & participation”. The part describing contextual factors is further divided into “environmental factors” and “personal factors”. The ICF uses the term ‘functioning’ to encompass all body functions, body structures, activities and participation [2]. Additionally, amputees can be classi- fied into subgroups based on their level of functioning. The Medicare Functional Classification Level system [3] (MFCL-0 through MFCL-4; or K0 through K4) is an ex- ample of such a classification. It classifies individuals based on their ability or potential ability to function with their prosthesis, as is indicated in Table 1. Such classification may aid clinicians in the selection of prosthetic knee joints, as the MFCL level of a patient is often associated with specific categories of prosthetic knee joints that are considered suitable for those patients [4]. HCFA = Health Care Financing Administration Search strategy The databases PubMed, CINAHL, Cochrane Library, Embase, Medline and PsychInfo were searched on May 1, 2013. The search focused on studies that compared the use of any type of MPK to the use of any type of mechan- ically controlled prosthetic knee joint. The following de- fault Boolean search strategy was used: “prosthe* AND knee AND (amput* OR disarticulation) AND (microproces- sor OR active OR electronic* OR magnetorheologic* OR in- telligent OR variable-damping OR computerized)”. The search was limited to articles written in English, Dutch, French, or German between January 1990 and April 2013. The first MPK became commercially available in 1993. In addition to the database search, the lists of references of all papers included were checked for eligible articles. Furthermore, it was specified for what particular pur- pose each body function, body structure, activity and par- ticipation, environmental factor, or personal factor was measured. The main topics of the papers included in the present review are described by the authors as: ‘quality of walking’, ‘energy cost’, ‘cognitive effort’, ‘safety’, ‘activity level’, ‘activities other than walking’, ‘prosthetic comfort’ and ‘other (e.g. health)’. In order to improve the clinical relevance of the present review these domains were used to classify the outcome of the literature search. Classification of outcome measures identified 1) to identify the outcome measures that have been uti- lized to assess the effects of using an MPK in comparison with a mechanically controlled prosthesis in persons with a knee disarticulation or transfemoral amputation, 2) to classify and structure the outcome measures identified ac- cording to the ICF framework, 3) to evaluate the charac- teristics and quality of the studies that have been performed to assess the differences between MPKs and mechanically controlled prosthetic knee joints, 4) to deter- mine possible gaps in the information that is available concerning the three ICF components ‘body functions’, ‘body structures, and ‘activities and participation’. Classification of outcome measures identified Main outcome parameters were identified in the selected studies. Each parameter was then categorised using the decision tree shown in Figure 1. This decision tree repre- sents the main structure of the ICF classification [2]. It was used as a tool to identify the particular aspect of func- tioning associated with each outcome parameter. Subse- quently, an ICF code was assigned following the general coding guidelines provided by the ICF (ICF annex 2 [2]) to further indicate the specific body structure, body function, activity, participation, personal factor or environmental factor the outcome parameter focused on. Also, a distinction was made between parameters that describe persons’ actual performance and persons’ self- perceived performance. Actual performance was defined as the objectively detectable level of functioning of the amputee in a given domain at a given moment. Per- ceived performance was defined as the level of function- ing subjectively experienced by the amputee in a given domain at a given moment (self-report). Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Page 3 of 12 optimally in daily life, it is vital that the clinician has ad- equate information about the effects of that particular prosthetic device on all ICF components that comprise persons’ level of functioning. Especially information con- cerning the ICF component “activities and participation” is of high importance, as that component closely de- scribes the person’s ability to function with the pros- thesis in daily life. Moreover, information is necessary that describes to what amputee subgroups (e.g. MFCL classes) those effects are applicable. A comprehensive overview of the scientific evidence concerning the differ- ences in the effects of MPKs and mechanically con- trolled prosthetic knee joints on the functioning of amputees is therefore warranted. A first step in creating such an overview is to investigate how and for which components of amputees’ functioning those effects have been determined thus far. assessing the full-text article and discussing on ultimate inclusion or exclusion. Subsequently, the full text of all pa- pers, found to be eligible, was reviewed. Papers were in- cluded when they met the following inclusion criteria: (1)Participants are persons with a transfemoral amputation or knee disarticulation; (1)Participants are persons with a transfemoral amputation or knee disarticulation; (2)Persons’ performance using a mechanically controlled knee joint is contrasted to persons’ performance using an MPK; Papers were excluded when one or both of the inclu- sion criteria were not met, when they focused on endo- prosthetic knee joints, or when the primary outcome parameters focused on performance of the prosthetic knee joint and not on the performance of the person using the prosthesis. The present review aims: Background However, if the patient is to re- ceive a prosthetic knee joint that enables him to function From the broad range of available components, rehabili- tation professionals have to choose a specific prosthetic knee joint that enables the patient to function optimally with the prosthesis in daily life. This choice is a compli- cated and challenging task, because every patient is unique in terms of age, gender, body weight, type of amputation, physical condition, cognition, ambulation-related goals regarding activities of daily living, and societal roles (s)he wishes to engage in. It is crucial that a prosthetic knee joint is selected that best meets the patient’s characteristics and needs, in order for the patient to reach an optimal Table 1 Medicare functional classification level (MFCL) [3] descriptions HCFA Modifier MFCL description K0 MFCL-0 Does not have the ability or potential to ambulate or transfer safely with or without assistance and a prosthesis does not enhance quality of life or mobility. K1 MFCL-1 Has the ability or potential to use a prosthesis for transfers or ambulation on level surfaces at fixed cadence. Typical of the limited and unlimited household ambulator. K2 MFCL-2 Has the ability or potential for ambulation with the ability to traverse low-level environmental barriers such as curbs, stairs, or uneven surfaces. Typical of the limited community ambulator. K3 MFCL-3 Has the ability or potential for ambulation with variable cadence. Typical of the community ambulator who has the ability to traverse most environmental barriers and may have vocational, therapeutic, or exercise activity that demands prosthetic utilization beyond simple locomotion. K4 MFCL-4 Has the ability or potential for prosthetic ambulation that exceeds the basic ambulation skills, exhibiting high impact, stress, or energy levels, typical of the prosthetic demands of the child, active adult, or athlete. HCFA = Health Care Financing Administration Table 1 Medicare functional classification level (MFCL) [3] descriptions MFCL-4 Has the ability or potential for prosthetic ambulation that exceeds the basic ambulation skills, exhibiting high im stress, or energy levels, typical of the prosthetic demands of the child, active adult, or athlete. Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Screening h Descriptive analysis of studies included A descriptive analysis of the studies included in the present review was performed. Information is provided about the characteristics of the participants included in the studies (i.e. age, aetiology of amputation, reported The papers found in the literature search were independ- ently screened by two researchers (PT and HS), based on title and abstract, and classified as ‘relevant’, ‘not relevant’, or ‘possibly relevant’. The two researchers resolved any disagreement regarding the classification of papers by Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Page 4 of 12 Figure 1 Decision tree utilized to assign an ICF code to the outcome parameters identified. Figure 1 Decision tree utilized to assign an ICF code to the outcome parameters identified. Classification of outcome measures identified A detailed description of the main outcome parameters extracted from the articles is shown in Table 2. A total of 72 different outcome parameters were identified. Thirty-eight parameters described participants’ actual performance, 30 parameters described participants’ self- perceived performance, and 4 parameters assessed both actual performance and perceived performance (indi- cated in Table 2 with “a-p”). functional level), and the characteristics of the studies included (sample size, study design, methodological quality). The methodological quality was assessed using the van Tulder’s quality assessment system. This scale scores the internal validity (maximum 11 points), the de- scriptive criteria (maximum 6 points) and the statistical criteria (maximum 2 points) [5,6]. Results F Classification according to the ICF framework demon- strated that 54% of all outcome parameters assessed the effects of using an MPK at the ICF component ‘body functions’, none of the outcome parameters focused on the ICF component ‘body structures’, and 35% at ICF component ‘activities and participation’. An additional 11% of the outcome parameters were classified as envir- onmental factors. In 7 cases, no specific ICF code could be ascribed to the parameter, because of the wide inter- pretability of those parameters. Figure 2 represents a schematic overview of the selection process of the studies. A total of 37 articles, in which the use of mechanically controlled prosthetic knee joints was contrasted to the use of MPKs, were included in the final analysis. A comprehensive overview of the characteristics of the papers included is presented in Additional file 1: Table S1. Papers are sorted in ascending order of publi- cation year. 7 additional papers identified 14 papers excluded: 2 reviews 5 focus not on patient performance 2 conference proceedings 5 unable to retrieve full text 37 papers selected and analysed 174 papers excluded by screening title and abstract for inclusion criteria 440 papers identified (Pubmed (125), Cinahl (71), Cochrane (15), Embase (132), Medline (91), PsycInfo (6)) 44 papers extracted for more detailed application of inclusion criteria 222 duplicate papers Figure 2 Schematic representation of article selection. Figure 2 Schematic representation of article selection. Figure 2 Schematic representation of article selection. ntified in studies included in review er ICF code [2] Actual or perceived performance Quality of walking Energy cost Cognitive effort Safety Activity level Activities other than walking Prosthetic comfort Other (e.g. Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 multiple papers. The mean age of participants was 44.9 years (sd 9.7 years; range = 15-85 years). The most common aetiology of amputation for the participants in the studies were trauma, congenital causes and malignancy (n = 28 studies) [7-13,15-19,23,25,26,28,30-38,40-42]. Participants with an amputation due to peripheral vascular diseases were included in 12 studies [18,25,26,30-32,34,37,38, 40,42,43]. In 26 [7-13,16-19,21,22,25-28,30-34,36,37,40,43] of the 37 studies the participants were described as active and fit individuals. One hundred and two participants (i.e. 12.6% of total number of subjects) with a reported lower functional level (≤MFCL-2) were included, divided over eight studies [18,25,30,34,37-39,42]. Five of those studies [18,34,38,39,42] specifically investigated the effects of MPKs in amputees classified as MFCL-2. In eight stud- ies [14,15,20,23,24,29,35,41] participants’ functional level was not clearly specified. Furthermore, 67% of all parameters that described per- sons’ actual performance targeted the ICF body functions component and 31% the ICF activities and participation component. For parameters that describe persons’ per- ceived performance, 32% measured the ICF component body functions and 44% the ICF component activities and participation. In thirty-two studies [7-11,13-18,20-36,39-41,43] (i.e. 87% of all studies), one or more parameters were utilized that focussed on ICF body functions to assess the differences in performance between an MPK and a mechanically con- trolled prosthesis. Fifty-one percent of all parameters assessing the ICF body functions component, divided over 18 studies [7,14-18,20,21,24-26,29,30,35,36,39-41] (i.e. 49% of all studies), targeted persons’ quality of walking with the prosthesis. These parameters typically encompass spatiotemporal parameters, kinematic, and kinetic param- eters, measured with standardised gait analysis. An add- itional 13% of all parameters assessing the ICF body functions component, divided over 14 studies [7-10,13, 14,16-18,20,24,27,29,31] (38% of all studies), focused on the energy cost of walking with the prosthesis. Further- more, 8% of the parameters assessing ICF body functions focused on cognitive effort (5 studies [11,18,22,25,34]), 15% on safety-related parameters (9 studies [7,18,25, 28,32-34,39,43]), 23% on activities other than walking (i.e. standing and ramp descent) (2 studies [26,39]), and 8% on other parameters (2 studies [18,23]). The mean sample size of participants for all studies in- cluded was 21.9 (sd 59.3; range 1–368). The sample size of the study of Berry et al [32] (n = 368) may be considered a far outlier. The mean sample size without this study was 12.3 (sd 9.9; range 1–42). Descriptive analysis of studies included Descriptive analysis of studies included The MPKs investigated in the studies were the C-leg (Otto Bock HealthCare, Duderstadt, Germany)(n = 27), the C-leg Compact (Otto Bock HealthCare, Duderstadt, Germany)(n = 3), Intelligent Prosthesis (Chas A. Blatch- ford & Sons Ltd, Basingstoke, UK)(n = 7), Rheo knee (Össur, Reykjavik, Iceland)(n = 1), Adaptive knee (Chas. A. Blatchford & Sons Ltd, Basingstoke, UK)(n = 1), Power knee (Össur, Reykjavik, Iceland)(n = 1), and a newly developed polycentric MPK prototype (n = 1). Ac- commodation time for the MPK ranged between 30 mi- nutes to 44 months. Results F health) 30,35,39] b770 a ● ● 29,36,40,41] b770 a ● n [15,35,39,41] b770 a ● ● 8,21,39] b770 a ● ● 7,18,21,26] b770 a ● b770 a ● b770 a ● b770 a ● [17,39,41] b770 a ● ● e [17,41] b770 a ● b770 a ● 5,39] b770 a ● ● ce [21,41] b770 a ● b770 a ● 35,39] b770 a ● ● ant [35] b770 a ● b770 a ● ttern [17,39] b760 a ● ● 4,25] b455 a ● 9] b755/b235 p ● te 7,31] b455 a ● 27] b455 a ● ndex [24,29] b455 a ● ated [31] b455 a ● 18,31] b469 p ● ention [18,22] b140 p ● 4] b140 p ● 11] b755 a ● nts 3] b770 a ● http://www.biomedcentral.com/1471 2474/14/333 p ergy st C ef p p dentified in studies included in review (Continued) ariability) [18] b770 a ● 32,34]/standing ,39,43]) b126 p/p/p ● y [18,32] b126 p ● el off [39] b770 a ● y [26] b755/ b235 a ● fected leg [18] b780 p ● body scheme [18] b180 p ● 3] b180 p ● 8]/ ability of walking d450 a-p/p ● k [10] d155 p ● ncy [25,29,30,34] p ● 25,29,30,34,43] p ● n [19,42] a ● 2] a ● y bouts of activity [42] a ● uency [19,25] d450 a ● ability [10,18,29,30]/ rs negotiation d455 a/p/p ● ability [10,18,29]/ negotiation d450 a/p/p ● 0,34]/effort [7] to ven terrain d450 a-p/p ● acle negotiation [27] d450 a ● ymmetry [37] of anding up d410 a/a ● l mobility d4* a ● rm daily life d4*/d649 a-p ● formance 42] p ● /QoL [25,27,34] p ● work/leisure d850/ d920 p ● ● ● ● ● ● ● ● alth. http://www.biomedcentral.com/1471-2474/14/333 p Page 8 of 12 Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 In 35 [7-34,36,38-43] of the 37 studies, a within-subject design was used to test the differ- ences between mechanically controlled prosthetic knees and MPKs. Two studies [35,37] compared the perform- ance of a cohort of amputees using mechanically con- trolled prostheses with one or two cohorts of amputees using a particular type of MPK. The methodological quality of all studies included, in- dicated by the mean total van Tulder score, was 9.2 (sd 1.7; range 6–12) out of a maximum score of 19. Mean score on the internal validity of the publications was 4.5 (sd 0.8) out of 11. The mean score on descriptive criteria was 3.2 (sd 0.9) out of 6, and the mean score for the statistical criteria was 1.5 (sd 0.5) out of 2. When using the cut-off value suggested by van Tulder (50% of the maximum score), the methodological quality was accept- able in 22 [13,15,17,19-23,25-27,30-34,36-40,42] of the 37 studies. Seventeen studies [7,10,12,16,18,19,25,27,29,30,32,34, 37-39,42,43] measured one or more parameters assessing the ICF component activities and participation to deter- mine the level of performance with the prosthesis. The majority (57%) of all parameters assessing ICF activities and participation described activities other than walking on level ground. These activities included a variety of daily life activities, including negotiation of stairs, hills, uneven terrain, and obstacles. Also, sitting down and standing up was assessed, as well as a variety of basic mobility tasks. Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Also, Seymour et al [27] developed a standardised walking obstacle course to determine persons’ ability to negotiate obstacles. Although little information is available about the psychometric properties of the aforementioned mea- sures, they seem ecologically valid. Tools that aim to measure participation are typically self-report measures. This is inherent to the definition of participation, as it in- volves social interaction, which is impossible to measure under standardised conditions. y The majority of the evidence that is available regarding the effectiveness of using an MPK is primarily based on measures covering the ICF body functions component. Such information is valuable to understand the princi- ples behind how an MPK may contribute to optimizing persons’ performance with a prosthesis. However, one of the main reasons to provide an above knee amputee with a prosthesis featuring a specific prosthetic knee joint is to enable that person to reach an optimal level of functioning in daily life. Yet, only few studies have inves- tigated the effects of an MPK on activities and participa- tion component of the ICF. Information about this ICF component is more closely related to the problems expe- rienced by amputees in daily life in comparison with measures concerning body functions. For instance, per- sons with an above-knee amputation are not necessarily interested in whether they consume less millilitres of oxygen per kilogram per meter walked on a treadmill at a constant speed. Instead, it is more important for them to know whether they are (more) able to perform their activities of everyday life, or be involved in life situations when using their prosthesis. The outcome measures utilized to measure functioning in the activities and participation domain were predomin- antly based on parameters on perceived performance. As this entails subjective information about persons’ perform- ance, it is more sensitive to bias (e.g. response or recall bias) compared with objective measurements. Persons may give answers that are influenced by their own expec- tations, or because they want to please the researcher. Also, subjective measures are more sensitive to changes in the participants’ frame of reference, i.e. their perception may change over time, for instance, because they have put a certain condition or state into perspective. Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Page 9 of 12 only 8 of the 31 studies, identified in the current review, parameters were assessed by measuring a combination of both actual and self-perceived performance. The assess- ment of a combination of both actual and perceived per- formance measures is strongly recommended for future prosthetic research. a majority (67 %) of all outcome measures, utilized to assess persons’ actual performance, targeted the ICF com- ponent ‘body functions’, whereas only few measures assessed the effects of using an MPK at activity and par- ticipation (31%). The outcome measures utilized to evalu- ate persons’ perceived performance with an MPK also typically described the effects on body functions and activ- ities and participation. Thus, the scientific knowledge that is currently available regarding the effects of using an MPK on persons’ functional abilities is limited. Additional information is necessary about how the use of an MPK may affect persons’ actual ability to perform activities in everyday life and how using an MPK may influence peo- ple’s participation in society. To objectively measure a person’s actual performance regarding ICF activity and participation is difficult, due to the fact that very few (validated) measuring tools are avail- able, that have been designed specifically to be used in persons with an amputation of a lower extremity [46]. Nevertheless, several original tools that objectively meas- ure activities were found in the present review. Theeven et al [38] have developed and used a test, the Assessment of Daily Activities Performance in Transfemoral amputees test (ADAPT), that includes 17 common daily activities that have to be performed by the person being measured. With ADAPT it is possible to reliably and objectively measure amputees’ functional abilities to perform daily ac- tivities [47]. Hafner et al [25,34] used three measures that aim to assess actual performance at ICF activities and par- ticipation. The Stair Assessment Index (SAI) [48] and Hill Assessment Index (HAI) [49], that measure persons’ over- all ability and quality of performance during stairs and hill negotiation, and a distracted walking test, that assesses the level of cognitive load during walking. During the latter test, persons walked outdoor around a busy city block while talking to a researcher on a cell phone. Participants had to repeat series of numbers back to the researcher in reversed order. Datta et al [16] used an original video ob- servation scale to evaluate persons’ overall gait quality. Discussion h The main aim of the present review was to provide insight in how the effects of using an MPK, in comparison with a mechanically controlled knee joint, have been determined thus far. To this end, an overview of the outcome mea- sures that have been utilized in comparative studies to de- scribe the effects of an MPK on patients’ were identified from the literature and subsequently structured according to the ICF framework to provide a comprehensive over- view of the components within the health and health- related domains that have been assessed. In addition, a descriptive evaluation of the study characteristics, the par- ticipant characteristics, and the overall quality of the stud- ies performed, was conducted. The findings indicated that In all studies a cumulative total of 810 participants was investigated, not taking into account the possibility that a same subset of participants may have been described in Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Page 9 of 12 Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Page 10 of 12 respectively, of the lower limb amputees are over 65 years old [50], and around 79% and 94% respectively, of the amputations are due to peripheral vascular disease [50,51]. The research attention concerning the evalu- ation of MPKs seems to be more focused on the high profile, well-performing subpopulation. However, it is important to recognise that the ageing of the general population and the increasing prevalence of obesity and cardiovascular disease, are likely to cause the relative number of older persons with an amputation for vascu- lar problems to grow. Persons with a lower functional level might also benefit from using a prosthesis featur- ing an MPK [18,24,34,38,39,42]. However, additional re- search is necessary to further increase our knowledge available about the effects of MPKs regarding this sub- population of prosthesis users. the investigations on negotiating stairs, slopes, and ob- stacles, only one study [38] assessed persons’ actual abil- ity to perform activities of daily living (ADL). Research should focus more on whether the effects measured in a human movement lab also apply to situations in persons’ everyday life, i.e. investigate the functional added value of using a prosthetic knee joint. Future research should focus on the development of tools that are able to objectively measure actual per- formance in persons with an above-knee amputation. More specifically, tools that measure activity and partici- pation are necessary to further increase our understand- ing about the possible effects of using an MPK on persons’ functioning. Moreover, a combination with pa- rameters on self-perceived performance is warranted. Prosthesis research should focus more on the effects of using an MPK in persons with a lower functional level (e.g. amputees classified as MFCL-2). Given their limited physical capacity, these persons might not benefit from using the adaptive swing phase control that should enable them to walk at varying speeds. However, they may pos- sibly benefit from the higher levels of stability of the knee due to the continuous adaptive stance phase control. The average quality of the studies identified in the re- view was moderate to low. In general, the effects of the MPKs were investigated in a small research population, leading to statistical power problems and affecting the generalizability of the results reported. Also, large vari- ation exists in the length of the accommodation time for the MPK (range 30 minutes to 44 months). Additional file Additional file 1: Table S1. Overview of study characteristics, outcome parameters identified, and results on main outcome parameters. Conclusions Much research has been done on the effects of MPKs. However, the information available provides insight into the effects of using an MPK on only a limited number of health and health-related domains. The effects have been predominantly investigated from the perspective of the body (ICF body functions), and so, objective information and scientifically valid evidence regarding the perform- ance of persons with an MPK in everyday life is still fairly limited. Research should therefore specifically focus on activity and participation, rather than body functions and body structures. Also, the information about the effects of MPKs on persons’ functioning cannot be generalised for the entire population of persons with an above-knee amputation, as testing of MPKs is almost only performed in healthy, fit, and active persons, who are relatively young. Future research should therefore also aim at the older, less ac- tive persons with an amputation to complement the in- formation available so far. Recommendations for future research The majority of the comparative studies have objectively investigated the effects of an MPK versus a mechanically controlled prosthesis on walking (quality, physical effort, and cognitive effort). Little information is available about other aspects of functioning with a prosthesis. Next to Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Intuitively, a longer accommodation time would result in a better per- formance on the tests. English et al [52] reported that at least one week of functional walking with the prosthesis is necessary for clinical decision making on the suitability of a knee joint, but that three weeks is recommended for re- search purposes. This recommendation, however, is based on the findings in one subject. To date, no consensus ex- ists on a suitable accommodation period. Additionally, the mean Van Tulder score for all studies incorporated in the review (9.1 out of 19), further underlines the moderate quality. According to Van Tulder [5,6] the methodological quality of a study is considered adequate with a score of at least 9.5. Especially, the internal validity is scored very low (4.4 out of 11), which may be associated with the lack of blinding in all studies. Blinding of the participants is prac- tically impossible to accomplish, as the different features of the MPKs have to be explained to enable subjects to use the MPK to its full potential. It should also be noted that the Van Tulder’s quality assessment system is typically designed to rate randomised controlled trials (RCT) or controlled clinical trials (CCT). The majority of the studies included in the present review were neither. This may have led to an underestimation of the quality of the papers in the review. Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Nevertheless, it is important that, in addition to information concerning persons’ actual performance level, the level of participants’ perceived performance is also included in the process of decision making for a prosthetic knee, as persons’ level of (dis)satisfaction with the prosthesis is strongly related to the level of use of the prosthesis [44,45]. Unfortunately, in Although this review focused on how the effects of an MPK were assessed, it should also be noted that the ma- jority (≈70%) of all studies that investigated the effective- ness of using an MPK included relatively young, fit, and active persons, with an amputation due to trauma (i.e. a classification of at least MFCL-3). In contrast, only twelve studies [18,25,26,30-32,34,37,38,40,42,43] included persons with an amputation due to peripheral vascular disease and in only five studies [18,34,38,39,42] the ef- fects of an MPK were specifically evaluated in amputees classified as MFCL-2. This does not seem to be repre- sentative of the total amputee population. The preva- lence numbers in, for instance, the United States and the Netherlands, indicate that an estimated 42% and 90% Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Page 10 of 12 Abbreviations ADAPT: Assessment of daily activities performance in transfemoral amputees; ADL: Activities of daily living; CCT: Controlled clinical trial; ESK: Endolite stabilised knee; HAI: Hill assessment index; HCFA: Health care financing administration; ICF: International classification of functioning, disability and Page 11 of 12 Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Received: 6 June 2012 Accepted: 5 November 2013 Published: 27 November 2013 Received: 6 June 2012 Accepted: 5 November 2013 Published: 27 November 2013 23. Bunce DJ, Breakey JW: The impact of C-Leg on the physical and psycho- logical adjustment to transfemoral amputation. J Prosthet Orthot 2007, 19 (1):7–14. Competing interest The authors declare that they have no competing interests. 17. Johansson JL, Sherrill DM, Riley PO, Bonato P, Herr H: A clinical comparison of variable-damping and mechanically passive prosthetic knee devices. Am J Phys Med Rehabil 2005, 84(8):563–575. Declaration of interest statement This study was made possible by a grant provided by Otto Bock HealthCare GmbH, Vienna, Austria. 20. Orendurff MS, Segal AD, Klute GK, McDowell ML, Pecoraro JA, Czerniecki JM: Gait efficiency using the C-Leg. J Rehabil Res Dev 2006, 43(2):239–246. Authors’ contributions f PT and HS performed the data acquisition. PT, BH, HS, and RS participated in the study design and data analysis. PT, BH, and HS have been involved in the initial drafting of the manuscript. All authors, i.e. PT, BH, HS, PB, and RS, participated in critically revising the manuscript and in the preparation of the manuscript. All authors have read and approved the final manuscript. 18. Wetz HH, Hafkemeyer U, Drerup B: [The influence of the C-leg knee-shin system from the Otto Bock Company in the care of above-knee amputees. A clinical-biomechanical study to define indications]. Orthopade 2005, 34(4):298, 300-314, 316-299. 19. Klute GK, Berge JS, Orendurff MS, Williams RM, Czerniecki JM: Prosthetic intervention effects on activity of lower-extremity amputees. Arch Phys Med Rehabil 2006, 87(5):717–722. Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 14. Perry J, Burnfield JM, Newsam CJ, Conley P: Energy expenditure and gait characteristics of a bilateral amputee walking with C-leg prostheses compared with stubby and conventional articulating prostheses. Arch Phys Med Rehabil 2004, 85(10):1711–1717. health; IP: Intelligent prosthesis; l/r: Left/right; MFCL: Medicare functional classification level; MPK: Microprocessor-controlled prosthetic knee joint; PEQ: Prosthesis evaluation questionnaire; PSPC: Pneumatic swing phase health; IP: Intelligent prosthesis; l/r: Left/right; MFCL: Medicare functional classification level; MPK: Microprocessor-controlled prosthetic knee joint; PEQ: Prosthesis evaluation questionnaire; PSPC: Pneumatic swing phase control; PVD: Peripheral vascular disease; QoL: Quality of life; RCT: Randomised controlled trial; SAI: Stair assessment index; sd: Standard deviation; SF-36: 36-item short-form health survey; VCO2: Rate of carbon dioxide production; VO2: Rate of oxygen consumption; VT: Van Tulder score. 15. 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Condie E, Scott H, Treweek S: Lower limb prosthetic outcome measures: a review of the literature 1995 to 2005. JPO 2006, 18(1S):13–45. 47. References Taylor MB, Clark E, Offord EA, Baxter C: A comparison of energy expenditure by a high level trans-femoral amputee using the Intelligent Prosthesis and conventionally damped prosthetic limbs. Prosthet Orthot Int 1996, 20(2):116–121. 8. Taylor MB, Clark E, Offord EA, Baxter C: A comparison of energy expenditure by a high level trans-femoral amputee using the Intelligent Prosthesis and conventionally damped prosthetic limbs. Prosthet Orthot Int 1996, 20(2):116–121. 29. Jepson F, Datta D, Harris I, Heller B, Howitt J, McLean J: A comparative evaluation of the Adaptive knee and Catech knee joints: a preliminary study. Prosthet Orthot Int 2008, 32(1):84–92. 9. Buckley JG, Spence WD, Solomonidis SE: Energy cost of walking: comparison of “intelligent prosthesis” with conventional mechanism. Arch Phys Med Rehabil 1997, 78(3):330–333. 30. Kahle JT, Highsmith MJ, Hubbard SL: Comparison of nonmicroprocessor knee mechanism versus C-Leg on Prosthesis Evaluation Questionnaire, stumbles, falls, walking tests, stair descent, and knee preference. J Rehabil Res Dev 2008, 45(1):1–14. 10. Datta D, Howitt J: Conventional versus microchip controlled pneumatic swing phase control for trans-femoral amputees: user’s verdict. Prosthet Orthot Int 1998, 22(2):129–135. 31. Kaufman KR, Levine JA, Brey RH, McCrady SK, Padgett DJ, Joyner MJ: Energy expenditure and activity of transfemoral amputees using mechanical and microprocessor-controlled prosthetic knees. Arch Phys Med Rehabil 2008, 89(7):1380–1385. 11. Heller BW, Datta D, Howitt J: A pilot study comparing the cognitive demand of walking for transfemoral amputees using the Intelligent Prosthesis with that using conventionally damped knees. Clin Rehabil 2000, 14(5):518–522. 12. Stinus H: Biomechanics and evaluation of the microprocessor-controlled C-leg. [German] Biomechanik und beurteilung des exoprothesenkniege- lenkes C-leg. Z Orthop Ihre Grenzgeb 2000, 138(3):278–282. 32. Berry D, Olson MD, Larntz K: Perceived stability, function, and satisfaction among transfemoral amputees using microprocessor and nonmicroprocessor controlled prosthetic knees: a multicentre survey. J Prosthet Orthot 2009, 21:32–42. 13. Schmalz T, Blumentritt S, Jarasch R: Energy expenditure and biomechanical characteristics of lower limb amputee gait: the influence of prosthetic alignment and different prosthetic components. Gait Posture 2002, 16(3):255–263. 33. Blumentritt S, Schmalz T, Jarasch R: The safety of C-leg: biomechanical tests. J Prosthet Orthot 2009, 21:2–15. Page 12 of 12 Page 12 of 12 Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Theeven PJR, Hemmen B, Stevens CHC, Ilmer EC, Brink PRG, Seelen HAM: Feasibility of a new concept for measuring actual functional performance in daily life of transfemoral amputees. J Rehabil Med 2010, 42(8):744–751. 48. Buell NC, Willingham LL, Allyn KJ, Hafner BJ, Smith DG: Evaluation of gait style to ascend and descend stairs for lower limb amputees. In 11th World Congress of the International Society of Prosthetics and Orthotics. Hong Kong; 2004:367. 49. Buell NC, Willingham LL, Allyn KJ, Hafner BJ, Smith DG: Evaluation of gait style for hill descent for lower limb amputees. In 11th World Congress of the International Society of Prosthetics and Orthotics. Hong Kong; 2004:53. 49. Buell NC, Willingham LL, Allyn KJ, Hafner BJ, Smith DG: Evaluation of gait style for hill descent for lower limb amputees. In 11th World Congress of the International Society of Prosthetics and Orthotics. Hong Kong; 2004:53. 50. Ziegler-Graham K, MacKenzie EJ, Ephraim PL, Travison TG, Brookmeyer R: Estimating the prevalence of limb loss in the United States: 2005 to 2050. Arch Phys Med Rehabil 2008, 89(3):422–429. 50. Ziegler-Graham K, MacKenzie EJ, Ephraim PL, Travison TG, Brookmeyer R: Estimating the prevalence of limb loss in the United States: 2005 to 2050. Arch Phys Med Rehabil 2008, 89(3):422–429. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit 51. Rommers GM, Vos LD, Groothoff JW, Schuiling CH, Eisma WH: Epidemiology of lower limb amputees in the north of The Netherlands: aetiology, discharge destination and prosthetic use. Theeven et al. BMC Musculoskeletal Disorders 2013, 14:333 http://www.biomedcentral.com/1471-2474/14/333 Prosthet Orthot Int 1997, 21(2):92–99. Submit your next manuscript to BioMed Central and take full advantage of: 51. Rommers GM, Vos LD, Groothoff JW, Schuiling CH, Eisma WH: Epidemiology of lower limb amputees in the north of The Netherlands: aetiology, discharge destination and prosthetic use. Prosthet Orthot Int 1997, 21(2):92–99. Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 52. English RD, Hubbard WA, McElroy GK: Establishment of consistent gait after fitting of new components. J Rehabil Res Dev 1995, 32(1):32–35. • Convenient online submission doi:10.1186/1471-2474-14-333 Cite this article as: Theeven et al.: Measures and procedures utilized to determine the added value of microprocessor-controlled prosthetic knee joints: a systematic review. BMC Musculoskeletal Disorders 2013 14:333.
https://openalex.org/W4212951384	https://peerj.com/articles/12920v0.4/submission	English	null	Peer Review #1 of "The effect of farmland on the surface water of the Aral Sea Region using Multi-source Satellite Data (v0.3)"	null	2,022	cc-by	15,233	The eﬀect of farmland on the surface water of the Aral Sea Region Using Multi-Source Satellite Data Jiancong Shi 1, 2 , Qiaozhen Guo Corresp., 1 , Shuang Zhao 1 , Yiting Su 3 , Yanqing Shi 1 1 school of geology and geomatis, Tianjin Chengjian University, Tianjin, China 2 Taiyuan Design Research Institute Group Co., Ltd for Coal Industry, Taiyuan, China 3 College of Geoscience and Surveying Engineering, China University of Mining and Technology-Beijing, Beijing, China Corresponding Author: Qiaozhen Guo Email address: gqiaozhen@tcu.edu.cn Background: The improper land utilization has brought tremendous pressure on the surface water of the Aral Sea Region in the past decades. It was seriously hindered for construction of the Green Silk Road Economic Belt by the fragile environment. Therefore, it is of great necessity for environmental protection and social development to monitor the change of surface water in the Aral Sea Region. Methods: In this study, LandTrendr algorithm was used on Landsat time-series data to characterize the change in farmland on the Google Earth Engine platform. Based on multi-source data, the water area changes of the Aral Sea were extracted based on the Google Earth Engine, and the mean method was utilized to extract the changes in water level and water storage. Finally, a water-farmland coupling degree model was utilized to evaluate the impact of farmland changes on the surface water in the Aral Sea Region. Results: As a result, the change of farmland is as follows: the farmland area of the Aral Sea Region has abandoned 3129 km 2 from 1987 to 2019, with overall accuracy of 85.3%. The farmland change had increased the drainage downstream of the Amu Darya River and the Syr Darya River. It has led area of the Aral follows: the farmland area of the Aral Sea Region has abandoned 3129 km 2 from 1987 to 2019, with overall accuracy of 85.3%. The farmland change had increased the drainage downstream of the Amu Darya River and the Syr Darya River. It has led area of the Aral Sea to decrease each year continuously. The area of the Aral Sea shrank by 1606.36 km 2 per year from 1987 to 2019. Furthermore, Aral Sea’s water level decreased by 0.13 m per year from 2003 to 2009. The amount of water storage in the Aral Sea Region also showed a downward trend from 2002 to 2016. There was a high-quality coupling coordination 0.903 relationship between surface water and farmland. It will increase the burden of water for people's normal daily life by the water loss resources caused by abandoned farmland. Manuscript to be reviewed The eﬀect of farmland on the surface water of the Aral Sea Region Using Multi-Source Satellite Data Jiancong Shi 1, 2 , Qiaozhen Guo Corresp., 1 , Shuang Zhao 1 , Yiting Su 3 , Yanqing Shi 1 This study emphasized threat of unreasonable farmland management to surface water of the Aral Sea Region. The ﬁndings contributed for decision makers to formulating eﬀective reasonable policies to protect surface water and use land of the Aral Sea Region. Meanwhile, the application of coupling degree model can provide a new method for PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Manuscript to be reviewed studying the connection of independent systems in the farmland, water, environment and more. PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Manuscript to be reviewed 1 The Effect of Farmland on the Surface Water of 2 Aral Sea Region Using Multi-Source Satellite D 3 4 Jiancong Shi1,2, Qiaozhen Guo1, Shuang Zhao1, Yiting Su3, Yanqing Shi1 5 6 1 School of Geology and Geomatics, Tianjin Chengjian University, Tianjin, China 7 2Taiyuan Design Research Institute Group Co., Ltd for Coal Industry, Taiyuan, Sha 8 3College of Geoscience and Surveying Engineering, China University of Mining an 9 Technology-Beijing, Beijing, China 10 11 Corresponding Author: 12 Qiaozhen Guo1 13 Jinjing Road, Xiqing District, Tianjin, 300384, China 14 Email address: gqiaozhen@tcu.edu.cn 15 16 Abstract 17 Background: The improper land utilization has brought tremendous pressure on the 18 of the Aral Sea Region in the past decades. It was seriously hindered for constructio 19 Silk Road Economic Belt by the fragile environment. Therefore, it is of great 20 environmental protection and social development to monitor the change of surfac 21 Aral Sea Region. 22 Methods: In this study, LandTrendr algorithm was used on Landsat time-s 23 characterize the change in farmland on the Google Earth Engine platform. Based on 24 data, the water area changes of the Aral Sea were extracted based on the Google Ear 25 the mean method was utilized to extract the changes in water level and water stor 26 water-farmland coupling degree model was utilized to evaluate the impact of farmla 27 the surface water in the Aral Sea Region. 28 Results: As a result, the change of farmland is as follows: the farmland area of the Ar 29 has abandoned 3129 km2 from 1987 to 2019, with overall accuracy of 85.3%. The far 30 had increased the drainage downstream of the Amu Darya River and the Syr Dary 31 led area of the Aral Sea to decrease each year continuously. The area of the Aral 32 1606.36 km2 per year from 1987 to 2019. Furthermore, Aral Sea’s water level decr 33 m per year from 2003 to 2009. The amount of water storage in the Aral Sea Region 34 downward trend from 2002 to 2016. There was a high-quality coupling coord 35 relationship between surface water and farmland. 4 Jiancong Shi1,2, Qiaozhen Guo1, Shuang Zhao1, Yiting Su3, Yanqing Shi1 13 Jinjing Road, Xiqing District, Tianjin, 300384, China Manuscript to be reviewed It will increase the burden of wat 36 normal daily life by the water loss resources caused by abandoned farmland 37 emphasized threat of unreasonable farmland management to surface water of the Ar 38 The findings contributed for decision makers to formulating effective reasonable pol 39 surface water and use land of the Aral Sea Region. Meanwhile, the application of co 1 The Effect of Farml 2 Aral Sea Region Us 3 4 Jiancong Shi1,2, Qiaozhen Guo1, S 5 6 1 School of Geology and Geomati 7 2Taiyuan Design Research Institu 8 3College of Geoscience and Surve 9 Technology-Beijing, Beijing, Chi 10 11 Corresponding Author: 12 Qiaozhen Guo1 13 Jinjing Road, Xiqing District, Tia 14 Email address: gqiaozhen@tcu.ed 15 16 Abstract 17 Background: The improper land 18 of the Aral Sea Region in the past 19 Silk Road Economic Belt by th 20 environmental protection and soc 21 Aral Sea Region. 22 Methods: In this study, LandT 23 characterize the change in farmlan 24 data, the water area changes of the 25 the mean method was utilized to 26 water-farmland coupling degree m 27 the surface water in the Aral Sea R 28 Results: As a result, the change of 29 has abandoned 3129 km2 from 198 30 had increased the drainage downs 31 led area of the Aral Sea to decrea 32 1606.36 km2 per year from 1987 33 m per year from 2003 to 2009. Th 34 downward trend from 2002 to 35 relationship between surface wate 36 normal daily life by the water 37 emphasized threat of unreasonable 38 The findings contributed for decis 39 surface water and use land of the A 1 The Effect of Farmland on the Surface Water of the 2 Aral Sea Region Using Multi-Source Satellite Data 4 Jiancong Shi1,2, Qiaozhen Guo1, Shuang Zhao1, Yiting Su3, Yanqing Shi1 42 Introduction 43 As the essential water resources in the Aral Sea Basin and even in Central Asia, the Aral Sea 44 could primarily ensure the stability of the surface water to be maintained (Micklin, 1988). 45 However, the area of the Aral Sea is currently only one-tenth of that in 1960 (Wu et al., 2020). As 46 impacted by the rapid shrinking of the Aral Sea, the surface water of the Aral Sea Basin has been 47 severely decreased (Eleni et al., 2020). The shrinking surface water caused land desertification and 48 land salinization (Parajuli et al., 2017), thereby seriously endangering the development of society. 49 Accordingly, it is essential for protection of the surface water and regional development to reveal 50 the root cause of the Aral Sea’s shrinkage. 51 The Aral Sea Basin refers to a severely arid area with annual precipitation of less than 300 52 mm (Elena, 2014). Thus, the level of the Aral Sea is determined by the inflow of two feeding 53 rivers, the Amu Darya and the Syr Darya (Philippe et al., 2007). The two rivers originate from the 54 melting of snow in the mountains of southeast of the Aral Sea Basin (Micklin, 2016). Scholars 55 have carried out researches on the upstream of Amu Darya and Syr Darya, as presented below. 56 Valentina et al. found that the snow depth in the rivers’ (e.g., the Amu Darya and the Syr Darya) 57 source tended to decrease after 1965, whereas the runoff downstream has been declining 58 (Valentina et al., 2009). Hagg et al. considered that the runoff of the upper reaches in the Amu 59 Darya River would gradually increase (Hagg et al., 2007). In a word, considerable researches 60 indicated a downtrend of snow depth in the source of the Amu Darya River and the Syr Darya 61 River, whereas the runoff downstream has been declining (Annina et al., 2012; Wang et al., 2016). 62 The above studies show that the upstream runoff of Amu Darya and Syr Darya increases each year, 63 which is contrary to the shrinkage of the Aral Sea. At the same time, researchers have found that 64 the shrinkage of the Aral Sea is directly related to the reduction of runoff downstream of rivers 65 (Shi et al., 2021; Micklin, 2007). Manuscript to be reviewed At the same time, researchers have found th 64 the shrinkage of the Aral Sea is directly related to the reduction of runoff downstream of rive 65 (Shi et al., 2021; Micklin, 2007). Therefore, this study will focus on the downstream area of rive 66 near the Aral Sea. 67 Over the past decades, unreasonable land utilization led to the decline of downstream runo 68 in the Amu Darya River and the Syr Darya River, which directly caused the shrinking of the Ar 69 Sea (Christopher et al., 2016). Primarily, neighbouring countries used a lot of lands to plan 70 farmland, and have done many radical measures to increase the value of agricultural output. Fo 71 instance, the former Soviet Union built the channel with 500 km in the Amu Darya River area an 72 the Syr Darya River area in 1960. Such a project took one-third of the water in rivers downstream 40 model can provide a new method for studying the connection of independent systems in the 41 farmland, water, environment and more. 40 model can provide a new method for studying the connection of independent systems in the 41 farmland, water, environment and more. 16 Abstract 17 Background: The improper land utilization has brought tremendous pressure on the surface water 18 of the Aral Sea Region in the past decades. It was seriously hindered for construction of the Green 19 Silk Road Economic Belt by the fragile environment. Therefore, it is of great necessity for 20 environmental protection and social development to monitor the change of surface water in the 21 Aral Sea Region. g 22 Methods: In this study, LandTrendr algorithm was used on Landsat time-series data to 23 characterize the change in farmland on the Google Earth Engine platform. Based on multi-source 24 data, the water area changes of the Aral Sea were extracted based on the Google Earth Engine, and 25 the mean method was utilized to extract the changes in water level and water storage. Finally, a 26 water-farmland coupling degree model was utilized to evaluate the impact of farmland changes on 27 the surface water in the Aral Sea Region. g 28 Results: As a result, the change of farmland is as follows: the farmland area of the Aral Sea Region 29 has abandoned 3129 km2 from 1987 to 2019, with overall accuracy of 85.3%. The farmland change 30 had increased the drainage downstream of the Amu Darya River and the Syr Darya River. It has 31 led area of the Aral Sea to decrease each year continuously. The area of the Aral Sea shrank by 32 1606.36 km2 per year from 1987 to 2019. Furthermore, Aral Sea’s water level decreased by 0.13 33 m per year from 2003 to 2009. The amount of water storage in the Aral Sea Region also showed a 34 downward trend from 2002 to 2016. There was a high-quality coupling coordination 0.903 35 relationship between surface water and farmland. It will increase the burden of water for people's 36 normal daily life by the water loss resources caused by abandoned farmland. This study 37 emphasized threat of unreasonable farmland management to surface water of the Aral Sea Region. 38 The findings contributed for decision makers to formulating effective reasonable policies to protect 39 surface water and use land of the Aral Sea Region. Meanwhile, the application of coupling degree PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Manuscript to be reviewed 40 model can provide a new method for studying the connection of independent systems in th 41 farmland, water, environment and more. 42 Introduction 43 As the essential water resources in the Aral Sea Basin and even in Central Asia, the Aral Se 44 could primarily ensure the stability of the surface water to be maintained (Micklin, 1988 45 However, the area of the Aral Sea is currently only one-tenth of that in 1960 (Wu et al., 2020). A 46 impacted by the rapid shrinking of the Aral Sea, the surface water of the Aral Sea Basin has bee 47 severely decreased (Eleni et al., 2020). The shrinking surface water caused land desertification an 48 land salinization (Parajuli et al., 2017), thereby seriously endangering the development of societ 49 Accordingly, it is essential for protection of the surface water and regional development to reve 50 the root cause of the Aral Sea’s shrinkage. 51 The Aral Sea Basin refers to a severely arid area with annual precipitation of less than 30 52 mm (Elena, 2014). Thus, the level of the Aral Sea is determined by the inflow of two feedin 53 rivers, the Amu Darya and the Syr Darya (Philippe et al., 2007). The two rivers originate from th 54 melting of snow in the mountains of southeast of the Aral Sea Basin (Micklin, 2016). Schola 55 have carried out researches on the upstream of Amu Darya and Syr Darya, as presented below 56 Valentina et al. found that the snow depth in the rivers’ (e.g., the Amu Darya and the Syr Dary 57 source tended to decrease after 1965, whereas the runoff downstream has been declinin 58 (Valentina et al., 2009). Hagg et al. considered that the runoff of the upper reaches in the Am 59 Darya River would gradually increase (Hagg et al., 2007). In a word, considerable researche 60 indicated a downtrend of snow depth in the source of the Amu Darya River and the Syr Dary 61 River, whereas the runoff downstream has been declining (Annina et al., 2012; Wang et al., 2016 62 The above studies show that the upstream runoff of Amu Darya and Syr Darya increases each yea 63 which is contrary to the shrinkage of the Aral Sea. Manuscript to be reviewed 80 investigated human irrigation-induced hydrological variations in the Aral Sea Basin by multiple 81 satellite data (Jin et al., 2017). Fabian et al. mapped abandoned farmland to support surface water 82 protection in Kyzyl-Orda, Kazakhstan (Fabian et al., 2015). However, existing studies could not 83 determine a quantitative model to assess the impact of farmland changes on surface water. 84 Accordingly, it is of great necessity to establish a method to assess the effect of farmland changes 85 on surface water. 86 For the complicated political issues and poor physical accessibilities of the Aral Sea Region, 87 there are severe limitations in collecting data required for large-scale and long-term change 88 detection and analyses (Fabian et al., 2015). As an alternative, remote sensing technology, 89 exhibiting the advantages in data of effective coverage of long-term span and multi-time scale, has 90 been extensively employed in natural disaster early warning, eco-environmental protection, 91 farmland protection, dynamic analysis of land use, social development, etc. (Tewkesbury et al., 92 2015; Li et al., 2003; Saha et al., 2019). Alka et al. used multi-source data to explore inter-annual 93 water storage variations in the Aral Sea (Singh et al., 2012). Jin et al. adopted multiple satellite 94 data to view environmental variations around the Aral Sea (Jin et al., 2017). At present, remote 95 sensing technology has become an essential measure of long-term monitoring variations in surface 96 water in large areas (Singh et al., 2012). However, the above researches are difficult to characterize 97 the impact of farmland on the surface water quantitatively. Thus, this study combined the 98 comprehensive variations of water and farmland changes to explore the internal relationship 99 between the surface water and farmland based on multi-source data. 100 The water area, water level and surrounding water storage of the Aral Sea act as critical 101 indicators to characterize the hydrological variations in the Aral Sea. Multi-source data efficiently 102 supports extracting the comprehensive variations of water. The LandTrendr (Landsat-based 103 Detection of Trends in Disturbance and Recovery) algorithm can capture both long-term gradual 104 and short-term drastic variations (Gorelick et al., 2017). Moreover, Kennedy et al. encapsulated 105 the LandTrendr on the GEE platform to consume considerable computation time in continuous 106 change detection, (Kennedy et al., 2018). Moreover, it has been broadly applied in most fields of 107 change detection (Zhu et al., 2019). Manuscript to be reviewed The coupling degree model is capable of reflecting the degree 108 of association between multiple subsystems. 109 This study aimed to examine the surface water attributed to land utilization change of the Aral 110 Sea Region. The specific steps are as follows: Firstly, the LandTrendr algorithm was run on 111 Landsat time-series of the GEE platform to detect the land utilization change modes. Second, the 112 variations of water area, water level and water storage were detected using multi-source data 113 (Landsat, ICESat, GRACE). Lastly, to explore the effect of farmland changes on the surface water, 114 the variations of water (water area, water level and water storage) and farmland were combined to 115 build a water-farmland coupling degree model. The mentioned efforts can provide explicit 116 cognitions regarding the effect of farmland on surface water. Furthermore, it can provide practical 117 implications for agricultural governance, water protection and surface water balance. 42 Introduction Therefore, this study will focus on the downstream area of rivers 66 near the Aral Sea. 67 Over the past decades, unreasonable land utilization led to the decline of downstream runoff 68 in the Amu Darya River and the Syr Darya River, which directly caused the shrinking of the Aral 69 Sea (Christopher et al., 2016). Primarily, neighbouring countries used a lot of lands to plant 70 farmland, and have done many radical measures to increase the value of agricultural output. For 71 instance, the former Soviet Union built the channel with 500 km in the Amu Darya River area and 72 the Syr Darya River area in 1960. Such a project took one-third of the water in rivers downstream 73 to farmland (Conant, 2006). The mentioned measure altered the rain-fed irrigation mode of 74 farmland in the Aral Sea Basin (Micklin, 2007). By 2010, the irrigated area of the Aral Sea Basin 75 was 7,895,600 ha, taking up 78.6% of planted land (data from the Central Asia Water Resources 76 Network: www.cawater-info.net). Inefficient irrigation systems and terrible management of land 77 utilization triggered the increase in water withdrawn from rivers. Thus, surface water protection 78 requires the exploration of the farmland variations in the Aral Sea Basin. Several studies have been 79 conducted on the surface water variations attributed to irrigation. For instance, Jin et al. PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) 119 Materials & Methods PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) 139 2.2.2 ICESat satellite altimetry data y 140 The ICESat mission was initiated in January 2003 and ended in February 2010 by National 141 Aeronautics and Space Administration (NASA) (Schutz et al., 2005). The Geoscience Laser 142 Altimeter System (GLAS) on ICESat has provided global measurements of polar ice sheet mass 143 balance, cloud and aerosol heights, land topography and vegetation characteristics with a surface 144 sampling diameter of 70 m as well as a spacing of 172 m (Zwally et al., 2008). ICESat/GLAS level 145 2 altimetry product (GLA14) consists of global land surface elevation data (Kwok et al., 2004). 146 The GLA14 has been broadly introduced in lake water level and land elevation and have reflected 147 accuracy over 10 cm (Zhang et al., 2011). Thus, the GLA14 can precisely contribute to the surface 148 elevation. The GLA14 from 2003 to 2009 were collected to extract the water level change in the 149 Aral Sea. 140 The ICESat mission was initiated in January 2003 and ended in February 2010 by National 141 Aeronautics and Space Administration (NASA) (Schutz et al., 2005). The Geoscience Laser 142 Altimeter System (GLAS) on ICESat has provided global measurements of polar ice sheet mass 143 balance, cloud and aerosol heights, land topography and vegetation characteristics with a surface 144 sampling diameter of 70 m as well as a spacing of 172 m (Zwally et al., 2008). ICESat/GLAS level 145 2 altimetry product (GLA14) consists of global land surface elevation data (Kwok et al., 2004). 146 The GLA14 has been broadly introduced in lake water level and land elevation and have reflected 147 accuracy over 10 cm (Zhang et al., 2011). Thus, the GLA14 can precisely contribute to the surface 148 elevation. The GLA14 from 2003 to 2009 were collected to extract the water level change in the 149 Aral Sea. Manuscript to be reviewed Manuscript to be reviewed 120 2.1 Study Area 121 The Aral Sea Region is located on the border between northwest Uzbekistan and south 122 Kazakhstan. It covers 130,000 km2, and includes North Aral, East Aral, West Aral and irri 123 farmland of the Aral Sea Region (Figure 1). This is a typical arid area with an average an 124 temperature of 11.62 ℃ and precipitation of 126.26 mm. 125 126 Figure 1. Research scope: (a) general location in the Aral Sea Basin and the Central 127 Asia; (b) 1987 land-use classification from Google Earth Engine (GEE) imagery 128 2.2 Data Preparation 129 2.2.1 Landsat data and Pre-processing 130 Landsat surface reflectance data (Landsat5, Landsat7 and Landsat8) were collected 131 1984 to 2019 on GEE platform. This study used the FMASK and neighboring position filli 132 improve extraction accuracy to remove the mentioned poor-quality (snow/ice, clouds, bui 133 shadows and scan-line corrector (SLC)-off gaps) images (Zhu et al., 2012). Accordingly 134 Landsat surface reflectance dataset was produced for our study area from 1987 to 2019 (A-LS 135 Lastly, the dataset (YA-LSTC) was synthesized into annual images from the A-LSTC (June 136 September 1). The normalized vegetation index (NDVI) was introduced to the dataset as a 137 band (Tucker, 1979). 130 Landsat surface reflectance data (Landsat5, Landsat7 and Landsat8) were collected from 131 1984 to 2019 on GEE platform. This study used the FMASK and neighboring position filling to 132 improve extraction accuracy to remove the mentioned poor-quality (snow/ice, clouds, building 133 shadows and scan-line corrector (SLC)-off gaps) images (Zhu et al., 2012). Accordingly, the 134 Landsat surface reflectance dataset was produced for our study area from 1987 to 2019 (A-LSTC). 135 Lastly, the dataset (YA-LSTC) was synthesized into annual images from the A-LSTC (June 1 to 136 September 1). The normalized vegetation index (NDVI) was introduced to the dataset as a new 137 band (Tucker, 1979). 𝑁𝐷𝑉𝐼= 𝜌𝑛𝑖𝑟‒ 𝜌𝑟 𝜌𝑛𝑖𝑟+ 𝜌𝑟 (1) (1) 138 Where and are the near-infrared band and red band in Landsat. ρnir ρr 138 Where and are the near-infrared band and red band in Landsat. ρnir ρr 162 163 Figure 2. Research Method: (a) The method of farmland change detection; (b) The method of 164 water change detection; (c) The method coupling degree model. 163 Figure 2. Research Method: (a) The method of farmland change detection; (b) The method of 164 water change detection; (c) The method coupling degree model. 163 Figure 2. Research Method: (a) The method of farmland change detection; (b) The method of 164 water change detection; (c) The method coupling degree model. 166 LandTrendr (Landsat-based Detection of Trends in Disturbance and Recovery) algorithm is 167 an approach to extract the spectral trajectories of land surface from Landsat time-series stacks 168 (Watts et al., 2014). This algorithm can reflect farmland variations by detecting the temporal- 169 spectral trajectory of each pixel. The input of every pixel for the algorithm is a time series with 170 one spectral value or index. This algorithm also needs to set parameters, such as noise-induced 171 spikes (outliers), potential vertices (breakpoints), fitting trajectories, and the optimal number of 172 segments (Watts et al., 2014). ( y) g 167 an approach to extract the spectral trajectories of land surface from Landsat time-series stacks 168 (Watts et al., 2014). This algorithm can reflect farmland variations by detecting the temporal- 169 spectral trajectory of each pixel. The input of every pixel for the algorithm is a time series with 170 one spectral value or index. This algorithm also needs to set parameters, such as noise-induced 171 spikes (outliers), potential vertices (breakpoints), fitting trajectories, and the optimal number of 172 segments (Watts et al., 2014). 173 LandTrendr algorithm on the GEE platform was used to detect the farmland change in the 174 Aral Sea Region (Figure 2-(a)). Before running this algorithm, the features of farmland variations 175 were analyzed, tested different combinations of parameter values. The NDVI was determined as 176 the spectral index, and other parameters were calculated (Table S1). NDVI of farmland ranged 177 from 0.45 to 0.69 in the Aral Sea Region and plummeted after abandonment to range from 0.2 to 178 0.3. Thus, NDVI of abandoned farmland was set between 0.2 and 0.3. The mentioned thresholds 179 were adopted to detect the change patterns of farmland, the change time of occurrence and end, 180 and duration. 181 Table S1. Operating parameters of LandTrendr 182 183 2.3.2. Manuscript to be reviewed 153 of Earth’s gravity by GRACE (Jin et al., 2017). Most gravity signals after de-noising of GRAC 154 reflect variations in water storage (Chen et al., 2019; Swenson et al., 2007). However, the orbit 155 altitude and resolution of GRACE limited the effective research range (< 200,000 km2) (Singh 156 al., 2012). Thus, the original research range was extended to 230,000 km2. Though the study are 157 is less than 230,000 km2, it can be assumed that the mass variations on long-time scales come from 158 the long-term storage change of water of the Aral Sea Region. Thus, the GRACE data wa 159 processed in the GEE platform from 2002 to 2016 (research range between June and Septembe 160 each year). 161 2.3. Detection Algorithm 162 163 Figure 2. Research Method: (a) The method of farmland change detection; (b) The method of 164 water change detection; (c) The method coupling degree model. 165 2.3.1. Detection Algorithm of Farmland Change 166 LandTrendr (Landsat-based Detection of Trends in Disturbance and Recovery) algorithm 167 an approach to extract the spectral trajectories of land surface from Landsat time-series stack 168 (Watts et al., 2014). This algorithm can reflect farmland variations by detecting the tempora 169 spectral trajectory of each pixel. The input of every pixel for the algorithm is a time series wit 170 one spectral value or index. This algorithm also needs to set parameters, such as noise-induce 171 spikes (outliers), potential vertices (breakpoints), fitting trajectories, and the optimal number o 172 segments (Watts et al., 2014). 173 LandTrendr algorithm on the GEE platform was used to detect the farmland change in th 174 Aral Sea Region (Figure 2-(a)). Before running this algorithm, the features of farmland variation 175 were analyzed, tested different combinations of parameter values. The NDVI was determined a 176 the spectral index, and other parameters were calculated (Table S1). NDVI of farmland range 177 from 0.45 to 0.69 in the Aral Sea Region and plummeted after abandonment to range from 0.2 t 178 0.3. Thus, NDVI of abandoned farmland was set between 0.2 and 0.3. The mentioned threshold 179 were adopted to detect the change patterns of farmland, the change time of occurrence and en 180 and duration. 181 Table S1. Operating parameters of LandTrendr 182 183 2.3.2. Manuscript to be reviewed Detection Algorithm of Waters Change 184 Via the GEE platform, the random forest algorithm was adopted to detect the variation o 185 water area (Figure 2-(b)). Random forest is a machine learning algorithm that is complies with th 186 decision tree. This algorithm uses CART clustering for prediction and classification (Breiman 187 2001). Two primary parameters should be set before implementing the random forest algorithm 188 To be specific, the first refers to the number of splitting times (mtry) of the branch tree in eac 153 of Earth’s gravity by GRACE (Jin et al., 2017). Most gravity signals after de-noising of GRACE 154 reflect variations in water storage (Chen et al., 2019; Swenson et al., 2007). However, the orbital 155 altitude and resolution of GRACE limited the effective research range (< 200,000 km2) (Singh et 156 al., 2012). Thus, the original research range was extended to 230,000 km2. Though the study area 157 is less than 230,000 km2, it can be assumed that the mass variations on long-time scales come from 158 the long-term storage change of water of the Aral Sea Region. Thus, the GRACE data was 159 processed in the GEE platform from 2002 to 2016 (research range between June and September 160 each year). 153 of Earth s gravity by GRACE (Jin et al., 2017). Most gravity signals after de-noising of GRAC 154 reflect variations in water storage (Chen et al., 2019; Swenson et al., 2007). However, the orbi 155 altitude and resolution of GRACE limited the effective research range (< 200,000 km2) (Singh 156 al., 2012). Thus, the original research range was extended to 230,000 km2. Though the study ar 157 is less than 230,000 km2, it can be assumed that the mass variations on long-time scales come fro 158 the long-term storage change of water of the Aral Sea Region. Thus, the GRACE data w 159 processed in the GEE platform from 2002 to 2016 (research range between June and Septemb 160 each year). 161 2.3. Detection Algorithm 162 163 Figure 2. Research Method: (a) The method of farmland change detection; (b) The method of 164 water change detection; (c) The method coupling degree model. 165 2.3.1. Detection Algorithm of Farmland Change 166 LandTrendr (Landsat-based Detection of Trends in Disturbance and Recovery) algorithm 167 an approach to extract the spectral trajectories of land surface from Landsat time-series stac 168 (Watts et al., 2014). 150 2.2.3 GRACE Data 151 The Gravity Recovery and Climate Experiment (GRACE) mission was launched in March 152 2002 and ended in October 2017 by NASA. The water mass variations were considered a change 151 The Gravity Recovery and Climate Experiment (GRACE) mission was launched in March 152 2002 and ended in October 2017 by NASA. The water mass variations were considered a change PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Manuscript to be reviewed This algorithm can reflect farmland variations by detecting the tempor 169 spectral trajectory of each pixel. The input of every pixel for the algorithm is a time series w 170 one spectral value or index. This algorithm also needs to set parameters, such as noise-induc 171 spikes (outliers), potential vertices (breakpoints), fitting trajectories, and the optimal number 172 segments (Watts et al., 2014). 173 LandTrendr algorithm on the GEE platform was used to detect the farmland change in t 174 Aral Sea Region (Figure 2-(a)). Before running this algorithm, the features of farmland variatio 175 were analyzed, tested different combinations of parameter values. The NDVI was determined 176 the spectral index and other parameters were calculated (Table S1) NDVI of farmland rang 162 Detection Algorithm of Waters Change 184 Via the GEE platform, the random forest algorithm was adopted to detect the variation of 185 water area (Figure 2-(b)). Random forest is a machine learning algorithm that is complies with the 186 decision tree. This algorithm uses CART clustering for prediction and classification (Breiman, 187 2001). Two primary parameters should be set before implementing the random forest algorithm. 188 To be specific, the first refers to the number of splitting times (mtry) of the branch tree in each 173 LandTrendr algorithm on the GEE platform was used to detect the farmland change in the 174 Aral Sea Region (Figure 2-(a)). Before running this algorithm, the features of farmland variations 175 were analyzed, tested different combinations of parameter values. The NDVI was determined as 176 the spectral index, and other parameters were calculated (Table S1). NDVI of farmland ranged 177 from 0.45 to 0.69 in the Aral Sea Region and plummeted after abandonment to range from 0.2 to 178 0.3. Thus, NDVI of abandoned farmland was set between 0.2 and 0.3. The mentioned thresholds 179 were adopted to detect the change patterns of farmland, the change time of occurrence and end, 180 and duration. Table S1. Operating parameters of LandTrendr Manuscript to be reviewed 189 model. This parameter should be set as the square root value of variables and 2 times or 1/2 of the 190 prediction’s open value. In this paper, the variables specifically refer to the bands of remote sensing 191 images (Table S2). The second refers to the number of generated trees (ntree) in model operation, 192 and the amount of model calculation is proportional to the value of ntree. The ntree was set as 500 193 because it will be pretty obvious with a bit of tinkering that the predictions from the model will 194 not change much after this value. 195 The accuracy of model depends more on quality of training samples. Therefore, we selected 196 200 pixels representing water and another 200 pixels representing non-water based on Landsat 197 imagery. In order to access accuracy of results, we visually examined all selected pixels (from 198 1987 to 2019) and used high-spatial-resolution Google Earth imagery for further manual 199 interpretation of land cover (Figure 3). Therefore, we determined and recorded whether and when 200 water pixels change occurred. 201 202 Table S2. Variables of Random Forest Algorithm 203 204 Figure 3. Examples areas of water change and farmland change near the Syr Darya and the Amu 205 Darya. Figures a1 and a2 represent the water change years 2009 and 2019; Figures b1, b2, b3 206 represent the water change years 2009, 2016 and 2019. 207 208 The ICESat data was adopted to analyze the alteration in water level in the Aral Sea. At first, 209 the data format of GLA14 was converted. Second, GLA14 was adopted to extract the water level 210 of the Aral Sea (Eq. 2). Lastly, the water volume was calculated according to the alteration in water 211 level and the variation of water area of the Aral Sea (Eq. 3). Meanwhile, researchers compared 212 ICESat data with airborne LiDAR data and DEM from GPS data. The error of ICESat data is less 213 than 3 cm (Kurtz et al., 2008; Fricker, 2005). 189 model. This parameter should be set as the square root value of variables and 2 times or 1/2 of the 190 prediction’s open value. In this paper, the variables specifically refer to the bands of remote sensing 191 images (Table S2). Manuscript to be reviewed This parameter should be set as the square root value of variables and 2 times or 1/2 of the 190 prediction’s open value. In this paper, the variables specifically refer to the bands of remote sensing 191 images (Table S2). The second refers to the number of generated trees (ntree) in model operation, 192 and the amount of model calculation is proportional to the value of ntree. The ntree was set as 500 193 because it will be pretty obvious with a bit of tinkering that the predictions from the model will 194 not change much after this value. 195 The accuracy of model depends more on quality of training samples. Therefore, we selected 196 200 pixels representing water and another 200 pixels representing non-water based on Landsat 197 imagery. In order to access accuracy of results, we visually examined all selected pixels (from 198 1987 to 2019) and used high-spatial-resolution Google Earth imagery for further manual 199 interpretation of land cover (Figure 3). Therefore, we determined and recorded whether and when 200 water pixels change occurred. 183 2.3.2. Detection Algorithm of Waters Change 184 Via the GEE platform, the random forest algorithm was adopted to detect the variation of 185 water area (Figure 2-(b)). Random forest is a machine learning algorithm that is complies with the 186 decision tree. This algorithm uses CART clustering for prediction and classification (Breiman, 187 2001). Two primary parameters should be set before implementing the random forest algorithm. 188 To be specific, the first refers to the number of splitting times (mtry) of the branch tree in each PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Manuscript to be reviewed The second refers to the number of generated trees (ntree) in model operation, 192 and the amount of model calculation is proportional to the value of ntree. The ntree was set as 500 193 because it will be pretty obvious with a bit of tinkering that the predictions from the model will 194 not change much after this value. 195 The accuracy of model depends more on quality of training samples. Therefore, we selected 196 200 pixels representing water and another 200 pixels representing non-water based on Landsat 197 imagery. In order to access accuracy of results, we visually examined all selected pixels (from 198 1987 to 2019) and used high-spatial-resolution Google Earth imagery for further manual 199 interpretation of land cover (Figure 3). Therefore, we determined and recorded whether and when 200 water pixels change occurred. 201 202 Table S2. Variables of Random Forest Algorithm 203 204 Figure 3. Examples areas of water change and farmland change near the Syr Darya and the Amu 205 Darya. Figures a1 and a2 represent the water change years 2009 and 2019; Figures b1, b2, b3 206 represent the water change years 2009, 2016 and 2019. 207 208 The ICESat data was adopted to analyze the alteration in water level in the Aral Sea. At first, 209 the data format of GLA14 was converted. Second, GLA14 was adopted to extract the water level 210 of the Aral Sea (Eq. 2). Lastly, the water volume was calculated according to the alteration in water 211 level and the variation of water area of the Aral Sea (Eq. 3). Meanwhile, researchers compared 212 ICESat data with airborne LiDAR data and DEM from GPS data. The error of ICESat data is less 213 than 3 cm (Kurtz et al., 2008; Fricker, 2005). 189 model. This parameter should be set as the square root value of variables and 2 times or 1/2 of the 190 prediction’s open value. In this paper, the variables specifically refer to the bands of remote sensing 191 images (Table S2). The second refers to the number of generated trees (ntree) in model operation, 192 and the amount of model calculation is proportional to the value of ntree. The ntree was set as 500 193 because it will be pretty obvious with a bit of tinkering that the predictions from the model will 194 not change much after this value. 189 model. Table S2. Variables of Random Forest Algorithm WLT = 𝑛 ∑ 𝑖= 1 𝑤𝑙 𝑛 (2) (2) 𝑊𝑉= 𝑊𝐿𝑇∗𝑊𝐴 (3) 𝑊𝑉= 𝑊𝐿𝑇∗𝑊𝐴 (3) 214 Where WLT is mean water level. wl is the land elevation of every ICESat point. WV is water 215 volume. WA is water area. 214 Where WLT is mean water level. wl is the land elevation of every ICESat point. WV is water 215 volume. WA is water area. 215 volume. WA is water area. 216 The GRACE dataset was used to extract the equivalent water height (EWH). The data 217 contained in this dataset are units of "Equivalent Water Thickness" which represent the deviations 218 of mass in terms of the vertical extent of water in centimeters (Watkins et al., 2015). The EWH 219 was processed at NASA Jet Propulsion Laboratory. Actually, this dataset was produced by three 220 institutions: CSR (U. Texas / Center for Space Research), GFZ (German Research Center for 221 Geosciences), and JPL (NASA Jet Propulsion Laboratory). However, since each institution 222 independently produces the coefficients, the results may be slightly different. And the mass change PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) 𝑊𝑆 = 𝐸𝑊𝐻𝑇∗𝐴 (4) 237 Where EWHT is the equivalent water height from GRACE dataset; WS is water storage; A 238 is the area of study region. 237 Where EWHT is the equivalent water height from GRACE dataset; WS is water storage; A 238 is the area of study region. 237 Where EWHT is the equivalent water height from GRACE dataset; WS is water storage; A 238 is the area of study region. A 240 Two hundred pixels were selected to build a confusion matrix (CM) to assess the accuracy 241 of the five surface features in 1987 (i.e., water, farmland, desert, shoal and irrigation land). 242 Meanwhile, we visually examined all surface features by high-spatial-resolution Google Earth 243 imagery. The overall accuracy, user accuracy, and producer accuracy for each feature were 244 concluded following the indexes defined by Foody and Green (Congalton et al., 1993; Foody, 245 2002). Two hundred validating pixels were selected to represent the stable area of farmland. In 246 addition, another 200 validating pixels represent the various area of farmland for each change 247 mode. The stable area refers to the pixels that remain unchanged as farmland for years. While the 248 various area represents the changed farmland pixels during the whole study period. The 20% pixels 249 of training samples in water extraction were adopted to produce a CM to access the accuracy of 250 water extraction results. Manuscript to be reviewed 223 maps of the three institutions have typically suffered from poor observability of east-west 224 gradients, resulting in “N-S stripes” that are conventionally removed via empirical smoothing 225 and/or “destriping” algorithms. In order to overcome these limitations, Watkins et al., developed 226 GRACE(-FO) mass concentration (mascon)solutions (Watkins et al., 2015; Wiese et al., 2016; 227 Landerer et al., 2020). The solutions allow for convenient application of a priori information 228 derived from near-global geophysical models to prevent striping. Significantly, over the land, 229 mascon solutions have more excellent resolution for smaller spatial regions, in particular when 230 studying secular signals. Meanwhile, for different application scenarios, the dataset has its 231 advantages. For instance, it is more suitable for water, ice mass, and ocean bottom pressure mass 232 change application to use JPL mascon (Watkins et al., 2016; Landerer et al., 2020). Therefore, JPL 233 data, which have been processed by the GRACE(-FO) mascon solutions, were used in this paper. 234 And the measurement accuracy of GRACE data is credible (5000km wavelength: 0.001mm; 235 500km wavelength: 0.01mm) (Zheng et al., 2009). Water storage variation in the Aral Sea Region 236 was analyzed (Eq. 4). 𝑊𝑆 = 𝐸𝑊𝐻𝑇∗𝐴 Manuscript to be reviewed C = { 𝑓1(𝑥) ∗⋯∗𝑓𝑖(𝑥) \| 𝑓1(𝑥) + ⋯+ 𝑓𝑖(𝑥) 𝑖 \| 𝑖} 1 𝑖 (6) 𝑓𝑖(𝑥) = 𝑗 ∑ 𝑏 𝑤𝑏𝑥𝑏 (7) C = { 𝑓1(𝑥) ∗⋯∗𝑓𝑖(𝑥) \| 𝑓1(𝑥) + ⋯+ 𝑓𝑖(𝑥) 𝑖 \| 𝑖} 1 𝑖 (6) 𝑓𝑖(𝑥) = 𝑗 ∑ 𝑏 𝑤𝑏𝑥𝑏 (7) (6) 𝑓𝑖(𝑥) = 𝑗 ∑ 𝑏 𝑤𝑏𝑥𝑏 (7) (7) 𝑏 259 Where is the comprehensive evaluation function of subsystem; is the weight of each 𝑓𝑖(𝑥) 𝑤b 260 index; is each index; is the number of subsystems to be access. There are two subsystems which 𝑥b 𝑖 261 need to be evaluated, so . ( ). If the C value is closer to 1, it will mean that the 𝑖= 2 𝐶∈[0,1] 262 subsystems are more related and coordinated (Congalton et al., 1993). 263 264 Results 265 3.1. Accuracy Assessment 266 267 The CM for the classification of 1987 imagery (Table S3) revealed that the overall accuracy 268 (OA) reached over 90%. The user accuracy (UA), and producer accuracy (PA) for each class 269 mainly were more than 86%. The accuracy of farmland is less than 90%. The reasons are elucidated 270 below. There were considerable irregular water bodies and farmland in irrigated land of the Aral 271 Sea Region; thus, the farmland often tended to mix with water and Irrigation land. Moreover, some 272 sparse vegetation was planted in the irrigation land. Thus, it was difficult to distinguish by 273 employing Landsat images with a spatial resolution of 30m. However, the overall accuracy reached 274 92.1%, suggesting the random forest model’s capability. A similar method was used to assess the 275 accuracy of extraction of water area in the Aral Sea from 1987 to 2019. The accuracy of extraction 276 of water area reached over 90%, demonstrating the credibility of the results. 277 Table S3. Confusion matrix for the 1987 classified image. 278 279 Table S4 show the farmland detection accuracy of abandon and replanting by LandTrendr. 280 The overall accuracy of 85.3% was low for abandoned farmland for the mixture of abandoned 281 farmland and sparse planting. For the replanting, the PA, UA, and OA are above 80%. Thus, it is 282 sufficient for farmland to detect the change model. In summary, the OA of abandoned farmland 283 change detection was more than 85%, demonstrating the detection method’s reliability based on 284 the LandTrendr. 285 Table S4. Confusion matrix for the change detection of abandoned farmland. 286 259 Where is the comprehensive evaluation function of subsystem; is the weight of each 𝑓𝑖(𝑥) 𝑤b 260 index; is each index; is the number of subsystems to be access. There are two subsystems which 𝑥b 𝑖 261 need to be evaluated, so . ( ). If the C value is closer to 1, it will mean that the 𝑖= 2 𝐶∈[0,1] 262 subsystems are more related and coordinated (Congalton et al., 1993). 263 264 Results 265 3.1. Accuracy Assessment 266 267 The CM for the classification of 1987 imagery (Table S3) revealed that the overall accuracy 268 (OA) reached over 90%. The user accuracy (UA), and producer accuracy (PA) for each class 269 mainly were more than 86%. The accuracy of farmland is less than 90%. The reasons are elucidated 270 below. There were considerable irregular water bodies and farmland in irrigated land of the Aral 271 Sea Region; thus, the farmland often tended to mix with water and Irrigation land. Moreover, some 272 sparse vegetation was planted in the irrigation land. Thus, it was difficult to distinguish by 273 employing Landsat images with a spatial resolution of 30m. However, the overall accuracy reached 274 92.1%, suggesting the random forest model’s capability. A similar method was used to assess the 275 accuracy of extraction of water area in the Aral Sea from 1987 to 2019. The accuracy of extraction 276 of water area reached over 90%, demonstrating the credibility of the results. 277 Table S3. Confusion matrix for the 1987 classified image. 278 279 Table S4 show the farmland detection accuracy of abandon and replanting by LandTrendr. 280 The overall accuracy of 85.3% was low for abandoned farmland for the mixture of abandoned 281 farmland and sparse planting. For the replanting, the PA, UA, and OA are above 80%. Thus, it is 282 sufficient for farmland to detect the change model. In summary, the OA of abandoned farmland 283 change detection was more than 85%, demonstrating the detection method’s reliability based on 284 the LandTrendr. 285 Table S4. Confusion matrix for the change detection of abandoned farmland. 286 259 Where is the comprehensive evaluation function of subsystem; is the weight of each 𝑓𝑖(𝑥) 𝑤b 260 index; is each index; is the number of subsystems to be access. There are two subsystems which 𝑥b 𝑖 261 need to be evaluated, so . ( ). If the C value is closer to 1, it will mean that the 𝑖= 2 𝐶∈[0,1] 262 subsystems are more related and coordinated (Congalton et al., 1993). 263 251 2.5. Water-Farmland Coupling Degree Model 252 The coupling degree model originated from the coupling coefficient model in physics. 253 Farmland and surface water were combined to build the water-farmland coupling degree model to 254 explore the impact of farmland on surface water. In such a model, four indicators were selected 255 related to surface water and farmland (Figure 2-(c)). At first, every indicator was normalized, and 256 then the entropy method was used to determine their weight. The index matrix is X = (xab)i × j 257 (where i is the evaluation index; j is the index, and is the b-th index of the a-th sample,). The 𝑥𝑎b 258 coupling degree model (C) is as follows (Chang, 2018). PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Manuscript to be reviewed 287 3.2. Temporal and Spatial Variations of Farmland 288 The distribution of abandoned farmland, in the Aral Sea Region during 1987-2019, 289 rendered the disturbance map with a gradient (Figure 4) according to the occurrent years. 290 abandoned region was mainly diffused around the Syr Darya and the Amu Darya, especi 291 around the Amu Darya (Table S5). Within 9 km of these Draya rivers, the percentage of abando 292 farmland reached 59.96% from 1987 to 2019. Overall, 3129 km2 of farmland was abandoned. 293 annual change peaked between 1987 and 1993 (Table S6), after which percentage of abando 294 farmland decreased under 3% from 2002 to 2016 (Table S6). However, the percentage 295 abandoned farmland was elevated to 5.7% in 2017 (Table S6), which resulted in the plumme 296 water volume in the Aral Sea. 297 298 Figure 4. Year of abandoned farmland in the Aral Sea Region with two areas shown in detail 299 Around the Syr Darya River; (b) Around the Amu Darya River. 300 301 Table S5. Area of Abandoned Farmland near the Amu Draya and the Syr Draya 302 303 Table S6. Area of Abandoned Farmland in the Aral Sea Region 304 305 3.3. Waters Variations of the Aral Sea Region 306 The Aral Sea has been shrinking at 1606.36 km2 per year from 1987-2019. In 2019, the a 307 of the Aral Sea took up only 15.6% of its size in 1987 (Figure 5). With an annual decreas 308 1306.18 km2, the East Aral (820.22 km2) had almost dried up by 2019. The area of the East A 309 in 2019, only accounted for 1.92% in 1987. On the contrary, as impacted by the construction 310 the Dike Kokaral dam in 2005, the North Aral showed a weak expansion trend with an aver 311 annual growth of 10.05 km2. The area of North Aral rose to 4658.773 km2 in 2019. Kazakh 312 Government built the Kokaral dam at the junction of the North Aral and East Aral (Figure 5) 313 order to keep the water level of North Aral and environment stable, the water flow from N 314 Aral to East Aral was artificially reduced. Though this policy effectively prevented the los 315 North Aral, it also reduced the water flowing into East Aral. Such a dam aggravated the dry-u 316 East Aral. The West Aral is small and far from the irrigated area. 264 Results 285 Table S4. Confusion matrix for the change detection of abandoned farmland. PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Manuscript to be reviewed Accordingly, the West Aral 317 shrunk more minor than the East Aral at 314.83 km2 each year. 318 319 Figure 5. Change of the Aral Sea area from 1987 to 2019: (a) the spatial distribution; (b) th 320 temporal distribution. 321 Likewise, the water level of the Aral Sea tended to decrease. The curve of the water leve 322 the North Aral significantly ascended as impacted by the construction of the dam (Figure 6). 323 water level of the North Aral was elevated near 0.32 m per year from 2003 to 2009. In the E 324 Aral, the water level drastically declined at 0.5 m per year. Moreover, the East Aral was only 0 287 3.2. Temporal and Spatial Variations of Farmland 298 Figure 4. Year of abandoned farmland in the Aral Sea Region with two areas shown in detail: (a) 299 Around the Syr Darya River; (b) Around the Amu Darya River. 300 Manuscript to be reviewed 325 m in 2019. Compared with the East Aral, declining West Aral was lower as impacted by its smaller 326 range and farther from the irrigated area. To be specific, the water level of the West Aral decreased 327 at 0.4 m per year and dropped to 2.1 m in 2019. 328 329 Figure 6. Change of the Aral Sea water level from 2003 to 2009. 330 Figure 7 illustrates the water mass change of the GRACE analysis in the study area. The curve 331 of equivalent water height shows a downward trend from 2002 to 2016. The change of equivalent 332 water height is same as the water level. The water mass was overall declining, marking a decrease 333 by 0.97 km3 each year. The decrease in water mass represented the descending of water storage, 334 such as the Aral Sea, rivers, reservoirs and groundwater. 335 336 Figure 7. Mass change of the Aral Sea Region from 2002 to 2016. 337 3.4. Construction of Water-Farmland Coupling Degree Model 338 According to the results of this study regarding water and farmland in the Aral Sea Region, 339 three indexes were selected to build a comprehensive evaluation index system (Table S7). This 340 system included two subsystems (surface water system, farmland system). Water subsystem had 341 two evaluation indexes, and farmland subsystem had one evaluation index (Table S7). 342 The entropy method was adopted to calculate the weight of each index. According to the 343 mentioned weights, the coupling degree was obtained between the two subsystems from 2003 to 344 2009. In addition, the results indicated that the annual coupling degree value was above 70%, and 345 the average coupling degree reached 0.903 (Table S8). By the criterion of coupling degree (Table 346 S9) (Chang, 2018), only 2003 and 2005 pertain to intermediate level coupling coordination. Other 347 years belong to high-quality coupling coordination. In addition, the average coupling degree 348 belongs to the high-quality coupling coordination. The mentioned coupling degree results 349 suggested that the greater coupling degree between the two subsystems, and farmland changes 350 affected the variations of surface water in the Aral Sea Region. 351 Table S7. Comprehensive evaluation index system of water and farmland coupling 352 degree. 353 354 355 Table S8. Weight of every index, coupling degree value of every year and average 356 coupling degree value. 357 358 Table S9. Table S6. Area of Abandoned Farmland in the Aral Sea Region 319 Figure 5. Change of the Aral Sea area from 1987 to 2019: (a) the spatial distribution; (b) the 320 temporal distribution. 321 Likewise, the water level of the Aral Sea tended to decrease. The curve of the water level of 322 the North Aral significantly ascended as impacted by the construction of the dam (Figure 6). The 323 water level of the North Aral was elevated near 0.32 m per year from 2003 to 2009. In the East 324 Aral, the water level drastically declined at 0.5 m per year. Moreover, the East Aral was only 0.05 PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Manuscript to be reviewed Criteria for Coupling Coordination Type 359 360 Discussion 325 m in 2019. Compared with the East Aral, declining West Aral was lower as impacted by its smaller 326 range and farther from the irrigated area. To be specific, the water level of the West Aral decreased 327 at 0.4 m per year and dropped to 2.1 m in 2019. Figure 6. Change of the Aral Sea water level from 2003 to 2009. Manuscript to be reviewed According to the results 384 section 3.2, we extracted the area of abandoned farmland (Table S6). The area of abandon 385 farmland reached per year 97.843 km2. And the abandoned farmland will cause a lot of wat 386 waste. Farmland in Aral Sea Region is concentrated on planting cotton, wheat and ri 387 (Chapagainet al., 2005). Table S10 shows that wheat is the thirstiest crop. And the water u 388 efficiency of cotton is the lowest (0.451108kg/km3). Table S11 shows the crop production 389 precious decades. 390 391 Table S10. Crop Water use efficiency in Aral Sea Region. 392 393 Table S11. Crop production in the Aral Sea Region. 394 395 Based on the above results, the loss volume of water loss by abandoned farmland can 396 extracted. If all the abandoned farmland were used to plant cotton in the past few years, the wat 397 loss would reach 0.028 km3 every year (Table S12). According to the current distribution 398 available water per capita from the World Bank (Uzbekistan, 2596 m3/ per person / annuall P J i i PDF \| (2021 07 63710 3 0 NEW 19 J 2022) 370 The coupling degree model was used for the first time to monitor the effect of farmland on 371 the surface water of the Aral Sea Region. Most previous researches regarded farmland and surface 372 water as independent objects, which could not acquire the natural connection between them. 373 Therefore, changes in farmland and surface water were put into a quantifiable system that can be 374 used to explore the substantial impact of farmland changes on the surface water. According to the 375 result, the coupling degree model showed good applicability in this field. In addition, it could 376 provide a new method for the internal connection between farmland, surface water, and ecology 377 in the Aral Sea Region and even the Aral Sea Basin. 378 Figure 7. Mass change of the Aral Sea Region from 2002 to 2016. Figure 7. Mass change of the Aral Sea Region from 2002 to 2016. Manuscript to be reviewed Manuscript to be reviewed 361 4.1. Benefits of Methods 362 The results shown that, the loss of water by abandoned farmland will increase the burden of 363 normal water use. During the past years, many scholars were committed to analyzing the reasons 364 for the decrease of surface water in the Aral Sea Region. Compared with previous researches, our 365 work has similarities and differences. For similarities, the surface water loss was caused by 366 improper irrigation management. On the contrary, our research was from the perspective of 367 farmland abandonment. Few studies have analyzed the loss of water caused by abandoned 368 farmland. They were analyzed effect of land water storage on surface water (Jin et al. 2017; 369 Gaybullaev et al, 2012; Parajuli et al. 2017). 365 work has similarities and differences. For similarities, the surface water loss was caused b 366 improper irrigation management. On the contrary, our research was from the perspective 367 farmland abandonment. Few studies have analyzed the loss of water caused by abandon 368 farmland. They were analyzed effect of land water storage on surface water (Jin et al. 201 369 Gaybullaev et al, 2012; Parajuli et al. 2017). 370 The coupling degree model was used for the first time to monitor the effect of farmland o 371 the surface water of the Aral Sea Region. Most previous researches regarded farmland and surfa 372 water as independent objects, which could not acquire the natural connection between them 373 Therefore, changes in farmland and surface water were put into a quantifiable system that can 374 used to explore the substantial impact of farmland changes on the surface water. According to t 375 result, the coupling degree model showed good applicability in this field. In addition, it cou 376 provide a new method for the internal connection between farmland, surface water, and ecolog 377 in the Aral Sea Region and even the Aral Sea Basin. 378 379 4.2. Effect of Abandoned Farmland on Surface Water in Aral Sea Region 380 The abandoned farmland will cause a lot of surface water loss. The main reason is th 381 irrigation canals of abandoned farmland are not blocked because of the negligence of managemen 382 It directly led to majority of the water was being soaked up by the desert and blatantly lo 383 (Ibragimov et al, 2007; Maksud et al, 2016; Jabbarov et al, 2013). 379 4.2. Effect of Abandoned Farmland on Surface Water in Aral Sea Region 379 4.2. Effect of Abandoned Farmland on Surface Water in Aral Sea Region 380 Th b d d f l d ill l f f l Th 380 The abandoned farmland will cause a lot of surface water loss. The main reason is that 381 irrigation canals of abandoned farmland are not blocked because of the negligence of management. 382 It directly led to majority of the water was being soaked up by the desert and blatantly loss 383 (Ibragimov et al, 2007; Maksud et al, 2016; Jabbarov et al, 2013). According to the results of 384 section 3.2, we extracted the area of abandoned farmland (Table S6). The area of abandoned 385 farmland reached per year 97.843 km2. And the abandoned farmland will cause a lot of water 386 waste. Farmland in Aral Sea Region is concentrated on planting cotton, wheat and rice 387 (Chapagainet al., 2005). Table S10 shows that wheat is the thirstiest crop. And the water use 388 efficiency of cotton is the lowest (0.451108kg/km3). Table S11 shows the crop production in 389 precious decades. Manuscript to be reviewed 399 Kazakhstan, 1943 m3/ per person / annually), the water loss can provide annual domestic water f 400 tens of thousands of people in the Aral Sea Region. The specific numbers are as follow 401 Kazakhstan are 14410 persons, and Uzbekistan are 10785 persons. Therefore, abandoned farmlan 402 caused additional loss of normal domestic water for people in the Aral Sea Region. 403 404 Table S12. Water Consumption for Planting Cotton in Abandoned Farmland. 405 406 The previous researches gave us much inspiration. Micklin macroscopically analyzed th 407 reason and effect of water changes in the Aral Sea (Micklin, 2010). Yoshihiro et al. found near 408 100% return flows of the applied irrigation water in the Aral Sea Drainage Basin (Yoshihiro et a 409 2007). Cretaux et al. analyzed effect of groundwater on the hydrological water budget of the Ar 410 Sea by remote sensing data (Cretaux et al., 2013). Compared with above researches, ou 411 contributions are as follows: 412 1. Based on the coupling degree model, we quantitatively characterized the effect of 413 farmland changes on surface water. 414 2. The coupling degree model was used for the first time to study the effect of farmland 415 changes on surface water. We believe that this attempt will help broaden the thinking of 416 researchers in the fields of remote sensing, environment and more. They can connect 417 independent systems through the coupling degree model. 418 3. The effect mechanism was analyzed on surface water changes by calculating the amount 419 of water loss in abandoned farmland. Moreover, the normal domestic water for people 420 caused by water loss was analyzed. 421 422 4.3. Limitations of Research 423 At first, vegetation indices can reduce other factors’ impacts in monitoring land cover chang 424 (Zhang et al., 2013). The NDVI can reduce the influence of external environmental factors on th 425 surface reflectance to some extent. Previous studies indicated that the accuracy of NDVI 426 detecting farmland is better than others (Tong et al., 2017). However, due to the disturbance b 427 other vegetation in the arid region, using NDVI to the LandTrendr algorithm has some limitation 428 (Watts et al., 2014). A typical example is that the sparse vegetation of the Aral Sea Regio 429 generated the same NDVI signal with farmland and affected the detection results. Therefore, it 430 necessary to add additional indices to improve accuracy. Table S11. Crop production in the Aral Sea Region. 395 Based on the above results, the loss volume of water loss by abandoned farmland can be 396 extracted. If all the abandoned farmland were used to plant cotton in the past few years, the water 397 loss would reach 0.028 km3 every year (Table S12). According to the current distribution of 398 available water per capita from the World Bank (Uzbekistan, 2596 m3/ per person / annually; PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) 422 4.3. Limitations of Research 432 However, this fusion of data yet offers valuable and unique information on the surface water. 433 Simultaneously, in the next step of our research, we need to improve data integrity of the surface 434 water. 431 Secondly, the data on water level and water storage cannot match the water area s time series. 432 However, this fusion of data yet offers valuable and unique information on the surface water. 433 Simultaneously, in the next step of our research, we need to improve data integrity of the surface 434 water. 432 However, this fusion of data yet offers valuable and unique information on the surface water. 433 Simultaneously, in the next step of our research, we need to improve data integrity of the surface 434 water. 433 Simultaneously, in the next step of our research, we need to improve data integrity of the surface 434 water. Manuscript to be reviewed 431 Secondly, the data on water level and water storage cannot match the water area’s time serie 432 However, this fusion of data yet offers valuable and unique information on the surface wate 433 Simultaneously, in the next step of our research, we need to improve data integrity of the surfac 434 water. 435 Conclusions 436 In this study, the farmland variations were detected by using LandTrendr algorithm based o 437 the GEE platform. Subsequently, multi-source data (Landsat, ICESat, GRACE) were employed Table S12. Water Consumption for Planting Cotton in Abandoned Farmland. 2 1. Based on the coupling degree model, we quantitatively characterized the effect of 3 farmland changes on surface water. 14 2. The coupling degree model was used for the first time to study the effect of farmland 15 changes on surface water. We believe that this attempt will help broaden the thinking of 16 researchers in the fields of remote sensing, environment and more. They can connect 17 independent systems through the coupling degree model. 417 independent systems through the coupling degree model. 418 3. The effect mechanism was analyzed on surface water changes by calculating the amount 419 of water loss in abandoned farmland. Moreover, the normal domestic water for people 420 caused by water loss was analyzed. 421 418 3. The effect mechanism was analyzed on surface water changes by calculating the amount 419 of water loss in abandoned farmland. Moreover, the normal domestic water for people 420 caused by water loss was analyzed. 422 4.3. Limitations of Research 423 At first, vegetation indices can reduce other factors’ impacts in monitoring land cover change 424 (Zhang et al., 2013). The NDVI can reduce the influence of external environmental factors on the 425 surface reflectance to some extent. Previous studies indicated that the accuracy of NDVI in 426 detecting farmland is better than others (Tong et al., 2017). However, due to the disturbance by 427 other vegetation in the arid region, using NDVI to the LandTrendr algorithm has some limitations 428 (Watts et al., 2014). A typical example is that the sparse vegetation of the Aral Sea Region 429 generated the same NDVI signal with farmland and affected the detection results. Therefore, it is 430 necessary to add additional indices to improve accuracy. 423 At first, vegetation indices can reduce other factors’ impacts in monitoring land cover change 424 (Zhang et al., 2013). The NDVI can reduce the influence of external environmental factors on the 425 surface reflectance to some extent. Previous studies indicated that the accuracy of NDVI in 426 detecting farmland is better than others (Tong et al., 2017). However, due to the disturbance by 427 other vegetation in the arid region, using NDVI to the LandTrendr algorithm has some limitations 428 (Watts et al., 2014). A typical example is that the sparse vegetation of the Aral Sea Region 429 generated the same NDVI signal with farmland and affected the detection results. Therefore, it is 430 necessary to add additional indices to improve accuracy. 423 At first, vegetation indices can reduce other factors’ impacts in monitoring land cover change 424 (Zhang et al., 2013). The NDVI can reduce the influence of external environmental factors on the 425 surface reflectance to some extent. Previous studies indicated that the accuracy of NDVI in 426 detecting farmland is better than others (Tong et al., 2017). However, due to the disturbance by 427 other vegetation in the arid region, using NDVI to the LandTrendr algorithm has some limitations 428 (Watts et al., 2014). A typical example is that the sparse vegetation of the Aral Sea Region 429 generated the same NDVI signal with farmland and affected the detection results. Therefore, it is 430 necessary to add additional indices to improve accuracy. 431 Secondly, the data on water level and water storage cannot match the water area s time series. Manuscript to be reviewed 438 extract the variations of water area, water level, water storage in the Aral Sea Region. Given the 439 mentioned results, the Water-Farmland assessment was developed. Such a system assessed the 440 impact of farmland on surface water in the Aral Sea Region. The specific results are elucidated 441 below: 442 Throughout the research, the abandoned farmland areas covered 98 km2 per year. The overall 443 accuracy of abandon took up 85.3%. There was most abandoned farmland near the rivers (the Amu 444 Darya River and the Syr Darya River) from 1987 to 2019 of the Aral Sea Region (Section 3.2). 445 The abandon of farmland had triggered a massive loss of water resources of the Aral Sea Region. 446 Second, the Aral Sea shrank by 1606.36 km2 per year from 1987 to 2019, in which the East 447 Aral shrank most seriously. Furthermore, the Aral Sea’s water level was down-regulated by 0.13 448 m per year from 2003 to 2009, especially the East Aral. However, as impacted by the construction, 449 Dike Kokaral dam in 2005, the water level and area curves in the North Aral started to elevate 450 after 2005. Moreover, the amount of water storage in the Aral Sea Region declined from 2002 to 451 2016. 452 The coupling degree model overall considers the variations in surface water and farmland. In 453 addition, the average coupling degree value exhibits high-quality coupling coordination, 454 demonstrating that farmland changes significantly impact the surface water. Meanwhile, the 455 volume of water loss by abandoned farmland caused additional loss of normal domestic water for 456 people. Such a method can be referenced to investigate the effect of farmland on surface water or 457 other fields. Moreover, the mentioned results are capable of helping relevant researchers gain 458 insights into the effect of variations in farmland on surface water deterioration in this region, and 459 offer sufficient support for subsequent governance and research. 435 Conclusions 436 In this study, the farmland variations were detected by using LandTrendr algorithm based on 437 the GEE platform. Subsequently, multi-source data (Landsat, ICESat, GRACE) were employed to 436 In this study, the farmland variations were detected by using LandTrendr algorithm based on 437 the GEE platform. Subsequently, multi-source data (Landsat, ICESat, GRACE) were employed to 36 In this study, the farmland variations were detected by using LandTrendr 437 the GEE platform. Subsequently, multi-source data (Landsat, ICESat, GRA PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) 463 References 464 Annina, S., Tobias, B., Markus, S., Olga, S. and Martin, B. 2012. Climate change impacts on 465 glaciers and runoff in Tien Shan (Central Asia). Nature Climate Change, 2(10), 725–731. 466 doi:10.1038/nclimate1592 464 Annina, S., Tobias, B., Markus, S., Olga, S. and Martin, B. 2012. Climate c 465 glaciers and runoff in Tien Shan (Central Asia). Nature Climate Change, 2 466 doi:10.1038/nclimate1592 467 Breiman, L. 2001. Random Forests. Machine Learning, 45(1), 5–32. 468 doi:10.1023/A:1010933404324 467 Breiman, L. 2001. Random Forests. Machine Learning, 45(1), 5–32. 468 doi:10.1023/A:1010933404324 469 Chang, Y. M. 2018. 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Long Term Monitoring of 606 Cropland Change near Dong ting Lake, China, Using the LandTrendr Algorithm with Landsat 607 Imagery. Remote Sensing, 11(10), 1234. doi: 10.3390/rs11101234 608 Zhu, Z. and Woodcock, C. E. 2012. Object-based cloud and cloud shadow detection in Landsat 609 imagery. Remote Sensing of Environment, 118, 83–94. doi: 10.1016/j.rse.2011.10.028 610 Zwally, H. J., Yi, D. H., Kwok, R. and Zhao, Y. H. 2008. ICESat measurements of sea ice 611 freeboard and estimates of sea ice thickness in the Weddell Sea, Journal of Geophysical 612 Research, 113(C2), C02S15, doi:10.1029/2007jc004284 610 Zwally, H. J., Yi, D. H., Kwok, R. and Zhao, Y. H. 2008. Manuscript to be reviewed Yuan, C. Boening, and F. W. Landerer. 2015. Improved 584 methods for observing Earth’s time variable mass distribution with GRACE using spherical cap 585 mascons. Journal of Geophysical Research-Solid Earth, 120, doi:10.1002/2014JB011547. 586 Watts, L.M. and Laffan, S.W. 2014. Effectiveness of the BFAST algorithm for detecting 587 vegetation response patterns in a semi-arid region. Remote Sensing of Environment, 154, 234– 588 245. doi: 10.1016/j.rse.2014.08.023 589 Wiese, D. N., F. W. Landerer, and M. M. Watkins. 2016. Quantifying and reducing leakage 590 errors in the JPL RL05M GRACE mascon solution, Water Resources Research, 52, 7490–7502, 591 doi:10.1002/2016WR019344. 592 Wu, T., Sang, S., Wang, S., and Yang, Y. Y. 2020. Remote sensing assessment and 593 spatiotemporal variations analysis of ecological carrying capacity in the Aral Sea Basin. Science 594 of the Total Environment 735 139562 doi: 10 1016/j scitotenv 2020 139562 592 Wu, T., Sang, S., Wang, S., and Yang, Y. Y. 2020. Remote sensing assessment and 593 spatiotemporal variations analysis of ecological carrying capacity in the Aral Sea Basin. Science 594 of the Total Environment, 735,139562. doi: 10.1016/j.scitotenv.2020.139562 592 Wu, T., Sang, S., Wang, S., and Yang, Y. Y. 2020. Remote sensing assessment and 593 spatiotemporal variations analysis of ecological carrying capacity in the Ar 594 of the Total Environment, 735,139562. doi: 10.1016/j.scitotenv.2020.139562 PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Research scope Research scope Manuscript to be reviewed ICESat measurements of sea ice 611 freeboard and estimates of sea ice thickness in the Weddell Sea, Journal of Geophysical 612 Research 113(C2) C02S15 doi:10 1029/2007jc004284 y, , , , , , 611 freeboard and estimates of sea ice thickness in the Weddell Sea, Journal of Geophysical 612 R h 113(C2) C02S15 d i 10 1029/2007j 004284 612 Research, 113(C2), C02S15, doi:10.1029/2007jc004284 PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Manuscript to be reviewed Figure 2 Research Method PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Figure 3 Examples areas of water change and farmland change near the Syr Darya and the Amu Darya. Figures a1 and a2 represent the water change years 2009 and 2019; Figures b1, b2, b3 represent the water change years 2009, 2016 and 2019 Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Manuscript to be reviewed Manuscript to be reviewed Figure 4 Year of abandoned farmland in the Aral Sea Region with two areas shown in detail Year of abandoned farmland in the Aral Sea Region with two areas shown in detail PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Change of the Aral Sea area from 1987 to 2019 PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Manuscript to be reviewed Figure 6 Figure 6 Change of the Aral Sea water level from 2003 to 2009 Change of the Aral Sea water level from 2003 to 2009 Manuscript to be reviewed PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) Figure 7 Figure 7 Mass change of the Aral Sea Region from 2002 to 2016 Mass change of the Aral Sea Region from 2002 to 2016 PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022) PeerJ reviewing PDF \| (2021:07:63710:3:0:NEW 19 Jan 2022)
https://openalex.org/W1989164068	https://www.scielo.br/j/sausoc/a/czK9mTJkHjvGT73t3S8s38p/?lang=pt&format=pdf	Portuguese	null	O "Sonho de Rose": políticas de saúde pública em assentamentos rurais	Saúde e Sociedade	2,006	cc-by	10,783	Rose’s Dream: public health policies in rural land settlements Diego Soares Mestre em Antropologia Social (PPGAS – UFRGS) Pesquisador Associado ao Núcleo de Antropologia e Cidadania (UFRGS) E-mail: soares2br@yahoo.com.br Diego Soares Mestre em Antropologia Social (PPGAS – UFRGS) Pesquisador Associado ao Núcleo de Antropologia e Cidadania (UFRGS) E-mail: soares2br@yahoo.com.br Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 57 O “Sonho de Rose”: políticas de saúde pública em assentamentos rurais Rose’s Dream: public health policies in rural land settlements Abstract In this paper, there is a reflection on the tension between the principle of universality and the cultural specificity of users of the Public Health system, inserting the discussion about collective health in rural land settlements in this axis. After presenting some statistical data about health conditions in ru- ral land settlements, ethnographic data on a project of university extension accomplished in a rural settlement located in the metropolitan area of Porto Alegre (RS) is presented. A dialogic model of interven- tion in the field of public health, respecting cultural differences and including alternative therapeutic practices to the biomedical model is discussed. As conclusion, some reflections are made on the different uses of the institutional devices starting from an opposition between the analysis of Foucault about the technologies of power and the critical observations introduced by Michel de Certeau, more concerned with analyzing the uses of the devices, the arts of doing that subvert the models of discipline. A preocupação com as condições de saúde das popu- lações, a efetivação de práticas e a emergência de ins- tituições e saberes sobre doença, o risco e a sua relação com a organização social e econômica das sociedades, pode ser descrita a partir de duas perspectivas com- plementares: de um lado, como tecnologias discipli- nares que agem sobre o corpo dos indivíduos (forças de produção), tendo como objetivo a promoção da saú- de do corpo social (conceito de biopolítica, de Michel Foucault); de outro, como o resultado de um conjunto de lutas sociais e táticas que acompanham o uso dos dispositivos institucionais (lutas pela conquista do direito à saúde, incluindo táticas de implementação de práticas terapêuticas alternativas ao modelo biomédico). Essas abordagens estão presentes nas discussões sobre a formação, no Brasil, de um siste- ma de seguridade social e, por força de extensão, das políticas de saúde pública: por um lado, a seguridade social seria o resultado da conquista de direitos soci- ais pelos assalariados; por outro, uma intervenção do Estado, tendo em vista a produção das condições ne- cessárias para o desdobramento da ordem social ca- pitalista (Noronha, 2005, p.33). Keywords: Public Health Policies; Social exclusion; Land Settlements; Traditional Knowledge; Land reform. Neste artigo, não se pretende negligenciar ne- nhum desses dois aspectos, pois, entende-se que uma política pública é, ao mesmo tempo, um conjunto de técnicas de intervenção e um campo de luta política. Abstract O objetivo aqui é desenvolver uma reflexão a partir desse pressuposto inicial, o que certamente influen- ciará na forma como será conduzida a discussão so- bre o tema da exclusão social e as políticas públicas na área de saúde. Resumo Este artigo partiu de uma breve reflexão sobre a ten- são entre os princípios de universalidade e a especifi- cidade cultural dos usuários das políticas de saúde pública, procurando inserir a discussão sobre saúde coletiva em assentamentos rurais dentro deste eixo reflexivo. Apresenta dados etnográficos sobre um pro- jeto de extensão universitária realizado no assenta- mento “19 de Setembro”, localizado nas imediações da região metropolitana de Porto Alegre (RS). A partir da experiência de implementação de uma “farmacopéia popular” nesse assentamento, discute-se um modelo dialógico de intervenção no campo da saúde pública, um modelo esteja mais apropriado ao diálogo com a alteridade e com a inclusão de práticas terapêuticas alternativas ao modelo biomédico. Para finalizar, de- senvolve algumas reflexões iniciais sobre os usos di- ferenciados dos dispositivos institucionais a partir de uma contraposição entre a análise de Foucault sobre as tecnologias de poder e as observações críticas intro- duzidas por Michel de Certeau, o qual analisa os usos dos dispositivos, as artes de fazer que subvertem os modelos disciplinares. Palavras-chaves: Políticas de saúde pública; Exclusão social; Assentamentos rurais; Saberes tradicionais; Reforma agrária. Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 57 58 Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 Como Pensar a Saúde Pública em Assentamentos Rurais regula as ações e serviços de saúde1, sendo uma das principais diretrizes da política nacional de seguri- dade social. Ao mesmo tempo, os princípios de parti- cipação da comunidade e descentralização adminis- trativa – regionalização e municipalização dos servi- ços – apontam para a necessidade de se administrar o sistema levando em conta as especificidades sociais, econômicas e culturais das diferentes regiões do país. Apesar de a luta no campo da reforma agrária tam- bém se fazer presente na produção e na interpretação dos dados estatísticos, existem algumas fontes confiáveis que podem fornecer um quadro geral, como é o caso do I Censo de Reforma Agrária do Brasil (Schmidt, 1998), que traz dados sobre assentamentos criados até 1996, abrangendo diferentes aspectos so- ciais e econômicos (até na área da saúde pública); e o extraordinário trabalho que vem sendo desenvolvido pelo Banco de Dados da Luta pela Terra (Dataluta, 2004), que traz estatísticas sobre número de assenta- mentos por região e ano, abrangendo um período que vai de 1942 a 2004. São essas as fontes que serão usa- das aqui para fornecer um quadro geral sobre os as- sentamentos rurais, procurando abordar brevemente algumas variáveis importantes. A mesma tensão entre o universal e o específico é expressa a partir de outras contradições que perpas- sam as políticas públicas na área de saúde: conflito entre a objetividade científica do modelo biomédico e a subjetividade fenomenológica do paciente e do terapeuta; entre diretrizes provenientes de modelos normativos que abordam a doença, mas não o doente, e a necessidade de levar em conta as particularidades próprias a cada sujeito (a sua cultura, o seu modo de vida); o conflito entre o ser humano (universal, indi- víduo biológico) percebido como objeto material de ciência e fechado em um quadro de valores normativos ou até mesmo considerado como mero consumidor de serviços (usuário), e o sujeito singular que emerge em um ambiente histórico, social, ecológico e cultural específico (Paul, 2005, p.73-75). A ação do modelo bio- médico que disciplina os corpos buscando a maximi- zação da sua utilidade produtiva não atua em um cam- po vazio de sentidos, mas entra em relação (conflituo- sa ou não) com um sujeito que escapa do seu olhar ordenador, dos seus sistemas de classificação. Isso ocorre porque o corpo não é apenas um corpo biológi- co, mas um corpo humanizado pela cultura. Políticas de Saúde Pública: entre o universal e o específico A partir da Constituição de 1988, a saúde passou a ser “direito de todos e dever do Estado, garantido me- diante políticas sociais e econômicas que visem à re- dução do risco de doença” (Brasil, 1988). A universa- lidade do acesso aos serviços públicos de saúde em todos os níveis de assistência está inscrita na lei que 1 Lei Nº. 8.080, de 19 de Setembro de 1990. Como Pensar a Saúde Pública em Assentamentos Rurais Os dados do censo foram produzidos a partir de uma amostra do número total de assentamentos cria- dos pelo Incra entre 1964 e 1996. É importante lem- brar que nem todos os assentamentos foram criados pelo governo federal, pois uma parte deles foi institu- ída pelos governos estaduais, principalmente duran- te o Governo Collor, quando houve uma estagnação da política nacional de reforma agrária e um aumen- to considerável das ações políticas no campo (Navarro e col., 1999). Os dados já estão um pouco desatualiza- dos, pois não levam em conta os assentamentos insti- tuídos a partir da segunda metade da década de 1990, no entanto, podemos superar essas limitações se es- tivermos dispostos a fazer uma leitura pouco preten- siosa desses índices. Ao invés de abordarmos os da- dos como um retrato fiel da realidade, vamos abordá- los como indicadores de um conjunto de elementos que devem ser pensados quando nos propomos abor- dar o problema da saúde pública nos assentamentos rurais. Um dos pontos positivos do censo é que os da- dos são organizados tendo em vista as diferenças re- gionais, o que já é uma grande conquista, pois permi- te analisar os itens de forma comparativa. Foi dentro deste debate que se inseriu toda a dis- cussão sobre políticas de saúde voltadas para os seto- res mais pobres da população urbana, assim como pa- ra as sociedades indígenas, comunidades negras (rema- nescentes de quilombo) e, ultimamente, para a popu- lação do meio rural. Como essas discussões abrangem as diferentes concepções de doença, risco e práticas de precaução vigentes numa sociedade complexa e heterogênea como a brasileira, também é uma boa forma de abordar a discussão sobre tecnologias de intervenção e lutas sociais. Este será o nosso ponto de partida para pensar as políticas públicas de saúde voltadas para o atendimento da demanda das popula- ções que vivem em assentamentos rurais. A idéia aqui é apresentar dados do censo que são pertinentes ao desenvolvimento do tema deste arti- Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 59 viços na área de saúde está disponível nos municípi- os e não dentro dos assentamentos, o que implica, por um lado, na necessidade de se negociar com o poder público local formas de acesso aos serviços já exis- tentes e, por outro lado, a necessidade de ampliação desses serviços tendo em vista um aumento da deman- da local. Como Pensar a Saúde Pública em Assentamentos Rurais Ainda na área de saúde, o estudo indica que as doenças mais comuns são: gripe (32%), verminose (14%) e diarréia (9,9%). Esse índice prevalece em to- das as regiões, com uma pequena alteração na região Norte, onde a malária é muito comum (15,46%) (Schmidt, p. 43). Essas doenças geralmente estão as- sociadas a más condições hidrossanitárias e ausên- cia de um sistema de saneamento adequado. go. Levando em conta que saúde pública não diz res- peito apenas ao atendimento médico e ao tratamento das doenças em nível individual, procuro interpretar alguns dados sobre condições hidrossanitárias, mo- radia, fonte de água e de energia utilizadas e, por últi- mo, a existência e a qualidade dos serviços de saúde nos assentamentos rurais. No que se refere à fonte de água utilizada nos as- sentamentos, o censo aponta que o quadro nacional se compõe de três tipos predominantes de fontes for- necedoras de água, o poço (46,07%), o riacho (14,17%) e a cacimba (13,02%), não sendo muito significativa a variação entre um estado e outro. As condições hidros- sanitárias, no geral, são precárias, sendo que uma parte significativa não respondeu à pergunta dos pes- quisadores, o que indica a ausência de qualquer uma das opções oferecidas no questionário na época em que os dados foram coletados. Conforme as conclusões do estudo, um grande número de assentados não tem acesso às condições mínimas de segurança hidros- sanitária, principalmente no norte e no nordeste do Brasil, onde o acesso à água encanada é menor do que nas demais regiões do país (Schmidt, 1998, p.39). Os dados apresentados pelo censo estão desatua- lizados, mas nos fornecem um quadro geral da situa- ção dos assentamentos implementados até a segun- da metade da década de 1990. Ao abordar questões tão complexas do ponto de vista quantitativo, perde-se, certamente, o sabor local dos dilemas enfrentados no dia-a-dia. Mais do que isso, uma análise como esta não consegue abordar o saber e as estratégias colocadas em práticas pelos próprios assentados para enfren- tar suas dificuldades na área de saúde. Uma visão ge- ral não consegue identificar os casos bem-sucedidos, pois a finalidade do censo é apontar tendências, su- gerir hipóteses que possam alimentar estudos quali- tativos, sejam eles etnográficos ou não. As pesquisas qualitativas nessa área são poucas ou quase inexis- tentes, a maioria dos estudos aborda esse tema como um dado complementar. 2 Habitações de madeira predominam no Centro-Oeste (49, 17%), Norte (50,89%) e Sul (73,46%). No Nordeste predomina a moradia de taipa (45,35%); e no Sudeste a de alvenaria (65,38%) (Schmidt, p. 37). 3 Energia elétrica: Sudeste (49,9%) e Sul (47, 9%); querosene e óleo diesel: Norte (64,3%), Centro-Oeste (50,7%) e Nordeste (48%) (Schmidt, p.41). 60 Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 3 Energia elétrica: Sudeste (49,9%) e Sul (47, 9%); querosene e óleo diesel: Norte (64,3%), Centro-Oeste (50,7%) e N p.41). Como Pensar a Saúde Pública em Assentamentos Rurais Muitos seriam os avanços se fosse possível relacionar dados mais abrangentes, como os que foram comentados aqui, com estudos de caso sobre estratégias locais, sejam elas promovidas pelo governo ou pelos próprios assentados. Os dados do censo apontam que as condições de habitação são melhores, com um baixo índice de as- sentados ainda vivendo em barracas de lona ou habi- tações provisórias. As moradias são predominante- mente de: madeira (31,90%), taipa (28,20%) e alvenaria (22,99%), com pouca diferenciação regional2. No que se refere às fontes de energia, existem diferenças re- gionais significativas: enquanto no Sul e no Sudeste prevalece o uso da energia elétrica, nas demais regiões é mais freqüente o uso de querosene e óleo diesel3. A área de saúde também foi avaliada em termos da existência e da qualidade de serviços básicos, como hospital, posto de saúde e agentes de saúde, tanto nos assentamentos como nos seus respectivos municípi- os. Esses dados foram prejudicados por um alto índi- ce de beneficiários que não responderam, mas, em geral, a avaliação foi negativa, sendo o difícil acesso aos hospitais mencionado como a carência maior. Ao mesmo tempo, o censo sugere que a maioria dos ser- O desenvolvimento de projetos na área de saúde voltados para a realidade dos assentamentos deve le- var em conta a diversidade de situações, tanto no que se refere às características socioculturais da popula- ção, mas também no que se refere às diferenças am- bientais, que mudam muito entre uma região e outra. Por outro lado, o Banco de Dados da Luta pela Terra tor A” e “setor B”. No “setor B”, está localizada a agro- vila, onde vive a maior parte das famílias e cujo aces- so é dado por uma estrada de chão, que perpassa as terras dos assentados. Foi nesse setor que as famílias se estabeleceram de forma definitiva, após uma ten- tativa infeliz de habitação no “setor A”, onde teve iní- cio o processo de humanização do território. Essa pri- meira migração do grupo ocorreu cinco meses após a chegada das famílias, quando a primeira agrovila, for- mada ainda por barracos de lona, foi alagada devido às chuvas de inverno. apresenta números significativos: entre 1942 e 2004, foram instituídos mais de seis mil assentamentos, abrangendo uma população de 800 mil famílias e uma área de quase 35 milhões de hectares. 4 A área ocupada pelo 19 de Setembro está mais precisamente localizada no km 297 da BR-116. 5 Essa diferença entre número de famílias e número de lotes ocorre porque muitas dessas famílias são extensas, compostas, muitas vezes, de mais de um beneficiário. 6 Conforme os dados fornecidos pela Secretaria de Planejamento da Prefeitura Municipal de Guaíba, a população total da cidade é de 100 mil habitantes, dos quais 40% são residentes nos bairros Cohab e Santa Rita. 7 A definição de zona rural assume aqui o sentido de área destinada à exploração extrativa agrícola, pecuária ou agroindustrial. Como Pensar a Saúde Pública em Assentamentos Rurais A maior parte desses assentamentos foi implementada na segunda metade da década de 1990 e está localizada em todas as regiões do Brasil. Também foi nesse período que ocorreu um aumento considerável das ocupações de terra e outras mobilizações políticas no campo da re- forma agrária. O que se percebe, portanto, é que o nú- mero de assentamentos rurais é significativo, e que a maioria foi implementada como resposta à pressão que os movimentos sociais vêm exercendo sobre o po- der público. Sabemos que o perfil da população envol- vida nessas mobilizações tem mudado consideravel- mente, principalmente em estados como São Paulo e Rio de Janeiro, onde a maioria é proveniente das gran- des cidades. No entanto, também não resta dúvida de que se trata de pessoas que se encontram numa situa- ção de pobreza, vivendo em condições de saúde coleti- va inadequadas, como é o caso dos assentados que são provenientes das favelas urbanas. A atual agrovila fica em uma região mais afasta- da do perímetro urbano, onde as terras são mais altas e as inundações menos freqüentes, constituindo-se um aglomerado de casas de alvenaria e madeira, dis- tribuídas em forma de semicírculo, tendo no seu inte- rior uma praça onde está localizada a “associação”. É ali que ocorrem as assembléias gerais, as reuniões da coordenação e do conselho comunitário, assim como as cerimônias religiosas e outras atividades e come- morações especiais. Essa associação também funcio- na, aos finais de semana, como “bar”, freqüentado à noite pelos moradores e por visitantes ocasionais dos bairros vizinhos. A praça central ainda conta com uma área aberta ocupada geralmente pelas crianças, prin- cipalmente na parte da manhã, quando é comum percebê-las correndo de um lado para o outro, subin- do em árvores, praticando esportes e jogos infanto- juvenis. O complexo também tem uma escola primá- ria, que fornece ensino fundamental (até a 4ª série), um campo de futebol e duas canchas de bocha, onde os homens se reúnem para jogar nos finais de semana. 8 As casas de alvenaria são em maior número do que as de madeira, contrariando a média habitacional dos assentamentos da região Sul, onde o material predominante utilizado na construção das habitações é a madeira. Ver Schmidt et al., 1998, p.38. 8 Esses dados indicam que o 19 de Setembro encontra-se, no que se refere às fontes de energia e de água, em uma situação favorável em relação à média da região Sul. Ver Schmidt et al., 1998, p. 84-85. 9 “Cultura de Sequeiro” são as culturas, ou produtos agrícolas, que só podem ser cultivados em solos secos, sem muita umidade (Ex: aipim, feijão, milho, batata-doce etc.). 10 O Cerrito é dividido em partes iguais por todos os beneficiários. 11 UNISOL; SESU/MEC. Relatório de pesquisa: diretrizes para revitalização do espaço sócio-produtivo do Assentamento 19 de Setem- bro em Guaíba, RS - 2ª Etapa, 2001. Porto Alegre: Pró-Reitoria de Extensão da UFRGS, 2001. Paisagem O 19 de Setembro está localizado nas imediações da zona urbana de Guaíba4, uma pequena cidade que fica nos arredores de Porto Alegre, capital do Rio Grande do Sul. São mais de 400 hectares, onde vivem atual- mente vinte e cinco famílias. A área, dividida em 37 lotes5 de onze hectares, está localizada a três quilô- metros dos bairros populares Cohab e Santa Rita6. Devemos considerar, portanto, que apesar de se tra- tar aqui de uma zona rural7, ela se encontra em inter- face com o perímetro urbano. No “setor B”, os terrenos onde se encontram as casas não ficam junto aos lotes, mas fragmentados em parcelas localizadas em diferentes regiões do as- sentamento, formando uma geografia de retalhos que só os próprios proprietários conseguem delimitar com precisão. No entanto, como os terrenos residenciais são grandes, muitas famílias possuem hortas, galpões e pequenos açudes em suas imediações. As terras do assentamento são divididas pela BR- 116, formando dois setores, denominados aqui de “se- O “setor A” fica do outro lado da BR-116, mais pró- ximo da zona urbana. O acesso a esse setor se dá atra- vés de uma estrada de chão, que cruza em frente a oito lotes de terra, distribuídos entre seis famílias. A ocu- pação definitiva desta parte do assentamento ocorreu um pouco mais tarde, em outubro de 1992, quando um grupo de beneficiários migrou da agrovila e se esta- beleceu ali. Nesse setor as casas estão localizadas jun- to aos lotes, possibilitando uma integração maior en- tre a morada e o ambiente de trabalho. revela um aspecto importante do processo de assen- tamento, pois os assentados são provenientes de uma região onde predominam terras bem drenadas, deno- minadas “terras altas”, onde eles plantavam culturas de sequeiro, como milho, aipim, feijão e soja. A mu- dança para o assentamento teve um forte impacto na relação dessas famílias com o trabalho e o meio am- biente, tanto do ponto de vista técnico como simbólico. Conforme o diagnóstico realizado pela equipe de extensão da universidade, os assentados também en- frentam sérios problemas ambientais devido à polui- ção causada pelas lavouras de arroz localizadas nas imediações, que deságuam herbicidas e adubos quí- micos de alta solubilidade no “Canal da Celupa”, sis- tema de drenagem que cruza pelo interior do assenta- mento. Paisagem Outra fonte de poluição tem sua origem nos afluentes domésticos dos núcleos urbanos vizinhos e do próprio assentamento, como substâncias orgâni- cas e detergentes, que também deságuam neste mes- mo canal11. O reservatório de água do “Arroio do Con- de”, regulado por uma barragem com comportas, en- contra-se nas terras do assentamento e está direta- mente interligado com o canal. Este complexo de drenagem abastece as grandes lavouras de arroz lo- calizadas na região e é um fator de conflito constan- te, pois a abertura das comportas da barragem, que são controladas pelos proprietários das terras contí- guas ao 19 de Setembro, ocasiona a inundação de uma área significativa do assentamento, utilizada como re- serva de pastagem para os animais. A grande maioria das casas do 19 de Setembro é de alvenaria, mas ainda persistem algumas residên- cias de madeira, construídas em mutirões realizados ainda nos primeiros anos de assentamento8. Hoje em dia, todas as famílias têm acesso à energia elétrica e à água encanada8. O ambiente interno dessas mora- dias é, com poucas exceções, ornamentado com os sig- nos da sociedade moderna, como os eletrodomésticos tão comuns nas residências urbanas, que convivem de forma harmoniosa com bandeiras e quadros do MST. As terras do 19 de Setembro estão localizadas, em termos geográficos, nas proximidades da planície aluvial do lago Guaíba. Segundo os técnicos da equi- pe de extensão da universidade, em mais de 90% da área predominam solos de várzea que apresentam sé- rios problemas de drenagem. Tendo em vista a quali- dade do solo, a sua vocação natural seria de cultivo do arroz irrigado. No caso de culturas de “sequeiro”9, se- ria necessária a implementação de sistemas de dre- nagem de alto custo econômico. Apenas uma peque- na parcela de 10%, denominada pelos assentados de “Cerrito”, está localizada em uma topografia mais ele- vada que apresenta solos bem drenados, mas de bai- xa fertilidade natural. Essa área é de extrema impor- tância para a subsistência das famílias, sendo desti- nada à produção de culturas de autoconsumo e forra- gem animal10,11 . Esse dado sobre a qualidade do solo O sistema de produção predominante entre os as- sentados é a pecuária de leite associada à horticultura. Juntas, essas duas atividades representam as princi- pais fontes de renda das famílias. 62 Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 Cenário Em 2001, existiam no Rio Grande do Sul duas cente- nas de assentamentos, onde viviam nove mil famíli- as, que juntas ocupavam uma área com mais de 150 mil hectares (Fernandes, 2004). O processo histórico de implementação desses assentamentos pode ser di- vidido em três fases históricas. A primeira delas teve início em 1978 e perdurou até 1984, caracterizando- se pela retomada das lutas sociais no campo. A segun- da fase ocorreu entre 1985 e 1988, período em que foi apresentado o “I Plano Nacional de Reforma Agrária”, marcado também por uma atuação mais intensa do Movimento dos Trabalhadores Rurais Sem Terra (MST). Conforme esta periodização, o 19 de Setembro foi implementado na terceira fase, que teve início no final da década de 1980, caracterizada por um reflu- xo da questão agrária em nível nacional e por um des- locamento geográfico dos assentamentos em direção à região metropolitana de Porto Alegre e ao noroeste do estado (Navarro e col., 1999). As mobilizações no campo aumentaram conside- ravelmente durante o início do Governo Collor (1991), devido ao seu posicionamento passivo em relação à reforma agrária, o que contrariou as expectativas de milhares de sem-terras. O que esses agricultores exi- giam era o reconhecimento, por parte do Estado, do que eles entendiam como um “direito constitucional”, ou seja, um direito de ter a sua própria terra para tra- balhar. Esse momento histórico ainda está vivo na memória das famílias do 19 de Setembro. Durante a pesquisa de campo, muitos deles comentaram que ti- nham “esperança” de que, com a promulgação da nova constituição, eles finalmente teriam os seus “direitos” reconhecidos pelo Estado. No entanto, nada disso ocorreu e, mais uma vez, o destino lhes reservou o gosto amargo do descaso governamental. Os dados sobre as ocupações de terra realizadas pelo MST revelam a intensificação do conflito agrá- rio no estado: se em 1988 foi realizada uma única ocupação, entre 1989 e 1991, o número de ocupações de terra subiu para doze. Essas mobilizações foram acompanhadas de perto pelos fazendeiros e mais de uma vez acabaram na primeira página dos jornais de grande circulação. 12 Novas linhas de financiamentos implementadas pelo Governo Lula. 13 Os dados levantados através da aplicação de questionários demonstram que mais de 90% das famílias produzem leite. Este percentual cai para 70% em relação à horticultura. A produção de arroz, em processo de evolução, já passa dos 30%. Culturas de autoconsumo são produzidas por mais de 70% das famílias. Paisagem Essa produção é complementada por culturas de autoconsumo, como o feijão, a mandioca, o milho e a batata-doce, que so- frem fortes restrições devido à qualidade dos solos. Recentemente, a partir de 2002, algumas famílias têm agrária organizadas por agentes do MST. Esses agen- tes circulavam pelas regiões interioranas do estado em busca de adeptos para os novos acampamentos que estavam sendo organizados. As reuniões eram secre- tas e, na maioria das vezes, realizadas na casa de al- gum simpatizante ou, quando possível, nos sindica- tos de trabalhadores rurais. Nessa época, conforme o relato dos assentados do 19 de Setembro, era comum se ouvir falar sobre peripécias dos colonos que parti- ciparam do acampamento na Encruzilhada Natalino, assim como de suas conquistas em Macali (1979), Bri- lhante (1979) e Annoni (1985). Essas histórias, que cir- culavam através de redes de parentesco e compadrio, acabaram incentivando a entrada na “luta” de muitos sem-terra, pois indicavam a possibilidade concreta de conseguir terra através do ingresso nas ações políti- cas promovidas pelo MST. investido na produção de arroz irrigado. Devido ao sucesso dessas experiências de vanguarda, apoiadas pelo diagnóstico técnico da equipe de extensão, assim como por uma conjuntura política favorável em nível nacional12, boa parte das famílias encontra-se em pro- cesso de elaboração de projetos voltados para a rizicul- tura13. A comercialização do excedente da produção do leite e das hortaliças é realizada pelos próprios as- sentados, diretamente ao consumidor. As entregas são realizadas de carroça, bicicleta, carro ou moto. A “cli- entela”, em alguns casos, inclui mercados, padarias e outros estabelecimentos localizados nos bairros po- pulares da cidade. Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 63 Cenário Foi somente após esse episódio, que teve como resultado dezenas de manifestantes feridos e a morte de um soldado, que o governo esta- dual resolveu atender a parte das reivindicações exi- gidas pelos manifestantes (SJP/RS, 1990; Fernandes, 2000). Esse conflito foi manchete nos principais jor- nais do país, além de ter sido matéria de romances, artigos científicos, teses acadêmicas e revistas popu- lares, como a Isto É e a Veja (Berger, 2003). A primeira conquista foi à visita do secretário de saúde do município, ainda nos primeiras semanas após o estabelecimento das famílias na área destina- da pelo governo estadual. Alguns meses depois, eles conseguiram que a prefeitura distribuísse água com o carro-pipa duas vezes durante a semana e uma re- serva de fichas para atendimento médico no posto de saúde de um bairro popular localizado nas imediações do assentamento. Ainda nos primeiros anos, após muita pressão por parte dos assentados, a prefeitura fez uma estrada de chão ligando a BR-116 e a agrovila, uma escola de ensino básico dentro do assentamento, além de ter contribuído na efetivação de algumas obras iniciais de saneamento básico, pois as terras sofriam inundações constantes durante o inverno, o que causava sérios problemas respiratórios, como bronquites, gripes e outras complicações. A demanda não foi dirigida apenas para o poder público local, pois o sistema de distribuição de água nas residências foi construído com o financiamento de uma Organização Não-Governamental (ONG) inter- nacional, tendo como instituição mediadora a igreja católica do município. As primeiras ações no que se refere à segurança alimentar foram fundamentais para a permanência dos assentados na área. Como o governo não ofereceu nenhum auxílio técnico inicial e esperou quase seis meses pra liberar os primeiros recursos, foi necessário garantir a alimentação de cerca de 60 pessoas, o que não é nada fácil. Os assen- tados foram buscar ajuda no Movimento dos Traba- lhadores Sem Terra (MST), nas organizações sindicais da localidade, igrejas e partidos políticos. Uma ampla rede foi mobilizada, o que permitiu superar o obstá- culo mais difícil e com conseqüências mais graves no processo de implementação de assentamentos de re- forma agrária: a segurança alimentar. Cenário Christa Berger realizou um le- vantamento sobre todos os títulos e manchetes do A década de 1980 foi marcada, com o final da dita- dura militar, por uma conjuntura de abertura política, com o surgimento de novas lutas pelo acesso a terra, moradia e outros direitos civis que culminaram na promulgação, em 1988, da nova Constituição Brasilei- ra (Gohn, 2002). Foi nesse contexto, de ampla mobili- zação popular, que as famílias que vivem no 19 de Se- tembro participaram de discussões sobre reforma Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 63 jornal Zero Hora que fizeram referência ao MST, pu- blicados entre 1990 e 1993. Durante 1990, por exem- plo, as ações do MST foram mencionadas 300 vezes. Esse número subiu para 463, em 1991 (2003, p.147). Muitas dessas notícias referem-se a ocupações que tiveram entre seus protagonistas as famílias que hoje se encontram assentadas em municípios locali- zados nas imediações da região metropolitana de Porto Alegre. ja, tudo o que dizia respeito aos interesses da comu- nidade política. Após a conquista da terra, o acesso aos serviços locais de saúde também passou a fazer parte da sua pauta de reivindicações diante do gover- no municipal. A negociação do acesso foi lenta e re- pleta de conflitos com setores contrários a reforma agrária, mas os assentados também encontraram ali- ados políticos na cidade, o que ajudou a levar adiante suas mobilizações. O nome do assentamento 19 de Setembro foi uma homenagem à data que marca a primeira ocupação realizada pelos assentados, ainda em 1989, junto com outras 1500 famílias14. Trata-se de um marco simbóli- co significativo que marca o início daquilo que eles denominam como a luta, palavra que sintetiza o con- junto de ações históricas das quais eles participaram: ocupações de terra e prédios públicos, longas cami- nhadas pelas rodovias do estado, acampamentos, gre- ves de fome, encontros e atividades internas do movi- mento, conflitos com as “forças ruralistas” e com a Brigada Militar, reuniões com representantes do go- verno estadual e federal, passeatas no centro de Porto Alegre e outras cidades do interior do estado. A situa- ção mais dramática ocorreu em agosto de 1990, quan- do um conflito no centro de Porto Alegre abalou a opi- nião pública gaúcha. 14 A terra ocupada foi uma fazenda localizada na região de Cruz Alta. 64 Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 a foi uma fazenda localizada na região de Cruz Alta. Cenário Foi nesse contexto que o então governador Alceu Collares, através da lei estadual 9.411, editada no dia 5 de novembro de 1991, cedeu à reforma agrária 70% das Estações Experimentais e outras propriedades agricultáveis do Estado que não estivessem tendo um aproveitamento adequado (Rio Grande do Sul, 1991). Entre essas terras estava uma área do Instituto de Previdência do Estado do Rio Grande do Sul (IPERGS), que foi destinada ao assentamento 19 de Setembro. Durante o processo de assentamento, as famílias passaram a valorizar os seus direitos coletivos, ou se- 64 Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 No último ano, o assentamento teve uma deman- da antiga atendida pela prefeitura municipal: a insta- lação de um Posto de Saúde na agrovila, com atendi- mento da equipe médica uma vez por semana. Com este novo recurso, já não é mais necessário deslocar- se até a vila Santa Rita, o entendimento ficou mais rápido e o acesso, facilitado. A inauguração do Posto foi comemorada com uma grande festa, que contou com a presença do representante do governo local, assentados da região, moradores da cidade de Guaíba e lideranças políticas locais. A mobilização por direi- tos coletivos continua, pois as famílias já estão tra- balhando numa nova bandeira de luta: um agente co- munitário de saúde, que possa acompanhar a situa- ção de cada uma das famílias, possibilitando a efeti- vação de um trabalho de prevenção. Comunidade Solidária. Segundo os seus idealiza- dores, esse programa teve como objetivo “mobilizar diferentes setores da sociedade e do Estado para tra- balhar em municípios pobres de todo o país, visando colaborar para a melhoria da qualidade de vida de suas comunidades, contribuindo para o seu desenvolvimen- to sustentável”. O trabalho de campo privilegiavam ações que envolviam as pessoas da comunidade, de- nominados “agentes multiplicadores”, tendo em vis- ta a conquista da “sustentabilidade dos trabalhos de- senvolvidos”. Os projetos aprovados em edital foram coordenados pelo setor de extensão das universida- des e envolveram os seus estudantes, docentes e fun- cionários. O apoio financeiro veio de instituições lo- cais, nacionais e internacionais17. O projeto realizado no 19 de Setembro teve como objetivo “capacitar a comunidade do assentamento” para “redirecionar a matriz produtiva visando recu- perar a [sua] sustentabilidade econômica” e “subsidi- ar a formulação de um Plano de Desenvolvimento Sus- tentável Integrado”. 15 UNISOL; SESU/MEC. Projeto: diretrizes para revitalização do espaço sócio-produtivo do assentamento 19 de Setembro em Guaíba, RS. Porto Alegre: Pró-Reitoria de Extensão da UFRGS, 2000. Habilitação ao Edital 01/2000 16 Título do Projeto: “Diretrizes para Revitalização do Espaço Sócio-Produtivo do Assentamento 19 de Setembro, Guaíba – RS”. Edital 01/2000 – SESU/MEC – Unisol. 17 UNISOL. Universidade Solidária. Disponível em <http://www.unisol.org.br/site/pagina.php?idconteudo=2> Acesso em: 14 out. 2006. 18 Esses professores prestaram consultoria em áreas específicas, como desenvolvimento rural, sustentabilidade, arquitetura, planeja- mento urbano e ambiental. A maioria deles (8) são doutores ou doutorandos. Cenário Na sua primeira etapa (01/2000), foram realizados cursos teórico-práticos envolvendo os seguintes temas: produção de arroz irrigado e pis- cicultura; formação de pomares domésticos; produção ecológica de hortaliças; e manejo integrado dos recur- sos naturais tendo em vista os seus impactos ambien- tais. Na segunda etapa (02/2000), foram realizadas novas atividades, como oficinas de educação ambien- tal, implementação de adubos verdes, produção agro- ecológica, saneamento, qualidade da água, doenças de veiculação hídrica e plantas medicinais. Também fo- ram realizadas visitas orientadas em unidades produ- tivas e experimentais, localizadas em assentamentos rurais do estado e na Estação Experimental da UFRGS. Participaram dessas atividades os “agentes multipli- cadores” já mencionados, escolhidos entre os mem- bros da “comunidade”, e os integrantes da equipe de extensão16. Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 65 O Projeto de Extensão Universitária A idéia inicial do projeto de extensão surgiu a partir do programa “Convivência Rural”, oferecido aos estu- dantes pela Pró-reitoria de Extensão da Universidade Federal do Rio Grande do Sul (PROREXT). Em 1996, foi criado um convênio com a Cooperativa de Assen- tamentos Rurais do Rio Grande do Sul (COCEARGS), “visando estimular o intercâmbio entre a Universida- de e assentamentos rurais do estado”15 . Em agosto de 1999, esse programa incluiu, “entre as comunidades parceiras”, o 19 de Setembro. Por um período de dez dias, durante as suas férias, estudantes de diferentes áreas do conhecimento visitaram o assentamento e realizaram um diagnóstico contemplando as áreas de saúde, educação e produção. Alguns meses depois, o Departamento de Educação e Desenvolvimento Social da PROREXT elaborou um projeto de extensão para ser desenvolvido no assentamento, tendo como refe- rência esse diagnóstico. Esse projeto foi encaminha- do ao “Programa Universidade Solidária”, de onde vi- eram os recursos econômicos para a sua execução16. O projeto contou com uma equipe executora for- mada por seis funcionários, 10 professores18 e 27 es- O “UniSol” foi criado em 1995 pelo Conselho da Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 65 tudantes19, todos da UFRGS, provenientes das mais diferentes áreas do conhecimento: arquitetura, agro- nomia, ciências sociais aplicadas, engenharia, enfer- magem, medicina e direito. As intervenções da equi- pe de extensão tiveram como referência uma visão multidisciplinar e participativa do processo de cons- trução do conhecimento, envolvendo a comunidade na definição dos problemas e na elaboração de estratégi- as de superação. Após um levantamento dos princi- pais obstáculos para a autosustentabilidade do assen- tamento, buscou-se discutir alternativas para superá- los Essas ações abrangeram áreas diferenciadas, a maioria delas relacionada, mesmo que indiretamen- te, à promoção da saúde coletiva do “19 de Setembro”. animais, devido às limitações dos recursos naturais (já comentados), e problemas jurídicos relacionados com a venda do leite in natura. No que se refere a produção de hortaliças, o projeto investiu em cursos de agroeco- logia, buscando agregar valor final ao produto. O Projeto de Extensão Universitária Considerando que a saúde e a qualidade de vida dos assentados depende da qualidade ambiental, e tendo em vista que boa parte das doenças mais co- muns no assentamento são causadas pela baixa qua- lidade da água, a equipe de extensão buscou aprofun- dar ainda mais o diagnóstico das condições de sanea- mento básico da agrovila, ao mesmo tempo em que procurou alternativas de tratamento dos afluentes do- mésticos do assentamento. Algumas experiências ini- ciais foram feitas neste sentido, com bons resultados até o presente momento. Essas alternativas exigem um investimento inicial de baixo custo na sua imple- mentação, utilizando-se de materiais de fácil acesso. No que se refere a qualidade do solo, fator que está interligado com a segurança alimentar dos assenta- dos e sua saúde individual, é possível pontuar algu- mas ações. Para proteger o solo mais seco da erosão e fortalecer a sua qualidade estrutural20, experiências com adubação verde foram implementadas em Unida- des de Teste e Validação (UTV’s), onde os agricultores e extensionistas tiveram a oportunidade de verificar a adequação dessas tecnologais ao meio ambiente lo- cal. Ao mesmo tempo, para resolver os problemas de alagamento da parte mais baixa do assentamento, o projeto introduziu um estudo de sistemas de drena- gem de baixo custo (as tecnologias disponíveis no mer- cado são muito caras). Em vez dos canos corrugados envolvidos em material do tipo bedim, foram cons- truídos drenos com materiais alternativos, como ta- quara e casca de arroz. O projeto de extensão conseguiu alcançar bons re- sultados, apesar de esbarrar em problemas relaciona- dos a um certo desconhecimento, por parte dos técni- cos, sobre a história social das famílias assentadas no 19 de Setembro, antes e após a conquista da terra. O principal obstáculo foi ampliar a utilização das tec- nologias introduzidas através do projeto para o con- junto de assentados, pois a maioria dos avanços fica- ram reduzidos às experiências implementadas nos lotes dos assentados interessados. Como veremos à seguir, a partir da descrição do projeto da farmaco- péia, esse problema está relacionado a dificuldades enfrentadas pelas famílias ainda nos primeiros anos de implementação do assentamento. No que se refere à matriz produtiva do assentamen- to, em um primeiro momento, procurou-se evidenciar os interesses dos assentados, para depois buscar al- ternativas de superação das limitações ambientais (ten- do em vista o aumento da qualidade do alimento e da geração de renda). O Projeto de Extensão Universitária Quando o projeto teve início, o sis- tema de produção do assentamento estava voltado pa- ra produção de leite, hortaliças e subsistência, mas atu- almente alguns assentados estão produzindo arroz para comercialização. Os principais fatores de restri- ção da pecuária leiteira são: falta de forragem para os 19 A equipe discente inicial era composta de dez estudantes de arquitetura; cinco de agronomia; quatro de ciências sociais aplicadas; dois de biologia; dois de engenharia; e quatro de medicina e enfermagem. A maioria deles estava cursando os últimos semestres de graduação. 20 O solo mais seco é muito importante para produção de alimentos para os assentados e animais (representa apenas 10% da área). 66 Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 66 Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 A Farmacopéia Popular Através da convivência prolongada com a comunida- de, os extensionistas perceberam que boa parte das famílias utilizava plantas medicinais para tratar pe- quenos problemas de saúde, foi assim que surgiu a idéia de implementar um horto medicinal no assenta- mento. Esse trabalho teve os seguintes objetivos: reu- nir o conhecimento dos assentados sobre fitoterapia gem, farmácia e medicina ficaram responsáveis pelo desenvolvimento do projeto junto à comunidade. e relacionar as plantas utilizadas em um único álbum herbário; organizar grupos de trabalho interessados em manter uma farmácia comunitária; aproximar o horto medicinal e a escola; integrar a farmácia comu- nitária e o horto medicinal com outros assentamentos da região. Estudantes das áreas de biologia, enferma- Ainda na fase inicial do trabalho, foi feito um le- vantamento do conhecimento etnobotânico das famí- lias, com a listagem do tipo de planta, uso medicinal, forma de utilização e cultivo. Tabela 1 - Plantas utilizadas no assentamento 19 de Setembro Planta Uso Medicinal Forma de Utilização Cultivo Abacate Rins, regular pressão Chá das folhas Sim Alcachofra Pressão alta, dor de estômago, colesterol, diabetes Chá com as folhas Sim Alecrim Calmante Tempero, chá Sim Arruda Para dor de cabeça e para simpatia Sim Babosa Cicatrizante Xarope Sim Bálsamo Calo inflamado, cicatrizante Pomada, lavagem com chá Sim Boldo Fígado, estômago Chá Sim Capim cidró Calmante, baixa pressão Chá Sim Calêndula Anti-alérgica, anti-séptica Camomila Calmante, cólica, estômago, garganta Flor com leite (para garganta), chá Sim e espontânea Carqueja Dor-de-barriga, diarréia Espontânea Cabelinho-de-porco Rins Espontânea Catinga-de-mulata Cólica, para estômago e fígado Frita no óleo e passa no umbigo Sim da criança, chá Chuchu Baixa a pressão Chá das folhas Couve Hemorragia Suco Confrei Cicatrizante, tóxica Pomada Sim Canela Cólica, estômago (gastrite), abortivo (altas doses) Endro intestino, estômago chá com folhas e sementes Sim Eucalipto Expectorante Inalação, chá, sabão (perfume) Erva cidreira Calmante, baixa a pressão Decocto ou chá com as folhas Sim Erva de Sta. Maria Vermífugo, tóxica, para zumbido no ouvido Tintura Espont / cultiv Funcho Cólica, gases Sim Gunchuma / Guanxuma Picada de inseto, rins Lavagem com as folhas, raiz Espontânea Guaco Expectorante Chá Sim Gervão Espontânea Hortelã Calmante, vermes Sim Laranjeira Calmante, gripe Chá (flor e folha) Sim Losna Estômago, fígado Chá Sim Manjerona Cólica, gases, dor-de-barriga. A Farmacopéia Popular Em altas doses Sim causa insônia em crianças Maracujá Calmante, baixa a pressão Sim Malva Infecção Pomada Sim Malva-cheirosa Infecção, afecções dos ovários Chá e banho de assento Sim Margarida Calmante Folha em chá Sim Tabela 1 - Plantas utilizadas no assentamento 19 de Setembro Tabela 1 - Plantas utilizadas no assentamento 19 de Setembro Tabela 1 (continuação) ç Planta Uso Medicinal Forma de Utilização Cultivo Mil-em-rama Cicatrizante e para cólicas Chá e pomada Sim Marcela Estômago Chá Espontânea Noz moscada Vômito (antiemético) Pata-de-vaca Diurético Pitangueira Diarréia, regulador de pressão Folhas em chá Picão Rins, reumatismo e para digestão chá da raiz Espontânea Penicilina Garganta, alergias Gargarejo e banho Sim Poejo Gripe, tosse Sim Ponta-alívio/Erva-da- Dor de cabeça Tintura Sim pontada (tb. novalgina) Salso chorão Salsa Estancar o sangue (), Diurético Folha, raiz C/ tempero Sálvia-da-gripe, Gripe chá das folhas Sim sálvia-da-tosse Sálvia Tristeza (cura o baço), excitante, emenagoga C/ tempero Serraia, serralha Digestivo Tintura Espontânea Tansagem Cicatrizante, infecção urinária, garganta, Pomada, gargarejo, chá Espontânea ataca o fígado Tiririca Anti-anêmico Espontânea Urtiga Reumatismo Espontânea Logo no início do projeto, uma especialista em fitoterapia, que estudou a utilização de plantas medi- cinais em comunidades amazônicas, ministrou um curso sobre técnicas de cultivo, colheita, secagem e armazenamento de plantas medicinais, assim como manipulação de tinturas, xaropes, pomadas, cremes e óleos. Durante o curso, os assentados entraram em contato com experiências bem-sucedidas na comercia- lização de medicamentos caseiros e passaram a ver na iniciativa a possibilidade de um retorno econômico a médio e a longo prazo. A partir do incentivo da equi- pe de extensão, formaram-se dois grupos diferenciados em torno do projeto: um composto de mulheres da “par- te baixa” do assentamento, “setor A”; e o outro forma- do de mulheres que residem na agrovila, “setor B”. a ruptura da cooperativa ainda nos primeiros anos de formação do assentamento. As mulheres do “setor A” (parte baixa do assentamento) faziam parte das primei- ras famílias que abandonaram a cooperativa para for- marem um grupo de trabalho menor, cujos membros eram ligados por relações de parentesco e compadrio. Mais tarde, aos poucos, outros grupos também aban- donaram a cooperativa. No entanto, o primeiro grupo ainda guarda a marca de ter sido o primeiro a romper com o projeto coletivo. Por outro lado, o grupo da parte baixa afirma que as mulheres da agrovila não conse- guem se organizar, pois são “só discurso” e “pouca prá- tica”. Como elas falaram diversas vezes, “com a farmacinha lá de cima (agrovila) é a mesma coisa que na época da cooperativa, fica só na conversa”. 68 Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 Saberes Tradicionais, Gênero e Saúde Pública Essa compreensão exige a consciência inicial de que o tempo da apropriação do projeto pela comuni- dade, mesmo numa situação em que o projeto é o re- sultado de um diálogo e de uma pesquisa-ação parti- cipativa, dificilmente acompanha o tempo das previ- sões orçamentárias das instituições financiadoras, que trabalham a partir de uma perspectiva de mudan- ça quase imediata (a maioria dos projetos tem previ- são de duração de um ano). Um dos maiores proble- mas das políticas públicas na área de saúde é que, na grande maioria dos casos, essas políticas não são con- cebidas considerando a necessidade de se estabelecer um diálogo com o sujeito social local, com a sua cul- tura e o seu modo de vida. A intervenção é geralmente pensada a partir da cultura dos interventores, suas próprias representações sobre o “outro”, na maioria das vezes construídas a partir de estereótipos. Talvez uma das especificidades da antropologia seja a forma de abordar as grandes questões humanas a partir de dados etnográficos sobre ambientes especí- ficos, buscando descobrir as contribuições que o en- tendimento das comunidades locais pode dar para a compreensão dos grandes temas existenciais. O olhar etnográfico, quando voltado para as pequenas sutile- zas do cotidiano, permite descrever como as “grandes questões” influenciam a vida de pessoas de carne e osso, seja nas aldeias, assentamentos ou em laborató- rios, departamentos governamentais e instituições totais. A narrativa do antropólogo emerge de um mer- gulho descritivo na vida do dia-a-dia, onde ele encon- tra material para sustentar suas reflexões mais ana- líticas. Para além de divergências escolares (ou de escolas teóricas), existe algo que nós não estamos dis- postos a abandonar: a tradição etnográfica. Seguimos, portanto, um saber fazer que é passado de geração para geração, algo que nos mantêm unidos como dis- ciplina, apesar de todas as diferenças (Peirano, 1995). No caso do projeto de extensão desenvolvido no as- sentamento, a equipe buscou auxílio na antropologia para entender as dificuldades de comunicação com o grupo local. Mesmo no caso da farmacopéia, onde foi feito todo um trabalho de registro do saber local dos assentados sobre as plantas medicinais, o dilema da comunicação intercultural esteve presente no momen- to de pensar a forma de organização dos grupos de trabalho. Cabe ao antropólogo mediar esses desenten- dimentos e construir um espaço onde a comunicação seja possível. Tabela 1 - Plantas utilizadas no assentamento 19 de Setembro Sem en- trar no mérito da veracidade dessas afirmações, é im- portante afirmar que os dois grupos de mulheres tam- bém desenvolvem atividades juntas, principalmente, na relação com os agentes externos ao assentamento (fes- tas, reivindicações coletivas e reuniões). Apesar da insistência inicial dos estudantes em for- mar um único grupo, logo ficou evidente que isso não seria possível, pois a formação de dois grupos expres- sava redes diferenciadas de afinidades. Essas diferen- ças estavam relacionadas a tensões que acompanharam Em outros projetos que foram desenvolvidos pela equipe de extensão, as atividades também só avança- ram a partir de grupos de afinidades (parentesco e compadrio), constituídos durante o processo de forma- ção histórica do assentamento e, em alguns casos, an- tes das famílias serem assentadas. A própria ruptura da cooperativa em grupos menores seguiu a lógica da afinidade, além de ser a expressão de uma série de ten- sões envolvendo uma desconfiança geral em relação à burocracia institucional necessária para manter uma cooperativa operando conforme as regras econô- micas e jurídicas (técnico contábil, presidente, secre- tários, advogado etc.). Nosso objetivo aqui não é dis- cutir a ruptura da cooperativa, mas indicar que os pro- jetos de saúde desenvolvidos em assentamentos ru- rais devem sempre levar em conta o passado das famílias assentadas, assim como os dilemas vivencia- dos durante o próprio processo de assentamento. extensão a entender melhor a forma como o projeto da farmacopéia foi apropriado pela comunidade: se por um lado os assentados não participavam das as- sembléias chamadas pelos estudantes para discutir a ampliação da prática de medicina alternativa para o “coletivo”, em um movimento lento e contínuo essas práticas e saberes permearam as relações sociais, com a troca de conhecimentos, medicamentos e saberes dentro dos grupos de afinidades, permitindo um mo- vimento reflexivo sobre o passado de cada sujeito en- volvido no projeto. O olhar é sempre disciplinar, por isso, é necessário saber olhar para entender as estra- tégias de apropriação diferenciadas, as formas de con- ceber a organização que seguem lógicas diferentes, mas que são tão ou mais eficazes que as nossas pró- prias formas de saber fazer. Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 69 Os Usos dos Dispositivos Institucionais e as Políticas de Acesso Diferenciado A descrição do projeto de extensão, mais especifi- camente do projeto de farmácia caseira, permitiu que algumas diferenças em relação à intervenção do go- verno fossem apontadas. A experiência com a produ- ção de medicamentos caseiros surgiu como uma táti- ca para superar os problemas relacionados ao acesso aos remédios utilizados no tratamento de doenças mais comuns, mas que não necessitam de um atendi- mento clínico especializado. Esse projeto foi incorpo- rado pelos assentados e implementado conforme a di- nâmica local, chegando a reproduzir as tensões oca- sionadas por conflitos que ocorreram durante o pro- cesso de assentamento. O sistema de atendimento clínico-hospitalar não é universal, mas se propõe a ser, o que é uma diferença importante. Nas sociedades complexas, as alternati- vas de assistência à saúde são inúmeras, constituin- do aquilo que Helman denomina de “pluralismo mé- dico”. No caso da sociedade brasileira, o modelo da bio- medicina pode ser denominado “oficial” na medida em que os seus pressupostos culturais e sociais estão le- gitimados pelo Estado. A maioria das políticas públi- cas e privadas voltadas para o setor de saúde dá prio- ridade ao tratamento médico hospitalar, legalmente sancionado como a alternativa “mais eficiente e obje- tiva” no que se refere ao tratamento de doenças. No entanto, além do sistema oficial existem outros, como é o caso, por exemplo, de uma série diversificada de práticas “informais” utilizadas por diferentes grupos sociais de nossa sociedade. A fabricação e o uso de medicamentos caseiros per- mitiram uma valorização do conhecimento tradicio- nal da comunidade sobre doença e práticas terapêuti- cas, incentivando um movimento reflexivo, que per- mitiu a valorização dos saberes tradicionais passados de geração para geração. Ao mesmo tempo, o envolvi- mento das mulheres no projeto possibilitou um espaço de discussão das questões de gênero, ao mesmo tem- po em que permitiu uma valorização do saber femini- no sobre o cuidado familiar. A segunda questão fundamental é que o modelo biomédico não pode ser abordado como um conjunto de práticas e saberes objetivos sobre a doença, como uma espécie de reconhecimento das leis da natureza, pois isso seria conceber o corpo humano, unicamen- te, como um corpo biológico, quando na verdade o cor- po é também um instrumento de construção simbóli- ca da sociedade, um signo a espera de significado. Saberes Tradicionais, Gênero e Saúde Pública No caso aqui analisado, procuramos for- necer subsídios que pudessem auxiliar a equipe de Durante a efetivação do projeto de extensão, aos poucos a lógica institucional foi sendo apreendida nos usos cotidianos dos assentados e da equipe de exten- são. O diferencial da extensão é que se trabalhou com a idéia de que o problema de saúde está relacionado a outros fatores, como segurança alimentar e produção agroecológica, qualidade do solo, cuidado com os ani- mais, tecnologias hidrossanitárias, meio ambiente e saúde coletiva. O problema da saúde foi abordado de forma interdisciplinar, procurando identificar as do- enças mais recorrentes e sua relação com as limita- ções de caráter ambiental. A forma de intervenção também foi diferenciada, os objetivos e as ações da equipe de extensão foram discutidos antes com a co- munidade, que teve participação na concepção das estratégias. O projeto foi concebido a partir de princí- pios de participação comunitária, adaptação técnica às limitações locais, pesquisa aplicada e formação in- tercultural (técnicos, estudantes e assentados). tos de acesso aos serviços. A intenção deste artigo é indicar que ações de inclusão na rede oficial de aten- dimento público na área de saúde – no caso dos assen- tamentos rurais – devem vir acompanhadas da imple- mentação de ações complementares que tenham como objetivo o reconhecimento da especificidade dos pro- blemas de saúde da comunidade, o que significa pro- blematizar a discussão sobre doença, saúde, meio ambiente e cultura. 70 Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 Os Usos dos Dispositivos Institucionais e as Políticas de Acesso Diferenciado As concepções de risco, as práticas de precaução, as di- ferentes formas de conceber a doença e a saúde são instrumentos simbólicos de fabricação da sociedade e não podem ser abordados como algo isolado do con- texto sociocultural mais abrangente. A principal diferença entre a ação do governo mu- nicipal e o projeto de extensão é que o primeiro consis- tiu em uma ação que facilitou o acesso a um sistema de atendimento que já existia, sem colocar em discus- são a forma terapêutica, as concepções de doença, assim como as características sociais, culturais e ambientais da comunidade local. A lógica vigente teve como referência principal uma ação de inclusão implementada a partir do pressuposto de que o servi- ço público de saúde está voltado para o atendimento universal da doença individual, tendo por base o dis- curso da igualdade dos usuários em termos de direi- Essas duas questões iniciais são fundamentais para pensar a tensão entre o universal e o específico e as políticas de saúde pública na área de assentamen- tos rurais21. A tensão entre a universalidade dos ser- viços e a alteridade dos usuários é o eixo principal de uma luta social em torno do controle dos dispositivos de poder que acompanham as intervenções nas con- dições de saúde das populações. Quando Foucault des- creve o surgimento da medicina social entre os sécu- los XVII e XIX, ele vai buscar identificar a lógica acio- nada pela intersecção entre as noções de “governabi- lidade” e “população”, e a sua relação na emergência de uma racionalidade própria do Estado moderno. Segundo o autor, essa nova “arte racional de governar” tem como principal objetivo o controle e a ordenação da população, pois dela depende a força do Estado. Para gerir essa população, é preciso, entre outras coi- sas, de uma política de saúde que possa intervir nas condições de vida, para modificá-las e impor normas (Foucaut, 1997, p. 81-86). Os Usos dos Dispositivos Institucionais e as Políticas de Acesso Diferenciado arquitetura urbana, que também estão relacionadas à economia urbana em geral – transportes, disposição dos bairros, praças e avenidas); a constituição de uma agência de pesquisa e estatística; as intervenções epidemiológicas e ações preventivas; o investimento no sistema clínico-hospitalar, com formação de par- ceria com o setor privado; a efetivação de políticas nacionais; e, finalmente, as ações mais recentes de ampliação do sistema e descentralização administra- tiva (municipalização da gestão e inclusão através dos conselhos comunitários), assim como as ações no campo de “atendimento diferenciado”. Uma breve des- crição geral desses serviços, suas continuidades e rupturas, as redes institucionais que perpassam o Estado e a sociedade, permitem efetivar de fato uma “arqueologia dos dispositivos de poder”, voltando o olhar da antropologia para o centro produtivo da eco- nomia simbólica das sociedades complexas. Foucault está interessado na articulação entre dis- positivos, técnicas e mecanismos disciplinares que organizam o espaço social a partir do século XVIII, definindo o funcionamento das instituições (exército, escola, prisão e hospital). Este movimento é legitima- do pelo discurso da universalidade dos procedimen- tos e sua aplicação uniforme. Essa análise considera o papel decisivo dos procedimentos e dispositivos tecnológicos na organização de uma sociedade, para depois evidenciar, a partir de uma operação historio- gráfica, os dispositivos disciplinares presentes numa tecnologia judiciária e científica que surge com o de- senvolvimento do Estado-nação moderno. Por outro lado, também precisamos de um outro pressuposto importante: é impossível reduzir os fun- cionamentos de uma sociedade a um tipo dominante de procedimentos. Devemos questionar qual é o esta- tuto de muitas outras séries de micro-práticas cotidi- anas que não deram lugar a uma configuração dis- cursiva nem a uma sistematização tecnológica. Esse questionamento inicial permitiu a Michel de Certeau levantar a hipótese “de que o sistema da disciplina e da vigilância, formado no século XIX a partir de pro- cedimentos anteriores, esteja sendo, ele mesmo, vampirizado por outros procedimentos”, menos visí- veis e mais dispersos, e por isso mesmo muito mais eficazes e silenciosos. Uma análise que não privilegia o aparelho produtor, mas os procedimentos propria- mente “populares”, dispersos, heterogêneos, anti-sis- temáticos, minúsculos e cotidianos, permitem abor- dar as maneiras de fazer, os usos subversivos dos pro- cessos que organizam a ordenação sociopolítica, for- mando a contrapartida dos consumidores. 21 Vale lembrar que a mesma problemática se aplica às políticas de saúde voltadas para o atendimento das sociedades indígenas, comu- nidades remanescentes de quilombos, classes populares, moradores de rua e outros tantos grupos sociais que colocam em questão a pretensão de universalidade do sistema de saúde coletiva a partir da sua alteridade. Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 71 Referências BERGER, Christa. Campos em confronto: a terra e o texto. 2. ed. Porto Alegre: UFRGS, 2003. transformá los em ocasiões (De Certeau, 1994, P. 41 5). O caso do projeto da farmacopéia popular ajuda a ilustrar as relações entre tecnologias de intervenção e as operações contrárias de subversão colocadas em práticas pelos sujeitos que são o alvo dessas políticas. A discussão sobre acesso diferenciado, alteridade cul- tural e desigualdade econômica consegue tencionar o paradigma do universalismo tecnocrático, pois per- mite o deslocamento do olhar do centro de produção econômica e simbólica (as instituições e seus opera- dores), para as margens (os usuários dos serviços). Os projetos de extensão universitária geralmente estão voltados para populações mais pobres, seja no campo ou na cidade. As concepções que orientaram a prática dos intervencionistas estão inseridas em uma políti- ca alternativa e complementar às ações institucionais implementadas pelo governo na área de saúde, como os serviços do Sistema Único de Saúde, ou até mesmo ações dos governos estaduais e municipais. BRASIL. Costituição Federal: outubro de 1988. 8. ed. Porto Alegre: Verbo Jurídico, 2004. CERTEAU, Michel de. A invenção do cotidiano. 6. ed. Petrópolis: Vozes, 1994. v. 1. FERNANDES, Bernardo Mançano. Núcleo de Estudos, Pesquisas e Projetos de Reforma Agrária (NERA). DATALUTA - Banco de Dados da Luta pela Terra: rela- tório, 2004. São Paulo: FCT/UNESP, 2006. Disponível em: http://www2.prudente.unesp.br/dgeo/nera/telas/ projetos.htm. Acesso em: 10 jun. 2006. FERNANDES, Bernardo Mançano. A formação do MST no Brasil. Petrópolis: Vozes, 2000. FOUCAULT, Michel. Segurança, território e população. In: ______. Resumos dos cursos do Collège de France (1970-1982). Rio de Janeiro: J. Zahar, 1997. p. 79-86. In: ______. Resumos dos cursos do Collège de France (1970-1982). Rio de Janeiro: J. Zahar, 1997. p. 79-86. O uso que foi feito da estrutura disponibilizada pelo projeto de extensão foi muito mais amplo e di- versificado do que os objetivos iniciais, pois o elemen- to dialógico e participativo aponta para uma “fusão de horizontes” entre o olhar do técnico e o sistema de conhecimento das populações, que são alvo de suas ações. Estamos falando aqui na capacidade de descen- tralizar o olhar da produção em direção aos usos coti- dianos, aos saberes e às práticas tradicionais, buscan- do encontrar soluções locais para superar obstáculos de ordem econômica, cultural e social. FOUCAULT, Michel. O nascimento da medicina social. In: ______. A microfísica do poder. 14. ed. Rio de Janeiro: Graal, 1999. p. 79-98. Os Usos dos Dispositivos Institucionais e as Políticas de Acesso Diferenciado Devemos analisar o uso dos dispositivos por si mesmos, as margens de manobra permitidas aos usuários pelas conjunturas, operações que proliferam no seio das estruturas tecnocráticas e alteram o seu funciona- mento por uma multiplicidade de táticas articuladas No Brasil, a saúde pública sempre foi uma preocu- pação estratégica do Estado, sendo um dos eixos fun- damentais de toda a ação governamental, pois a pró- pria noção de “governabilidade” só é possível a partir de uma preocupação racional com a gestão das “po- pulações”. Trata-se de uma economia governamental que produz um espaço disciplinar onde a razão de Estado predomina, seja através de decretos legisla- tivos, seja através de operadores que atuam como interventores legítimos. Podemos mencionar, por exemplo, as ações que visam à institucionalização e fiscalização da profissão médica; as condições de sa- neamento urbano (incluindo as transformações da Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 71 rais e sociais, permite abordar a saúde coletiva a par- tir de sua complexidade, reconhecendo o sujeito social como portador de valores culturais. sobre os detalhes do cotidiano. Esses modos de proce- der, essas artes de fazer, esses usos poéticos formam uma rede antidisciplinar, que é importante mencionar na análise das políticas públicas”. O “usuário” do siste- ma é um microoperador de discursos e práticas, a sua arte consiste em jogar com os acontecimentos para transformá-los em ocasiões (De Certeau, 1994, P. 41-5). 72 Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 Recebido em: 07/08/2006 Aprovado em: 20/10/2006 Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 73 Referências GOHN, Maria da Glória. Teoria dos movimentos soci- ais: paradigmas clássicos e contemporâneos. 3. ed. São Paulo: Loyola, 2002. NAVARRO, Zander et al. Pequena história dos assenta- mentos rurais no Rio Grande do Sul: formação e desen- volvimento. In: MEDEIROS, Leonilde (Org.). A formação dos assentamentos rurais no Brasil: processos e po- líticas públicas. Porto Alegre: UFRGS, 1999. p. 19-53. A escolha pela fitoterapia foi feita tendo em vista a sua situação econômica e a possibilidade de apro- veitar um conhecimento disperso colocado em práti- ca no espaço privado da família. A metodologia parti- cipativa e as noções de pesquisa-ação que acompanha- ram as atividades desenvolvidas pela equipe de exten- são permitiram uma valorização do conhecimento tradicional, contribuindo para legitimar um saber que historicamente foi estigmatizado pelos agentes “ofi- ciais” de saúde. A abordagem da doença em relação a um conjunto mais amplo de fatores ambientais, cultu- NORONHA, José Carvalho de. Os rumos do Estado Bra- sileiro e o SUS: a seguridade social como política pú- blica da sociedade e estado. Saúde e Sociedade, São Paulo, v. 14, n. 2, p. 31-38, maio/ago. 2005. NORONHA, José Carvalho de. Os rumos do Estado Bra- sileiro e o SUS: a seguridade social como política pú- blica da sociedade e estado. Saúde e Sociedade, São Paulo, v. 14, n. 2, p. 31-38, maio/ago. 2005. PAUL, Patrick. Transdisciplinaridade e antropoforma- ção: sua importância nas pesquisas em saúde. Saúde e Sociedade, São Paulo, v. 14, n. 3, p. 72-92, set./dez. 2005. PEIRANO, Mariza. A favor da etnografia. Rio de Janei- ro: Relume Dumará, 1995. 72 Saúde e Sociedade v.15, n.3, p.57-73, set-dez 2006 SCHMIDT, Benício V.; MARINHO, Nolasco; ROSA, Su- eli. Os assentamentos de reforma agrária no Brasil. Brasília, DF: UnB, 1998. SINDICATO DOS JORNALISTAS PROFISSIONAIS DO RIO GRANDE DO SUL. Nos bastidores da batalha. In: GÖRGEN, Frei Sérgio (Org.). Uma foice longe da terra: a repressão aos sem-terra nas ruas de Porto Alegre. 2. ed. Petrópolis: Vozes, 1991. p. 67-92. Recebido em: 07/08/2006 Aprovado em: 20/10/2006
https://openalex.org/W4393941413	https://singipedia.singidunum.ac.rs/preuzmi/44474-the-future-development-of-rural-areas-through-the-application-of-slow-tourism-a-case-study-of-western-serbia/5449	English	null	The Future Development of Rural Areas through the Application of Slow Tourism - A Case Study of Western Serbia	null	2,023	cc-by-sa	4,707	SITCON 2023 MANAGING THE FUTURE BY LEARNING FROM THE PAST – CONTEMPORARY TRENDS IN TOURISM & HOSPITALITY SITCON 2023 MANAGING THE FUTURE BY LEARNING FROM THE PAST – CONTEMPORARY TRENDS IN TOURISM & HOSPITALITY CONFERENCE PAPERS CONFERENCE PAPERS Keywords: Keywords: slow tourism, rural tourism, Western Serbia, SWOT analysis. y slow tourism, rural tourism, Western Serbia, SWOT analysis. Abstract: Tamara Surla1, Tatjana Pivac1, Darija Lunić2, Jovana Miljković1 Tamara Surla1, Tatjana Pivac1, Darija Lunić2, Jovana Miljković1 Western Serbia, as a crossroad of different cultures and an area of rich material and cultural heritage imbued with exceptional natural values, has rich potential and developed rural tourism. However, rural tourism can be significantly improved and used in a more adequate way. The chance for the possible development of rural areas can be reflected in changes in the way of thinking and habits of modern tourists, who pay more and more attention to authenticity. Also, there is a chance to define a new horizon for tourism based on the discovery of traditional food and rural communities. This type of tourism can represent slow tourism. The main goal of the paper was to show the link between rural and slow tourism, as well as the possibility of applying the concept of slow tourism in the rural areas of Western Serbia. 1University of Novi Sad, Faculty of Sciences-Department of Geography, Tourism and Hotel Management, Novi Sad, Serbia 2Singidunum University, Belgrade, Serbia INTRODUCTION Rural tourism represents one of the alternative forms of tourism which generates an increasing number of tourists (Košić, 2009). As Serbia has an exceptional potential for the development of rural tourism, which has not been adequately utilized (Bratić et al., 2021), it is necessary to define new horizons for tourism that will be based on traditional food and discoveries in rural communities. The potential for rural development can be observed in the changing mindsets and behaviors of modern travelers, who value authenticity more and increasingly— particularly when choosing food for consumption by tourists. The idea of slow tourism, which has emerged as a response to the fast-paced lifestyle of today, is one of these solutions. It can be understood as a new travel niche or as an approach of traveling in general. Slow tourism is characterized as a type of tourism that emphasizes slowness, unhurriedness, simplicity, but also disconnection from the hustle and bustle of everyday life (Serdane, 2020). In this connection, there are destinations that can be called “slow”, which highlights their character that makes them suitable for slow tourism (Noor et al., 2015). A rural area can have certain characteristics of a “slow” destination because it belongs entirely to the natural environment, with the presence of a traditional community structure that is reflected in a lifestyle that fascinates tourists and drives them to participate in it (Kastenholz & Lima, 2013). Sitcon 2023 www.sitcon.singidunum.ac.rs Correspondence: Tamara Surla e-mail: tamara.bozovic@dgt.uns.ac.rs 118 DOI: 10.15308/Sitcon-2023-118-125 SITCON 2023 MANAGING THE FUTURE BY LEARNING FROM THE PAST – CONTEMPORARY TRENDS IN TOURISM & HOSPITALITY Western Serbia is a vast region that includes a few geographical and traditional micro-regions, which represent a geographical and natural-anthropogenic complex, i.e. a space of intertwined actions of nature, people, and a rich inventory (Markovic, Pavlovic, 1995). It is a predominantly rural region that uses its potential in the development of rural tourism (Perić et al., 2020). Western Serbia is important for the development of tourism in Serbia in general, because 43.7% of the total number of tourists who spent the night in Serbia in 2021 stayed in Western Serbia and Šumadija (Statistical Office of the Republic of Serbia, 2022). Therefore, it is very important to research the topic of the possibility of further development of tourism in this region, primarily in literature, so that planners in the tourism industry can be guided by it. INTRODUCTION While numerous studies have been done on the relatively new concept of slow tourism (Mavrić et al., 2022), not much has been done on the relationship between slow and rural tourism (Farhadi Uonaki et al., 2022) or how slow tourism is perceived in rural areas and destinations. In a similar manner, slow tourism is a relatively new concept in Serbia, and our study contributes significantly to the repository of knowledge on this topic. Therefore, the goal of this paper is to investigate the possibility of applying the concept of slow tourism in the rural areas of Western Serbia, as well as to show all the benefits of this development. LITERATURE REVIEW Similarities in the concepts of slow tourism and rural tourism. Table 1. Similarities in the concepts of slow tourism and rural tourism. Slow tourism Rural tourism Small scale (Markwell et al., 2012) Small scale (Blanco, 1996) Authentic (Markwell et al. 2012) Authentic (Marie & Vilard, 1977) Environmental sustainability (Lipman & Murphy, 2012) Environmental sustainability (Mansfield & Jonas, 2006) Against modern consumption (Conway & Timms, 2012) Against modern consumption (Sharpley, 2002) Psychological (Gardner, 2009) Psychological (Voase, 1995) Source: Noor et al., 2015. As slow tourism overlaps with different types of tourism, the spectrum of activities associated with slow tourism is very wide and depends only on the imagination of the offer and the wishes of the consumer, i.e. tourist. Thus, a comprehensive framework was created, and unique criteria were established for distinguishing the activities that most fully describe slow tourism (Figure 1). As shown in Figure 1, the authors established two axes to classify tourism activities. These are the “passive-active” and “individual-mass” axes. Slow tourism activities appear when “active” activities meet “individual” activities. It should be noted that “individual” activities refer not only to individual travelling but also to travelling in small groups. The activities advocated by slow tourism are characterized as all those activities that are in line with eco-tourism, rural tourism, staying in nature, trekking and the like. Figure 1. Activities included in slow tourism. Individual SLOW TURISM Active Passive Wellness tourism Cruise Winer mountain Wine&Food Sun&Beach Entertainment Relax mountain Mass Nutrical tourism River Rural tourism Horse riding City break Trekking Cycling tourism Nature tourism Ecotourism Source: Dall’Aglio, 2011, p. 6. Although the topic of rural tourism has been researched both in Serbia (Bratić et al., 2021; Demirović et al., 2020) and in Western Serbia (Perić et al., 2020; Vesić et al., 2022), the topic of slow tourism is still in the beginning (Božović et al., 2021; Božović et al., 2022; Marić et al., 2021; Lunić et al., 2020). On the other hand, only one research was conducted that connects slow and rural tourism in Western Serbia (Božović, 2023). Therefore, this paper makes a significant contribution to fill the gap in the scientific literature on this developing topic. Figure 1. Activities included in slow tourism. SLOW TURISM Wellness tourism River Cycling tourism Rural tourism Passive Winer mountain Wine&Food Sun&Beach Entertainment Relax mountain Mass Source: Dall’Aglio, 2011, p. 6. Conference papers LITERATURE REVIEW Slow tourism is a type of tourism in which travelers who want to experience quality travel experiences through getting to know the culture, history, tradition, and gastronomy of the local population participate. Through slow tourism, tourists explore destinations at their own pace without a predetermined schedule or imposed time limits. Slow tourism supports staying in sustainable rural, cultural, or eco-destinations, where the social, economic, and environmental components of a destination’s sustainability have the same impact and should all be considered equally. Therefore, slow tourism takes place predominantly in rural, less populated areas, which are rich both in natural and cultural values (Božović et al., 2022). It is possible that rural areas are the most effective places to implement slow tourism because of the socially constructed perception of them as having lower levels of consumption than urban locations (Noor et al., 2011). A rural destination, with a slow way of life, could potentially be considered an “avenue for slow tourism” (Noor et al., 2015), given that rural tourism fosters the preservation of the environment, culture, as well as a slow way of life (Joffe et al., 2004). The style of rural areas is characterized by a slower pace of life that presents tourists in rural destinations with experiences that are closer to the natural environment and traditional heritage (Liu, 2006). It is the “slow” experiences that reflect the unique characteristics of the local community, highlighting the importance of heritage and community lifestyle (Lumsdon & McGrath, 2011). In addition to the preservation of tradition and culture, the ecological characteristics of the rural area and green practices that can be enabled through the implementation of slow tourism, the characteristics of the rural area in the service of slow tourism are important (Noor et al., 2011). In their research, Noor and colleagues (2015) state the similarities between rural and slow tourism (Table 1), considering the relevant literature on the mentioned forms of tourism. Seeing the similarities between both types of tourism, they introduce a working definition of slow tourism in a rural destination: “Slow tourism is a type of tourism in a rural environment that is small-scale and consists of a psychological approach to travel emphasizing authentic experiences, ecological sustainability opposed to the consumption of modern society” (Noor et al., 2015, p. 4). Therefore, rural destinations represent an ideal place for implementing slow tourism. 119 Conference papers Table 1. RESULTS After a detailed review of the literature on rural, slow tourism and their linking, priority tourism products common to all destinations in the territory of Western Serbia were investigated, to gain knowledge about the potential and future direction of tourism development. Also, current models of tourism management in destinations were investigated, and examples of good practice that can be used as an example for further tourism development were listed. LITERATURE REVIEW Although the topic of rural tourism has been researched both in Serbia (Bratić et al., 2021; Demirović et al., 2020) and in Western Serbia (Perić et al., 2020; Vesić et al., 2022), the topic of slow tourism is still in the beginning (Božović et al., 2021; Božović et al., 2022; Marić et al., 2021; Lunić et al., 2020). On the other hand, only one research was conducted that connects slow and rural tourism in Western Serbia (Božović, 2023). Therefore, this paper makes a significant contribution to fill the gap in the scientific literature on this developing topic. Although the topic of rural tourism has been researched both in Serbia (Bratić et al., 2021; Demirović et al., 2020) and in Western Serbia (Perić et al., 2020; Vesić et al., 2022), the topic of slow tourism is still in the beginning (Božović et al., 2021; Božović et al., 2022; Marić et al., 2021; Lunić et al., 2020). On the other hand, only one research was conducted that connects slow and rural tourism in Western Serbia (Božović, 2023). Therefore, this paper makes a significant contribution to fill the gap in the scientific literature on this developing topic. 120 SITCON 2023 MANAGING THE FUTURE BY LEARNING FROM THE PAST – CONTEMPORARY TRENDS IN TOURISM & HOSPITALITY METHODOLOGY METHODOLOGY The paper is aimed at building a theoretical basis based on which it is possible to see the possibilities of linking rural and slow tourism, as well as the implementation of slow tourism in rural areas. For this reason, through qualitative criteria, a framework was built that will check whether slow tourism has the characteristic elements of a rural destination. To that end, the authors, after a detailed review of the literature, singled out the elements characteristic of slow tourism and which are associated with rural tourism. To determine which human, natural, cultural, and social resources are most involved in slow tourism, as well as what predominates in tourism activities and services in the destination, various local values were analyzed (Nunes & Cooke, 2021). For this reason, a SWOT analysis of the application of slow tourism in the rural areas of Western Serbia was conducted. The priority tourist product of Western Serbia The tourism development strategy of the Republic of Serbia for the period from 2016 to 2025 defines 18 priority tourist destinations that are important for completing the tourist product of Serbia. Tourist destinations are defined according to the infrastructure and superstructure developed so far, accessibility and realized traffic. In the territory of Western Serbia, rural tourism is a type of tourism that is common to all priority tourist destinations. The interest in rural tourism in Western Serbia is constantly increasing, given the existence of still attractive and “unspoiled” rural areas. To an increasing extent, the interests of tourists in staying in nature are highlighted. More and more visitors show interest in getting to know the culture, customs and living conditions of the place and surroundings where they stay during their vacation. Motives for coming to the countryside, such as peace, pure nature, interaction with new people (hosts), healthy food, “slow food” (enjoying food), easy pace, physical activity, are responsible for the emergence and survival of rural tourism as tourism oriented to the individual guest and his needs. Western Serbia has great tourist potential for the development of this type of tourism, however, despite the exceptional perspective in the region of Podrinje and Valjevo, it is still modest in scope. Both tourism regions in their tourism development strategies state plans to support the development of rural tourism as an important potential of the regions. Therefore, the possibility of opening new tourist facilities, improvement of tourist infrastructure, opening of local handicraft facilities, branding of rural products, etc. is mentioned (Bozovic, 2023). On the other hand, the tourist region of Western Serbia has many households on its territory that meet the high standards and requirements for the provision of catering services prescribed by current regulations (Table 2). 121 Conference papers Table 2. Accommodation capacities in the villages of the Western Serbia region. Municipality/city Number of facilities Number of rooms Number of beds Prijepolje 13 35 77 Arilje 14 40 90 Bajina Bašta 7 27 76 Čajetina 46 74 258 Kosjerić 13 44 117 Ivanjica 49 119 312 Lučani 7 41 108 Nova Varoš 51 167 758 Požega 9 31 84 Užice 32 71 168 Sjenica 4 15 60 In total 245 664 2.108 Source: Author- based on the tourism development program of the Western Serbia tourist region from 2020 to 2025. Table 2. Accommodation capacities in the villages of the Western Serbia region. The priority tourist product of Western Serbia Many households in this region own buildings of characteristic traditional architecture, such as sculptures, dairies, mills, and the like. There are many marked cycling routes in the region, and routes that link several destinations are of special quality. Most accommodation providers offer hiking trails. Some households own family workshops for woodworking, making souvenirs, pottery workshops, workshops for making traditional folk costumes, tasting centers, which can only be improved in terms of a better-quality offer within the wider local community. As for other contents in accommodation facilities in the territory covered by the tourist or- ganization of the region of Western Serbia, most households are engaged in the production of organic/healthy and high-quality food (gardens, greenhouses, fields). Therefore, the link between agriculture and rural tourism is significant. In this manner, the placement of domestic products through the preparation and serving of meals is greatly facilitated. Many households process agricultural products (sweets, juices, jams, ajvar...), some of them even organize the preparation of winter food, but not cooking schools. The processing of these products provides insignificant additional household income. Production of meat, meat products and dairy products on farms is limited, which is compensated by other agricultural farms in the local community, producers of the same (Tourism Development Strategy of the Republic of Serbia, 2016). Conference papers Tourism management authorities For tourism to develop in an adequate way and to follow modern trends in tourism, it is necessary to have permanent tourism management bodies that will monitor this development. An example of good domestic practice is the existence and successful work of the tourist organization of the region of Western Serbia, which has existed since 2006 and is the only regional tourism organization in the Republic of Serbia, formed in accordance with the Law on Tourism as a DMO (Destination Management Organization) in the process of establishment. In the management structures, in addition to representatives of the public sector - local tourist organizations, there are TOS (Tourist Organization of Serbia), the association of hoteliers, associations of travel agencies, representatives of the Regional Development Agency Zlatibor and the Regional Chamber of Commerce of Užice. Such an organization should stimulate the formation of Regional Tourism Organizations in other parts of the Republic of Serbia, which would eventually grow into regional DMOs (Tourism Development Strategy of the Republic of Serbia, 2016). Cities and municipalities in the tourist region of Western Serbia are characterized by different levels of tourism development. The municipality of Čajetina is a dominant destina- tion that participates with more than 50% in tourist traffic. It is followed by Bajina Bašta, Užice and Nova Varoš, while other destinations in the region have tourist potential, but little tourist traffic is realized in them. 122 SITCON 2023 MANAGING THE FUTURE BY LEARNING FROM THE PAST – CONTEMPORARY TRENDS IN TOURISM & HOSPITALITY SITCON 2023 MANAGING THE FUTURE BY LEARNING FROM THE PAST – CONTEMPORARY TRENDS IN TOURISM & HOSPITALITY This step improved the management of the destination, created a new tourism product at the regional level, contributed to better marketing, visibility, and positioning of the destination on the tourist map of Serbia and the region (Bozovic, 2023). SWOT ANALYSIS SWOT analysis is intended as a set of the most interesting suggestions, proposals and ideas that can be found in scientific papers or obtained based on an overview of the researched area of Western Serbia. Based on the previous studies conducted in Western Serbia (Bozovic, 2023; Ivanovic, 2013) as well as based on field research and study of the region, the conclusions presented in the SWOT analysis were reached. Table 3. Swot analysis of the application of slow tourism in rural areas of Western Serbia. Strengths Weaknesses • geographical position, • great cultural potential, • great natural potential, • many rural tourist households, • authentic architecture, • authentic gastronomy • insufficiently developed infrastructure (roads, connectivity...), • insufficient education about modern forms of tourism Opportunities Threats • networking of destinations • creating a unique tourist product, • attracting new segments of tourists, • desire of the local population to get involved and support the further development of tourism • focusing only on individual attractions in the area • lack of financial resources • departure of young people from rural areas, • lack of support staff • slow administration at the state level • absence of a clear strategy for further sustainable development Source: Author’s Table 3. Swot analysis of the application of slow tourism in rural areas of Western Serbia. SITCON 2023 MANAGING THE FUTURE BY LEARNING FROM THE PAST – CONTEMPORARY TRENDS IN TOURISM & HOSPITALITY SITCON 2023 MANAGING THE FUTURE BY LEARNING FROM THE PAST – CONTEMPORARY TRENDS IN TOURISM & HOSPITALITY DISCUSSION AND CONCLUSION Worldwide tourism trends highlight the need and desire of modern society to return to tradition, the natural world, and an unspoiled environment, with a focus on rural places. Slow tourism has a propensity to take up a sizeable portion of the market. By adhering to sustainable development principles and fostering the local way of life, slow tourism emerges as one of the most important and practical avenues for the long-term development of rural areas. The territory of Western Serbia is a predominantly rural area that uses its potential to the greatest extent for the development of rural tourism, which is also the priority type of tourism in this region. It is also important to note that rural destinations such as Western Serbia, with a slow lifestyle, could potentially be considered an ideal place for the development of slow tourism based on the characteristics of this type of tourism. There is also a need for better cooperation between all communities in the territory of Western Serbia. Cooperation is necessary to create a brand and to better market the products of slow tourism as a unique tourist product of Western Serbia and the whole of Serbia. Such a united region, apart from representing a space for building competitiveness, would have a greater chance of being recognized as key to cooperation with European funds that would contribute to the further development of tourism. The creation of a unique tourist product can be implemented through networking and education aimed at raising the quality and competitiveness of the entire region of Western Serbia. Based on the creation of a unique tourist product and the development of slow tourism, a new business model can be developed. Namely, personalized trips, due to their value and acquired experiences, often lead tourists to spend more (Valls et al., 2019). As emotions increase and material value decreases, consumption undoubtedly increases (Kazeminia et al., 2016). This may mean that a higher quality service rich in experiences could enable better profitability. As stated by Lowry and Back, if we are what we eat, the same could be said: we are represented by the choices of our travels (Lowry & Back, 2015). ACKNOWLEDGMENT The authors gratefully acknowledge the financial support of the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Grant no. 451- 03-47/2023-01/200125 and Grant no. 451-03-23/2023-01). Source: Author’s. The main strengths of Western Serbia are reflected in its favorable geographical position. Important roads and tourist routes pass through the territory of Western Serbia, which represent a necessary condition for the survival and development of a tourist region (Romelić, 2008). Western Serbia has a favorable geographical and touristic position because it is located at the intersection of roads that connect Western and Southern Europe, Vojvodina, and Belgrade with the coast (Montenegro and southern Dalmatia). Also, a great strength is reflected in the fact that Western Serbia is characterized by a high complementarity of natural and anthro- pogenic values that have a favorable effect on its tourism position. Natural geographical units that influence the favorable tourist position of Western Serbia are certainly mountains and rivers, which are suitable for rural and slow tourism. Authentic architecture and recognizable gastronomy that can be seen in many rural tourist households is a factor that attracts many tourists (Bratić, 2021). The potential for collaboration areas and developing a distinctive tourism offering that would draw in new tourist demographics represents the opportunity for future growth. International travel trends highlight the need and motivation of contemporary individuals to return to tradition, the natural world, and an unspoiled environment, with a focus on rural areas. Slow tourism has the potential to become one of the most important and strategic avenues for the future development of rural areas when it complies to sustainable development principles, preserves local customs and culture, and occupies an important part of the market. Eventually, improved cooperation is required between all communities within the Western Serbian area. Collaboration is essential to building a name for slow tourism and promoting it as a distinct travel option for Western Serbia and all of Serbia (Bozovic, 2023). Weaknesses and threats for future development are insufficiently developed infrastructure that makes life difficult for the local population, as well as access to tourist destinations, as well as the increasing migration of young people from rural to urban areas. Also, insufficient education in tourism can be a threat, as well as focusing on only individual attractions in Western Serbia, thereby neglecting lesser-known destinations with a lot of potential. 123 123 Conference papers REFERENCES Božovic, T., Miljkovic, J., & Mikulic, K. (2021). Goals and outcomes of slow tourism: case study of Vojvodina Province. Tourism in Southeast Europe, Conference: 6 the International Scientific Conference ToSEE - Tourism in Southern and Eastern Europe 2021, 125-135, doi: 10.20867/tosee.06.9 Bozovic, T., Vujičić, M. D., Kovačić, S., Jovanović, T., & Pivac, T. (2022). Tourist motivation for slow travel: A case study of the Vojvodina Region, Serbia. Acta geographica Slovenica, 62(3), 33-45. 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Econstat Strategies for the Travel Industry. https://docplayer. net/40624488-Slow-tourism-seminar-stefano-dall-aglio-econstat-strategies-for-the-travel-industry.html Demirović Bajrami, D., Radosavac, A., Cimbaljević, M., Tretiakova, T. N., & Syromiatnikova, Y. A. (2020). Determinants of residents’ support for sustainable tourism development: Implications for rural communities. Sustainability, 12(22), 9438, https://doi.org/10.3390/su12229438 124 Conference papers SITCON 2023 MANAGING THE FUTURE BY LEARNING FROM THE PAST – CONTEMPORARY TRENDS IN TOURISM & HOSPITALITY Farhadi Uonaki, M., Rabani, R., & Khorasani, M. (2022). The Key Drivers of Slow Tourism Brand in Rural Destinations (Case Study: Javaherdeh Village-Mazandaran Province). Journal of Rural Research, 12(4), 716-733. Gardner, N. (2009). A manifesto for slow travel. Hidden Europe Magazine, 25, 10–14. Available at: h hiddeneurope.eu/a-manifesto-for-slow-travel (accessed: 10.07.2023). Ivanovic, М. (2013). Stanje i mogućnosti razvoja kulturnog turizma u Zapadnoj Srbiji (Doctoral d University of Novi Sad (Serbia)) Joffe, H., Yardley, L., & Marks, D. (2004). Research methods for clinical and health psychology. Content and thematic analysis, 56-68. Kastenholz, E., & Lima, J. (2013). Co-creating quality rural tourism experiences–the case of a Schist Village in Portugal. International Journal of Management Cases, 15(4), 193-204. Kazeminia, A., Hultman, M., & Mostaghel, R. (2016). Why pay more for sustainable services? The case of ecotourism. Journal of Business Research, 69(11), 4992-4997. Košić, K. (2009). Ruralni turizam Vojvodine i održivi razvoj. REFERENCES Doktorska disertacija, Novi Sad: Prirodno- matematički fakultet, Departman za geografiju, turizam i hotelijerstvo. Lipman, M. B., & Murphy, L. (2012). Make haste slowly: environmental sustainability and willing workers on organic farms. Tourism: Experiences and Mobilities. Bristol, 84-98. Liu, A. (2006). Tourism in rural areas: Kedah, Malaysia. Tourism management, 27(5), 878-889. https://doi. org/10.1016/j.tourman.2005.05.007 Lowry, L. L., & Back, R. M. (2015). Slow food, Slow Tourism and sustainable practices: A conceptual model. Sustainability, Social Responsibility and Innovation in Hospitality-Tourism, 71-89. Lumsdon, L. M., & McGrath, P. (2011). Developing a conceptual framework for slow travel: A grounded theory approach. Journal of Sustainable Tourism, 19(3), 265-279h Lunić, D., Micera, R. & Stanišić, N. (2020). The Covid-19 Pandemic and Slow Tourism. SITCON 2020 Marić, D., Leković, K., & Tomić, S. (2021). Slow tourism consumers’ recommendations. Teme, 1429-1 Mavrić, B., Öğretmenoğlu, M., & Akova, O. (2021). Bibliometric analysis of slow tourism. Advances in Hospitality and Tourism Research (AHTR), 9(1), 157-178. https://doi.org/10.30519/ahtr.794656 Mansfeld, Y., & Jonas, A. (2006). Evaluating the socio‐cultural carrying capacity of rural tourism communities: a ‘value stretch’approach. Tijdschrift voor economische en sociale geografie, 97(5), 583-601.l Markwell, K., Fullagar, S., & Wilson, E. (2012). Reflecting upon slow travel and tourism experiences. Slow tourism: Experiences and mobilities, 227, 233. Noor, F. A. M., Nair, V., & Mura, P. (2011). Conceptualizing a Framework for Slow Tourism in a Rural Destination in Malaysia. Noor, F., Nair, V., & Mura, P. (2015). Towards the conceptualization of a Slow Tourism theory for a rural destination. Hosp. Tour, 2015, 175-179. Nunes, S., & Cooke, P. (2021). New global tourism innovation in a post-coronavirus era. European Planning Studies, 29(1), 1-19. Romelic, Ј. (2008). Tourist Regions of Serbia, Novi Sad: Faculty of Science, Department of Geography, Tourism and Hotel Management.i Serdane, Z., Maccarrone-Eaglen, A., & Sharifi, S. (2020). Conceptualising slow tourism: A perspective from Latvia. Tourism Recreation Research, 45(3), 337-350.Sharpley, R. (2002). Rural tourism and the challenge of tourism diversification: the case of Cyprus. Tourism management, 23(3), 233-244. i Tourism development strategy of the Republic of Serbia for the period from 2016 to 2025, “Službeni glasnik RS“, no. 98/2016. Valls, J. F., Mota, L., Vieira, S. C. F., & Santos, R. (2019). Opportunities for slow tourism in Madeira. Sustainability, 11(17), 4534. Vesić, M., Savić, M., Pavlović, S., & Bolović, J. (2022). Sustainability-focused rural tourism development in Western Serbia. Glasnik Srpskog geografskog drustva, 102(1), 87-106. Voase, R. (1995). Tourism: the human perspective. REFERENCES Hodder and Stoughton 125 Conference papers
https://openalex.org/W2909285378	http://www.imedpub.com/articles/rural-community-gardens-capacity-to-increase-accessibility-and-affordability-of-healthy-foods-in-alabama.pdf	English	null	Rural Community Gardens Capacity to Increase Accessibility and Affordability of Healthy Foods in Alabama	Journal of Food Nutrition and Population Health	2,018	cc-by	5,207	Tel: (334)844-3780 Results: At conclusion of the three-month growing season, the nine rural community gardens in this study yielded 19 different varieties of vegetables weighing a total of 2,708.71 pounds and valued at $3,788.22. This equated to 15,110.64 servings of vegetables, which is enough for 33 adults to meet the USDA daily vegetable recommendations during the three-month growing season. Citation: Henning M, Brock R, Struempler B, Parmer S, Funderburk K, et al. (2018) Rural Community Gardens Capacity to Increase Accessibility and Affordability of Healthy Foods in Alabama. J Food Nutr Popul Health Vol.2 No.2:14 Conclusions: Findings from this study demonstrate by growing fresh vegetables, rural community gardens can increase the accessibility and affordability of healthy foods. This data suggests rural community gardens also may have a role in improving overall health and nutrition of rural populations. Keywords: Rural; Community gardens; Production capabilities; Harvest values; Accessibility and affordability Received: December 07, 2018; Accepted: December 19, 2018; Published: December 26, 2018 in the United States having some type of community garden program [1]. In 2018, the American Community Gardening Association reported more than 18,000 community gardens in the United States and Canada [3]. While this is far fewer than the amount of victory gardens, these modern community gardens have been shown to improve accessibility and affordability of healthy foods [4-9], increase fruit and vegetable consumption [4,8-13], increase physical activity [9-14] and provide numerous social [9,15,16] and psychological [9,13,14] benefits for both the gardeners and the community; these myriad of benefits suggest Citation: Henning M, Brock R, Struempler B, Parmer S, Funderburk K, et al. (2018) Rural Community Gardens Capacity to Increase Accessibility and Affordability of Healthy Foods in Alabama. J Food Nutr Popul Health Vol.2 No.2:14 Research Article Research Article 2018 Journal of Food, Nutrition and Population Health ISSN 2577-0586 iMedPub Journals www.imedpub.com Vol.2 No.2:14 Megan Henning1, Ruth Brock2, Barb Struempler2, Sondra Parmer2, Katie Funderburk2 and Alicia Powers3* Megan Henning1, Ruth Brock2, Barb Struempler2, Sondra Parmer2, Katie Funderburk2 and Alicia Powers3* Megan Henning1, Ruth Brock2, Barb Struempler2, Sondra Parmer2, Katie Funderburk2 and Alicia Powers3* Rural Community Gardens Capacity to Increase Accessibility and Affordability of Healthy Foods in Alabama *Corresponding author: Alicia Powers Setting: Six geographically dispersed counties throughout Alabama during one fall production cycle Setting: Six geographically dispersed counties throughout Alabama during one fall production cycle Participants: Nine established rural community gardens Director Strategy and Policy, Hunger Solutions Institute, College of Human Sciences, Auburn University, 334B Spidle Hall Auburn University, AL 36849, USA Main outcomes measured: Production capabilities were measured as harvest weight, in pounds and ounces, which also were converted to edible portion serving sizes. Harvest values were calculated using harvest weights and the average retail prices for harvested produce. Tel: (334)844-3780 1 Tri-County Health Department, USA 2 Alabama Cooperative Extension System, Duncan Hall Auburn University, USA 3 Hunger Solutions Institute, College of Human Sciences, Auburn University, USA , © Under License of Creative Commons Attribution 3.0 License \| This article is available in: http://www.imedpub.com/food-nutr Abstract 1 Tri-County Health Department, USA 2 Alabama Cooperative Extension System, Duncan Hall Auburn University, USA 3 Hunger Solutions Institute, College of Human Sciences, Auburn University, USA , Context: Urban community gardens have been shown to make healthy foods more accessible and affordable. There is limited research available, however, regarding the influence of rural community gardens, where populations consume less fruits and vegetables and often travel further and pay more for healthy food. Objective: To evaluate rural community gardens’ abilities to increase accessibility and affordability of healthy foods through measurement of harvest weights and conversions to edible portion serving sizes and harvest values Design: Longitudinal pilot study mons Attribution 3.0 License \| This article is available in: http://www.imedpub.com/food-nutrition-and-population-health/ Introduction Similarly, produce from community garden plots in Laramie, Wyoming, had an average value of $401 to $459 per plot in a single growing season [5]. In New York City, the value of yielded produce was even greater; yields from 67 community gardens in New York City had an estimated value of more than $200,000 [6]. Self-reported financial benefits were indicated by 58.9% of rural Missouri participants working in a community garden at least once a week stating they subsequently spent less money on food [7]. By providing a space to grow fruits and vegetables rather than purchase from food stores, community gardens provide a means to making healthful food more accessible and affordable. Community gardens offer a possible way to address barriers and increase accessibility and affordability of healthy foods, and ultimately, increase daily fruit and vegetable consumption of rural residents. However, the majority of research regarding benefits of community gardens has focused on urban areas. The purpose of this pilot study is to describe production capabilities and harvest values of rural community gardens in Alabama during one fall production cycle. Findings of this study will inform the capacity of rural community gardens in addressing barriers to healthy eating and guide future studies in determining the impact rural community gardens may have in improving fruit and vegetable consumption of rural populations. Interestingly the increase in accessibility and affordability to healthy foods provided by community gardens is not restricted just to gardeners. Both urban [13] and rural [5,7,8] studies reported gardeners sharing their produce with neighbors, friends and local charity organizations. Thus, not only are community gardens making a wide variety of healthy foods more accessible and affordable for gardeners, gardens also are making these foods more available for all members of the community. Therefore, the benefits of increased accessibility and affordability of healthy foods extends to non-garden members as well. Introduction Community gardens have existed for more than 250 years. They are thought to be direct descendants of the allotment gardens in Europe; however, the modern movement is more rooted in the victory gardens of World Wars I and II [1]. In 1942, more than 15 million victory gardens yielded more than 7.5 billion pounds of food [2]. As time passed and memories of the depression and wars faded, so did victory gardens. In the 1970s, the community garden movement experienced a resurgence, with every state 1 2018 Journal of Food, Nutrition and Population Health ISSN 2577-0586 Vol.2 No.2:14 Vol.2 No.2:14 community gardens may be a promising strategy in addressing many public health problems [17]. have an 8.6% higher prevalence of diabetes and 38.8% higher prevalence of heart disease than urban residents (P=0.001 and P<0.001, respectively). Similarly, an analysis of NHANES data [18] found significantly more rural adults were obese compared to urban adults (39.9% and 33.4% respectively, P<0.01). However, research has shown higher fruit and vegetable consumption is associated with decreased risk of chronic disease and obesity [19,20]. Unfortunately, rural residents consume less fruits and vegetables than urban adults [21] perpetuating their health problems. Furthermore, rural residents often have to travel farther to food stores [20], and often the closest food store is a convenience store [22,23]. Unlike grocery stores, convenience stores usually have limited quantities of healthy foods; healthy foods available are often significantly more expensive than the same product in a grocery store [23,24]. Documented evidence indicates community gardens increase access to healthy foods [4-9]. In one growing season, garden plots in Laramie, Wyoming, produced an average of 128 lbs of vegetables per 253 sq ft, which provided enough daily vegetable servings for one adult for nine months [5]. Increase in access to healthy foods also was seen in San Jose, California, where 10 community garden plots, varying from 100 to 600 sq ft in size, produced an average of 292 lbs of produce in one growing season [4]; and in New York City where 67 gardens, totaling 1.7 acres, produced more than 87,000 lbs of produce in a season [6]. An additional benefit of community gardens is increased affordability of fresh produce [4-6]. In 10 community garden plots in San Jose, California, each plot’s produce yield had an average value of $435 in a four-month growing season [4]. Population/Sample Researchers used a purposive sample of nine, established, rural community gardens in six counties throughout Alabama. Rural was defined, using the US Census Bureau’s definition, as an area with a core metropolitan area of less than 50,000 people [25]. The recruited gardens represented diversity based on: geographical location, size and type of garden and variety of crops grown. Gardens which did not have a fall production cycle were excluded. Research design This longitudinal pilot study measured production capabilities and harvest values of rural community gardens in Alabama for the fall 2017 production cycle. Production capabilities were measured as harvest weight, in pounds and ounces, which also were converted to edible portion serving sizes. Harvest values were calculated using harvest weights and the average retail prices of produce. A growing body of evidence demonstrates the increase in accessibility and affordability of fresh fruits and vegetables provided by community gardens contributes to increased fruit and vegetable consumption [4,8-13]. A study in Flint, Michigan [10], found household members who participated in community gardens consumed 1.4 times more fruits and vegetables than those who did not. A study in Denver, Colorado [11], found 56% of community gardeners met daily recommendations for fruit and vegetable consumption compared to 25% of non-gardeners (P<0.05). Similar increases were seen in rural populations. A study in rural Iowa [8] found access to a garden of any type significantly increased the likelihood of meeting the daily vegetable recommendations (P=0.002). These findings have implications on many public health problems prevalent in today’s society, specifically chronic disease and obesity. This article is available in: http://www.imedpub.com/food-nutrition-and-population-health/ Procedures The university’s Institutional Review Board deemed the study protocol as not constituting “human subjects research”. Garden Information Forms were distributed to community garden leaders in July 2017. The community garden leaders were given two weeks to complete and return the form to the researchers. Each garden’s vegetable production varied. On average, each garden produced 301 lbs of unprocessed vegetables valued at $470.82 ($0.91/lb). When converted to edible yield, each garden produced an average of 223.66 lbs of raw, ready to cook or eat vegetables which equates to an average of 1,678.96 servings of vegetables per garden. Table 3 summarizes the average production capabilities and harvest values during a fall production cycle for the nine community gardens in this study. Harvested crop weights were collected from the middle of September to the middle of December. Harvesters received training on weighing crops harvested and using the Harvest Information Forms to record crop weights. Blank Harvest Information Forms were kept in a weather proof box at each garden and were to be completed each time crops were harvested. Once completed, the forms were placed in a separate weather proof box. Community garden leaders retrieved completed forms and submitted them to a local data entry clerk on a monthly basis. Table 1 Descriptive characteristic of nine rural community gardens. Table 2 Total weight for each type of vegetable grown at nine rural community gardens. Garden Type of garden Size (acers or Sq ft ) Number of vegetables grown 1 Raised Bed (n=4) 128 sq ft 6 2 Raised Bed (n=8) 256 sq ft 3 3 Raised Bed (n=10) 320 sq ft 5 4 Raised Bed (n=53) 1,696 sq ft 10 5 Row 0.05 acres 1 6 Row 0.25 acres 4 7 Row 0.25 acres 1 8 Row 0.50 acres 1 9 Row 0.75 acres 4 Table 1 Descriptive characteristic of nine rural community gardens. Researchers developed a spreadsheet for each garden where data entry clerks entered data from Harvest Information Forms. Weight data were entered by garden, date and crop. To ensure accuracy of data entry, the data entry clerks participated in a virtual training on how to access the spreadsheet on the organization’s internal website and properly enter data. To calculate serving size, researchers converted each vegetable’s harvest weight to edible portion by multiplying by yield percentage from USDA’s Food Buying Guide for Child Nutrition Programs [27]. Procedures Once the edible portion weight was calculated, researchers used USDA’s Food Composition Database [28] to determine the number of servings provided by a specific weight for each vegetable. Table 2 Total weight for each type of vegetable grown at nine rural community gardens. Table 2 Total weight for each type of vegetable grown at nine rural community gardens. Vegetables Weight (lbs) Beans 4.5 Beets 2.63 Broccoli 12.5 Brussel Sprouts 60 Cabbage 8.63 Carrots 9.38 Cauliflower 1.5 Greensa 1,058.13 Lettuce 15.19 Okra 106 Onion 1 Peas 280 Peppers 64.5 Pumpkins 182 Radishes 2.5 Squash 3 Sweet Potatoes 815 Tomatoes 62.13 Turnips 20.13 aGreens included collard greens, mustard greens, turnip greens, Swiss chard and kale. To tabulate harvest value, data for price per pound of a specific vegetable during the harvest season were determined from the USDA’s Agricultural Marketing Services for the Southeast United States [29]. The average prices for okra, peas and turnips were not available on this resource; therefore researchers went to a local retail outlet to determine the cost rural residents paid per pound. Information on harvest values were tabulated by multiplying collected harvest crop weights by average price per pound. Statistics Appropriate descriptive statistics were used to summarize data. Determination of harvest weights, serving sizes and values were determined for each garden, as well as, for the aggregate and average of included gardens. Instrumentation General characteristics of rural community gardens were obtained using a Garden Information Form developed by the researchers. The form included nine questions and was completed by the community garden leader. Chronic disease and obesity are complex problems and are especially widespread in rural communities. Data from the US Centers for Disease Control and Prevention’s (CDC) Behavioral Risk Factor Surveillance System [17] have shown rural residents Each garden used a Rubbermaid® Dial Hanging Scale with Tare and a Taylor® 20lb Hanging Scale Platform to weigh produce to 2 2018 Journal of Food, Nutrition and Population Health ISSN 2577-0586 Vol.2 No.2:14 Vol.2 No.2:14 Among the gardens, four were raised bed gardens, and five were row gardens (Table 1). Throughout the fall production cycle, partnering gardens produced 19 different vegetables, with greens yielding the greatest weight followed by sweet potatoes and peas (Table 2). the nearest ounce. Accuracy of the scale was predetermined by Rubbermaid® at the checkpoint of 10lbs with a tolerance of +/- 2oz [26]. The scale and scoop were maintained in a permanent weigh station or on a portable scale stand where both were locked to support calibration maintenance. The nine community gardens in this study yielded 2,708.71 lbs of vegetables valued at $3,788.22 in a single three-month fall growing season. Edible yield (74%) totaled 2,012.91 lbs. When converted to serving sizes, this equated to 15,110.64 servings of edible vegetables. Table 3 provides details on total harvest weights, serving sizes and values. Harvest data were recorded using Harvest Information Forms created by the researchers. Information collected included collection date, crop harvested and weight in pounds and ounces. aGreens included collard greens, mustard greens, turnip greens, Swiss chard and kale. Results aGreens included collard greens, mustard greens, turnip greens, Swiss chard and kale. Nine gardens totaling 1.85 acres were included in this study. © Under License of Creative Commons Attribution 3.0 License 3 2018 Vol.2 No.2:14 Journal of Food, Nutrition and Population Health ISSN 2577-0586 2018 Vol.2 No.2:14 Table 3 Weight, edible portion, servings and cost savings of nine rural community gardens. Table 3 Weight, edible portion, servings and cost savings of nine rural community gardens. Garden Weight Produced (lbs) Edible Portion Produced (lbs)a Total Edible Servingsb Total Value ($) Value/lb ($) Raised Bed 1 20.44 15.32 128.22 23.68 0.86 2 21.64 17.29 115.21 21.67 1 3 27.82 21.52 167.91 31.34 0.89 4 175.81 144.37 936.03 216.13 0.81 Subtotalc 245.71 198.49 1,347.37 292.82 3.56 Row 5 81 59.94 485.51 76.14 1.06 6 735 538.59 4,444.31 830.94 0.88 7 110 81.4 659.33 103.4 1.06 8 229 169.46 1,372.60 215.26 1.06 9 1,308.00 965.03 6,801.00 2,269.67 0.58 Subtotalc 2,463.00 1814.42 13,763.27 3,495.41 4.65 Totalc 2,708.71 2,012.91 15,110.64 3,788.22 8.22 Mean (SD) 300.97 (438.7) 223.66 (322.48) 1,678.96 (2,348.70) 470.82 (772.7) 0.91 (0.16) aRaw edible weights were calculated using edible yield percentage. bOne serving is a one-half cup for all vegetables except leafy greens, which is one full cup. The USDA recommendation for adults is two and one-half cups of vegetables or five cups of leafy greens per day. cMay not sum to total due to rounding. Table 3 Weight, edible portion, servings and cost savings of nine rural community gardens. aRaw edible weights were calculated using edible yield percentage. bOne serving is a one-half cup for all vegetables except leafy greens, which is one full cup. The USDA recommendation for adults is two and one-half cups of vegetables or five cups of leafy greens per day. cMay not sum to total due to rounding. aRaw edible weights were calculated using edible yield percentage. bOne serving is a one-half cup for all vegetables except leafy greens, which is one full cup. The USDA recommendation for adults is two and one-half cups of vegetables or five cups of leafy greens per day. cMay not sum to total due to rounding Compared to a study [6] of 67 community gardens totaling 1.7 acres in New York City [6], the current study’s harvest weight production capabilities were much less despite having a greater total area of 1.85 acres. Results A factor which could contribute to this difference is urban community gardens usually employ bio intensive farming methods [4,6,31]. Bio intensive farming methods emphasize soil quality, utilize raised beds and high production techniques and promote the growth of high yield vertical crops, like tomatoes and beans [4,6,31]. These methods allow gardens to grow crops more efficiently in limited space and have been shown to increase average yields per square foot [4,6,31]. The rural raised bed and row gardens in the current study, however, more closely resemble large-scale industrial farming [31]. With more open space available in rural areas [32], there is less emphasis on producing high yields in a small area, even in the raised bed gardens. High availability of open space permits the growth of crops which require more space, like collard greens and sweet potatoes, therefore decreasing the yields per square foot. This is supported with the current study’s results only reporting an average of 0.10 lb/sq ft of produce in the raised bed gardens and 0.03 lb/sq ft in the row gardens, for a total average of 0.05 lb/sq ft. In comparison, the urban studies in New York City [6] and in California [4], which both utilized bio intensive farming methods, reported 1.2 lb/sq ft of produce and 0.75 lb/sq ft of produce, respectively. Discussion The results from this longitudinal pilot study demonstrate the potential production capabilities and harvest values of rural community gardens. During a single three-month growing season, the nine community gardens in this study yielded more than 2,700 lbs of vegetables with an estimated value of more than $3,750. Results also suggest production capabilities of rural community gardens can have potential nutritional implications for residents. When converted to serving sizes, this study’s yield equated to more than 15,000 servings of vegetables which is enough for 33 adults to meet the USDA daily recommendations of two and one-half cups of vegetables or five cups of leafy greens per day [30] throughout the three-month growing season. This article is available in: http://www.imedpub.com/food-nutrition-and-population-health/ Harvest value trimmed and cleaned to remove any pits, stems or other inedible portions. Edible yield provides researchers with the percent of a food item which consumers can eat and allows researchers to calculate how much of a raw food item is able to be consumed. By utilizing the edible yield, the serving size calculations in the current study represent edible servings. This makes the results more applicable to the potential nutrition implications of rural community gardens. Like production capabilities, there also were limited rural studies which reported specific data on the value of produce for rural community gardens. The current study’s harvest value was $0.91/ lb compared to $3.62/lb reported in the rural Wyoming study [5]. This difference is most likely due to different methods used to obtain price per pound data. In the current study, researchers used the USDA’s report of average, nonorganic retail prices of produce in the Southeastern United States [29] or local, nonorganic store prices. In comparison, researchers in the Wyoming study [5] used local farmer’s market prices or organic grocery store prices. Using farmers market and organic store food prices mostly likely increased the value of price per pound in the Wyoming study [5] as prices in these settings are typically higher compared to the nonorganic groceries used in the current study [33]. The current study’s harvest value per pound also was less than other urban community garden studies who reported harvest values [4,6]. The New York City study [6] reported an approximate value of $2.44/lb during their growing season while the San Jose, California, study [4] reported $1.53/lb. Researchers in the New York City study [6] used similar methods to the Wyoming study [5], and obtained price per pound values from local, organic grocery stores which most likely explains the difference from the current study’s results. In the San Jose, California, study [4], researchers used the Bureau of Labor statistics for the Western region to determine price per pound of vegetables for four of the vegetables and then local, nonorganic grocery store prices for all other vegetables [4]. These methods are similar to the current study’s, therefore the differences observed could be due to the difference in geographic locations where food costs are higher in the Western United States compared to the Southeastern United States [34]. Conclusions The results from this pilot study suggest rural community gardens, by growing fresh vegetables, can increase accessibility and affordability of healthy food. During a three-month growing season, nine gardens in this study yielded 2,708.71 lbs of vegetables valued at $3,788.22. This equated to 15,110.64 servings of vegetables, which is enough for 33 adults to meet the daily USDA recommendations throughout the three-month fall growing season. This data suggest rural community gardens also may have a role in improving overall health and nutrition of rural populations. Future studies are needed to determine rural community gardens’ impact on fruit and vegetable consumption of the rural population who are more obese [18] and at higher risk for chronic disease [17] than their urban counterparts. Acknowledgements Given the current study was a pilot study involving applied research, limitations are inherent. Unlike previous studies [4- 6] which reported spring and summer production cycles, this current study used a fall production cycle. This most likely limited this study’s crop variety, value and production capacity. Other limitations included diversity in garden size and use of both row and raised bed gardens, which made comparisons among gardens and other studies difficult. While the current study’s garden diversity was a limitation, it is also the strength of the study. Unlike previous research [4-6], results from the current study are from six geographically different counties throughout the state rather than a single city. This increases representativeness and generalizability of the current study’s results of rural community gardens’ role in positively influencing accessibility and affordability of healthy food. The authors gratefully acknowledge the contributions to this work by the Alabama Cooperative Extension System, Alabama Extension’s Supplemental Nutrition Assistance Program- Education (SNAP-Ed) at Auburn University, Alabama Extension’s High Obesity Program entitled Alabama Preventing and Reducing Obesity: Helping Engage Alabamians for Long-Term Health (ALProHealth) and the rural community gardens which participated. Harvest weights Researchers found few rural studies available for comparison. Nevertheless, the current study’s harvest weight production capabilities were higher than the other rural study [5] found. In the current study, the average yield was 300.97 lbs of produce in a single three-month growing season while the other rural study [5] reported an average yield of 128.00 lbs of produce in a single four and a half month growing season. The current study’s production capabilities also were greater than a previous urban community garden study in San Jose, California [4], which reported an average yield of 292.10 lbs of produce in a single four-month growing season. These differences in harvest weights are most likely due to the greater size of rural gardens included in this study compared to those in the previous studies [4,5]. The gardens in this study averaged 8,978 sq ft while those in the rural Wyoming study [5] averaged 253 sq ft. The California study [4] did not report an average size, but their garden plot sizes ranged from 100 to 600 sq ft, which is less than the currents study’s. The current study’s pounds per square foot results also were less than the other rural study [5], which reported 0.51 lb/sq ft of produce. This difference could be due to the gardens in the Laramie, Wyoming, study [5] utilizing growing methods more similar to the bio intensive methods utilized by urban community gardens to help maximize their production capabilities in a challenging growing environment. In addition, this other rural study’s [5] growing season was four and a half months, while the current study’s was only three-months. 4 2018 Vol.2 No.2:14 Journal of Food, Nutrition and Population Health ISSN 2577-0586 2018 Journal of Food, Nutrition and Population Health ISSN 2577-0586 Vol.2 No.2:14 Vol.2 No.2:14 Harvest value In addition, in the both of the urban studies [4,6] tomatoes, which have a national nonorganic retail average price of $1.22/lb [29], were the highest yielded crop while in the current study greens, which have a national nonorganic retail average price of $0.98/lb [29] had the highest yield which could further explain the differences observed. Increasing accessibility and affordability of healthy foods is especially important for rural residents who often have to travel further [20] and pay more for healthy food [22,23]. To further describe the capabilities of rural community gardens, future studies should seek to include a greater number of rural community gardens. With more gardens included, researchers should separate raised bed and row gardens as well as recognize if bio intensive farming methods were used to further compare the capabilities of these diverse types of gardens. Also, data should be collected across an entire year to understand seasonal and annual production capacities and harvest values. A better understanding of production capabilities and harvest values of rural community gardens is the first step to understanding community gardens’ potential role in impacting rural population’s overall nutrition and health. © Under License of Creative Commons Attribution 3.0 License Funding/Financial Disclosures Funding for this research was provided by the Alabama Cooperative Extension System, the US Department of Agriculture’s Supplemental Nutrition Assistance Program– Education and the Centers for Disease Control and Prevention High Obesity Program. An additional strength of the current study was unlike the other rural study [5] which determined production capabilities in terms of serving sizes, the current study utilized edible yields when calculating serving sizes. Most harvested produce is not in the form it will be consumed. Often these foods need to be Conflict of Interest Disclosure References 1 Fowler MLC (1978) Growing with Community Gardening. 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https://openalex.org/W4312067069	https://joppp.biomedcentral.com/counter/pdf/10.1186/s40545-022-00502-3	English	null	Patient medication counselling in community pharmacy: evaluation of the quality and content	Journal of pharmaceutical policy and practice	2,022	cc-by	11,070	Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 https://doi.org/10.1186/s40545-022-00502-3 Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 https://doi.org/10.1186/s40545-022-00502-3 (2022) 15:103 Open Access © 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. Segun Johnson Showande* and Monioluwa Wonuola Laniyan Segun Johnson Showande* and Monioluwa Wonuola Laniyan Abstract Correspondence: sj.showande@gmail.com Correspondence: sj.showande@gmail.com Department of Clinical Pharmacy and Pharmacy Administration, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria Department of Clinical Pharmacy and Pharmacy Administration, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria Abstract Background: Patient medication counselling (PMC) is a pharmaceutical care service targeted at optimizing patient drug use, safety and improving treatment outcomes. This study assessed the content and quality of PMC from the community pharmacists’ (CPs) and pharmacy customers’ (PCs) perspectives. Methods: A cross-sectional questionnaire-guided survey was conducted in Ibadan, Nigeria, among 125 CPs and 612 PCs. The 35-counselling items validated United States Pharmacopeia Medication Counselling Behaviour Guideline scale with 10-point graded responses (1 = poor to 10 = excellent) was used. Self-reported medication counselling information content provided by CPs and received by PCs was assessed and expressed in median and interquar- tile ranges. The quality of PMC was evaluated and graded as poor (1–29.9%), unsatisfactory (30–59.9%), satisfactory (60–79.9%) and excellent (80–100%). Associations between demographic variables and overall quality of counselling were determined with Mann–Whitney U and Kruskal–Wallis tests at p < 0.05. Results: The response rate was 92.5% and 97.6% for PCs and CPs, respectively. The PCs’ opinions on the individual content of the PMC provided by the CPs were significantly different from the pharmacists’ self-report (p < 0.05). Some of the PMC content included how to take the medicine PC = 6.00 (2.00) vs CP = 8.00 (2.00), information on possible side effects PC = 6.00 (2.00) vs CP = 8.00 (2.00), taking history of allergies and other medications PC = 6.00 (6.00) vs CP = 7.00 (1.00), and how to incorporate drug regimen into daily routine PC = 5.00 (6.00) vs CP = 8.00 (3.00). The quality of PMC purportedly provided by CPs and received by the PCs was satisfactory (75%) and unsatisfactory (55%), respectively. The quality of communication counselling offered by CPs trained in Nigeria (Mean rank = 62.49) was higher than those trained outside Nigeria (Mean rank = 26.40), U = 228.00, p = 0.024. The PC’s age, marital status, and highest educational qualification were significantly associated with their opinion on the quality of counselling received. Conclusions: Both the community pharmacists and pharmacy customers reported the provision of patient medica- tion counselling on side effects, drug usage, medication history and allergies among others. However, the quality of counselling provided by the pharmacists was satisfactory, but the quality of counselling received by the pharmacy customers was unsatisfactory. Pharmacists may need to engage pharmacy customers more during medication counselling. Backgroundh The pharmacy profession has evolved into a more patient-oriented practice involving the provision of care, advice, and medication counselling [1]. Pharmacists are the third largest healthcare professional group in the world [2] and community pharmacists are visited daily by millions of people across the globe. On average, patients visit community pharmacists (CPs) nine times more than they do primary care physicians in a year [3, 4]. They are mostly the first point of contact for some patients and for few others, the only contact point [5]. This pro- vides the CPs with the opportunity to leverage on the interaction with individual patients, intervene in health- related matters, facilitate public health services, and pro- vide other health and well-being services [6]. Both the Omnibus Budget Reconciliation Act of 1990 (OBRA’90) and the code of ethics of the Royal Pharmaceutical Soci- ety of Great Britain enjoined pharmacists to be actively involved in patients care by providing information and advice on safe and effective medicine use. [7, 8]. Many professional organizations in the United States of America and Australia have published guidelines on counselling [18, 27, 28] with varying content [29]. Various studies have examined the use of counselling guidelines by pharmacists [30, 31], rate of verbal counselling with author-defined counselling content [32–35], and the con- tent of verbal counselling [36, 37]. Pharmacists behaviors were evaluated in these studies, but others have observed both pharmacists and PCs’ PMC services [32, 38]. As much as it is pertinent to know the number of patients or consumers who received a pre-defined counselling con- tent or medication information, it is equally important to know how comprehensive is the content of the PMC or the quality of the counselling received by the patients or offered by the pharmacists. Most of these cross-sec- tional studies did not evaluate the quality of pharmacist counselling which considered empathic understanding, acceptance, and the demonstration of genuine feeling during the counselling process [19]. This study, there- fore, evaluated the community pharmacists’ and phar- macy customers’ opinions on the content and quality of PMC provided by community pharmacists to PCs using the United States Pharmacopeia Medication Counselling Behaviour Guideline (USP-MCBG) [29]. f Patient medication counselling (PMC) may be described as “providing medication information orally or in written form to the patients or their representatives or providing proper directions of use, advice on side effects, storage, diet and lifestyle modifications” [9]. Keywords: Patient medication counselling, Pharmacist, Consumer, United States Pharmacopeia Medication Counselling Behaviour Guideline, Nigeria the Pharmacy Council of Nigeria (PCN) [24], and the National Universities Commission [25] made provision for inclusion of communication skills and patient coun- selling in the undergraduate curriculum with experiential training at community pharmacies and hospitals during the externship and clerkship programs, respectively. This is also included in one of the modules offered to commu- nity and hospital pharmacists in the PCN organized Man- datory Continuing Professional Development [26]. Backgroundh The provi- sion of appropriate and adequate PMC by pharmacists could help pharmacists identify and resolve drug therapy problems [10, 11] engage patients in self-management of diseases [12, 13], and prevent treatment failure and limit resource wastage [14, 15]. In developing countries like Nigeria, the provision of such patient-oriented services by pharmacists is still evolving compared to developed nations [16, 17].h Study design and settingh This cross-sectional questionnaire-guided survey was carried out in community pharmacies in Ibadan, Nigeria. The city with an estimated population of 3,649,000 as of 2021 is located in the south-western part of the nation in Oyo state [39]. The study lasted for 6 months, from June to November 2021. Participants involved in the study included community pharmacists and customers who visited the pharmacies during the period of the study and who may be patients, caregivers or customers herein referred to as pharmacy customers (PCs). The provision of PMC is however compulsory and backed by enabling laws in some countries [18, 19], including monetary penalties for failure to provide PMC for prescription drugs [20]. Yet, pharmacists have often failed to deliver appropriate and detailed medication information to patients [12], despite increased interest in the quality of patient counselling, its propriety, and acceptability in community pharmacies [21]. Though pharmaceutical care is fast becoming the mode of prac- tice in Nigeria, most pharmacists still provide inadequate patient-oriented services including PMC [22]. This may be due to professional complacency and conservatism, extrinsic system failures, and inadequate human resources for health as suggested by Abdu-Aguye et al. [23]. There is no PMC policies, guidelines, incentives or standards in the country. However, to enhance the quality of PMC, © 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. Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Page 2 of 14 Keywords: Patient medication counselling, Pharmacist, Consumer, United States Pharmacopeia Medication Counselling Behaviour Guideline, Nigeria Sample size and sampling techniques A total of 158 pharmacies were registered within Ibadan metropolis, Oyo state, as of December 2020 according to the Pharmacy Council of Nigeria register. Taro Yamane formula was employed to determine the sample size with Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Page 3 of 14 a 0.05 margin of error and 95% confidence interval with a 10% provision for non-response. The calculated sample size for the community pharmacies was 125. applicable” grading was not included in the scale used for this study. Thus, the USP-MCBG scale graded responses was from 1 to 10, for this study. The USP-MCBG scale contained four subscales or components; needs assess- ment, precaution and warnings, management of treat- ment and communication with 9, 8, 13, and 5 items, respectively. y p For the PCs, the sample size was determined by modi- fying the method described by Showande and Babalola in a study carried out in the same city [1]. Briefly, a conserv- ative estimate of PCs who visited and bought drugs from the selected community pharmacies between 9.00 am and 12.00 pm daily (Monday to Friday) were observed by the authors to be an average of 120. The sample size for each pharmacy was calculated with the 120 PC’s sample frame using Taro Yamane formula to be 95. Furthermore, allowance for a 10% non-response was added and the eventual PCs’ sample size for each community pharmacy was 102. Six community pharmacies with high patronage were purposively selected from six local government area (LGA) out of the 11 LGAs in Ibadan for the PCs’ sur- vey. One community pharmacy per LGA. Four commu- nity pharmacies were selected from the five urban LGAs while two were selected from the six semi-urban LGAs in Ibadan. Thus, the total sample size of the PCs surveyed in the six selected community pharmacies was 612. The same USP-MCBG scale was used for the commu- nity pharmacists’ survey but in addition, a 7-item Lik- ert scale with 5 graded responses (strongly disagree—1, strongly agree—5) on the pharmacists’ understanding of the goals of patient medication counselling was included beside the respondent characteristics. Since the pharma- cist-participants were literates, translation to a local lan- guage was not required. The questionnaire for PCs was translated from English language to Yoruba language and back translated for consistency because some PCs were non-literate in English. Inclusion and exclusion criteriah The survey included the superintendent pharmacists, staff pharmacists, non-intern locum pharmacists and pharmacists undergoing the compulsory 1-year National Youth Service Corps (a year mandatory national program for all graduates less than 30 years old) who gave writ- ten informed consent to participate in the study. Intern pharmacists and pharmacy students posted to commu- nity pharmacies for externship were excluded from the survey. The PCs, as defined under the study design and setting section, who were 18 years and above, had given verbal informed consent and could read and understand either English or Yoruba language were included in the study. Pharmacy customers who were indisposed to attend to the questionnaire were excluded. One hundred and twenty-five pharmacies were con- veniently selected and one pharmacist per pharmacy who consented to participate was administered the question- naire which was retrieved after completion on the same day. Where the pharmacist denied verbal consent at the time of visit, another pharmacist on duty was approached or in cases where there was only one pharmacist on duty the next community pharmacy on the list was visited. In another situation where there was no pharmacist on duty on two different visits, the next pharmacy on the list was visited. Study procedure Th h The pharmacy customers were selected through conveni- ence sampling. A day in a week was picked to conduct the survey in each of the six selected community phar- macies among the pharmacy customers from 9 am to 12 noon. Pharmacy customers exiting the community phar- macy who had purchased a drug were approached, the study was explained to them and consent sort. Those who gave verbal informed consent to participate were given the questionnaire written in English language, but those who understood only Yoruba were assisted by a trained research assistant (a final year pharmacy student) to fill the questionnaire. The questionnaire took about 15 min to fill. If the consent was denied, the next customer com- ing out of the pharmacy was approached and the sam- pling continued until the estimated sample size from the pharmacy was reached.i Sample size and sampling techniques The Cronbach alpha reliability coefficient of the scales used in the study ranged from 0.701 to 0.937. Study instrumenth The PCs’ questionnaire contained sections on respondent characteristics, what the PCs understood about medica- tion counselling and what is expected from pharmacists during medication counselling. It also contained the USP-MCBG scale to evaluate the content and quality of medication counselling provided to PCs by community pharmacists. The USP-MCBG has been used to evaluate patient medication counselling by pharmacists and was found reliable even after modifications [29]. There are 35 items on the scale and the rating of the items utilized 11-point graded responses divided into six categories: 0—not applicable, 1—not done, 2—poor, 3–5—unsat- isfactory, 6–7—satisfactory, 8–10—excellent. The “not But before the main study, the study instruments were reviewed by four pharmacy researchers for relevance, appropriateness, and acceptability. A pre-test was con- ducted in five pharmacies for all the participants in the Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Page 4 of 14 females slightly higher 282 (50.4%). Approximately half, 241 (43.7%), of the PCs had a tertiary education and 116 (21.0%) were employees while 145 (26.3%) were students. Other respondent characteristics are listed in Table 1. study. Based on the comments of the researchers the wordings of the items in the USP-MCBG for the partici- pants were modified to reflect the type of participants being assessed. For example, for item 30 in the USP- MCBG scale, it was captured as “The pharmacist probed me for additional information” for PCs, and for commu- nity pharmacists, it was written as “I usually probe for additional information from the patient/customer”. Five PCs per pharmacy and one pharmacist per community pharmacy were surveyed during the pre-test. No modifi- cation was made to the instrument after the pre-test. The data collected during the pre-test were not included in the final analysis. Some of the PCs, 194 (34.3%), claim to visit the phar- macies at least once a month. Others visited the phar- macies at least once a week 67 (12.1%), once in 2 weeks 84 (15.1%) while some were visiting the pharmacies for the first time 41 (7.4%). The purpose of the visit included complaining about illness 72 (13.4%), purchasing drugs 372 (69.8%), for medical advice 38 (7.1%), refilling pre- scriptions 32 (6.0%) and for a scheduled appointment with the pharmacist 10 (1.9%). Most of the PCs, 497 (90.0%), knew that they needed to be counselled on the use of their medications. Pharmacy customer assessment Six hundred and twelve questionnaire were distrib- uted and all were retrieved. Forty-six of these question- naire were unusable due to missing valuable data in the Likert scale. Thus, 566 questionnaire were analyzed. The response rate was therefore 92.5%. The mean age of the PCs was 34.16 ± 13.35 years (range 18–82 years). There was an almost equal distribution of gender with Study instrumenth The PCs, 437 (79.5%), also believed that medication counselling should be provided in community pharmacies. However, few of the PCs, 190 (34.4%), believed that only the pharmacist is in the best place to give information on medications obtained from the pharmacy. The PCs opined that some of the informa- tion the pharmacists should provide during medication counselling should include indications 495 (90.0%), what to do with leftover medications 276 (50.5%) and the cost of the medication 386 (70.8%) (see Table 2). Data analysis y Descriptive statistics of sample characteristics and ques- tionnaire items were used to summarize part of the data obtained using mean and standard deviation, median and interquartile range, and proportions with the aid of the Statistical Package for Social Sciences Windows version 26.0 (IBM corp. New York, USA). The quality of coun- selling was estimated as a percentage: 100 (Mean USP- MCBG scale score or subscale score)/(Total obtainable USP-MCBG scale score or subscale score). For the four subscales or components; needs assessment, precaution and warnings, management of treatment and communi- cation, the total obtainable scores were 90, 80, 130, and 50, respectively. The total obtainable USP-MCBG scale score was 350 (item 34 on the scale has four questions, the average of the scores was considered for item 34). The quality of counselling was further categorized as: poor (1–29.9%), unsatisfactory (30–59.9%), satisfactory (60–79.9%); and excellent (80–100%) based on the graded responses of the USP-MCBG scale. The differences in the USP-MCBG scale content score between the PC’s and the community pharmacist’s self-reports were deter- mined with independent sample t-test with consideration for equal variance assumed or not assumed. The associa- tion between the socio-demographic variables of the par- ticipants and the USP-MCBG subscales and total scores was assessed using Mann–Whitney U and Kruskal–Wal- lis tests. The level of significance was set at p < 0.05. Pharmacist self‑evaluation One hundred and twenty-five questionnaire were dis- tributed and 122 were retrieved and analyzed. Three pharmacists were unable to complete and submit the questionnaire. The response rate was 97.6%. The aver- age age of the pharmacists was 34.54 ± 12.07 years (range 23–85 years), 76 (63.3%) were males, and only 5 (4.1%) were trained outside Nigeria (Table 3). The mean year of community pharmacy experience was 6.31 ± 8.69 years. The community pharmacists agreed that patient counsel- ling should comprise both written and oral provision of medication information to PCs 113 (92.6%) and it should be a one-on-one interaction 112 (91.8%). Pharmacists opined that medication counselling should provide infor- mation on the appropriate use of medication 115 (94.3%), it should entail being involved in the patient’s social, die- tary, psychological and emotional needs 102 (83.6%) and good communication is a vital tool to achieve optimal patient medication counselling 115 (94.3%). Community pharmacists are stakeholders concerning medication and medication-related information 116 (95.1%). Pharmacists’ and pharmacy customers’ opinions on the content and quality of patient medication counsellingh The PCs’ opinions on the individual content of the PMC provided by community pharmacists were significantly different from the pharmacists’ self-report (p < 0.05); see Page 5 of 14 Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Page 5 of 14 Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Page 5 of 14 Table 1 Demographic characteristics of pharmacy customers * The pharmacy customers’ age was binned based on an equal percentile of scanned cases Demographic information Variables Frequency (percentage %) Mean ± SD Age, years, (n = 566) 34.16 ± 13.35 Age group ≤ 23 192 (33.9) 26–44 257 (45.4) 45–63 102 (18.0) 64+ 15 (2.7) Gender (n = 560) Male 278 (49.6) Female 282(50.4) Religion (n = 533) Christianity 376 (70.5) Islam 155 (29.1) Traditional practice 2 (0.4) Marital status (n = 557) Single 283 (50.8) Married 246 (44.2) Widowed 17 (3.1) Divorced 5 (0.9) Separated 1 (1.1) Highest academic qualification obtained (n = 551) No formal education 15 (2.7) Primary education 23(4.2) Secondary education 180 (32.7) Tertiary education 241 (43.7) Postgraduate education 92 (16.7) Working status (n = 552) Self-employed 132 (23.9) Employee 116 (21.0) Unemployed 27 (4.9) Civil/public servant 41 (7.4) Artisan 18 (3.3) Student 145 (26.3) Farmer 4 (0.7) Trader 28 (5.1) Businessman/woman 37 (6.7) Others 4 (0.7) Table 1 Demographic characteristics of pharmacy customers Demographic information Variables Table 1 Demographic characteristics of pharmacy customers 34.16 ± 13.35 The pharmacy customers’ age was binned based on an equal percentile of scanned cases * The pharmacy customers’ age was binned based on an equal percentile of scanned cases to prevent or manage side effects 7.00 (2.00). The PCs were also satisfied with the provision of these counsel- ling. In terms of the provision of counselling on the management of treatment, the PCs were not satisfied that the pharmacists did not factor their drug regimen into their daily routine 5.00 (6.00), however, they were satisfied with the counselling that the pharmacists pro- vided on when the effect of the drug will be seen 6.00 (4.00) and how to take the medications 6.00 (2.00). The community pharmacists nevertheless claimed that all the content of the counselling under the management of the treatment was provided to the PCs in detail. Pharmacists’ and pharmacy customers’ opinions on the content and quality of patient medication counsellingh 5) 49 (8.9) 64 (11.6) Only the pharmacist is in the best place to give information as regards medications gotten from the pharmacy (n = 553) 190 (34.4) 200 (36.4) 163 (29.5) I believe that medication counselling involves the pharmacist providing information about: What the medication is supposed to do (550) 495 (90.0) 9 (1.6) 46 (8.4) How long the medication is to be taken (n = 549) 486 (88.5) 15 (2.7) 48 (8.7) How to properly store the medication (n = 550) 389 (70.7) 83 (15.1) 78 (14.2) The way the medication was manufactured (n = 547) 120 (21.9) 287 (52.5) 140 (25.6) The company that produced the medication (n = 546) 135 (24.7) 266 (48.7) 145 (26.6) What to do with leftover medications (n = 546) 276 (50.5) 163 (29.9) 107 (19.6) Proper diet and how to adjust my lifestyle (n = 547) 415 (75.9) 66 (12.1) 66 (12.1) The cost of the medication (n = 555) 386 (70.8) 83 (15.2) 76 (13.9) where 70% of the pharmacists considered the USP- MCBG useful in PMC [29]. Our study evaluated the opinion of the PMC provider (the community pharma- cists, CPs) and the receiver (the pharmacy consumers, PCs) on the content of medication counselling and sub- sequently determined the quality of the medication infor- mation given or received. To the best of our knowledge, this is the first study in Nigeria focusing on this perspec- tive of PMC. medication counselling provided by the community pharmacists are outlined in Table 4. Relating to the quality of PMC provided by the com- munity pharmacists, Table 5 shows that the PCs were not satisfied with the overall counselling. The mean percent quality counselling for the entire USP-MCBG scale was 54.78 ± 18.44% compared with the pharma- cist 75.00 ± 12.41% which was satisfactory (p < 0.001). In terms of the communication component of the USP- MCBG scale, the PCs were satisfied with the quality of counselling provided under this section 66.20 ± 15.62%. The findings in this study showed that both the pro- vider and the recipient of PMC agreed that the com- munity pharmacist asked about the patient’s allergies, medical, family, and social history. In Saudi Arabia, phar- macists showed little or no concern about taking the his- tory of drug allergy [40]. Pharmacists’ and pharmacy customers’ opinions on the content and quality of patient medication counsellingh Both community pharmacists and PCs agreed that the pharmacists provided satisfactory medication counsel- ling by communicating in understandable language and with nonverbal behaviors. The opinions of the PCs and the community pharmacists on other contents of the Table 4. In terms of needs assessment, the community pharmacists claim to ask the PCs for the history of aller- gies or other medications 7.00 (1.00)–[Median (inter- quartile range)], respond with empathy to PC’s concerns 8.00 (3.00), and ask about medical, family and social his- tory 7.00 (1.00). However, the PCs claimed that the pro- vision of this counselling was satisfactory, but the use of appropriate counselling aids to support medication counselling by the community pharmacists was poor 2.00 (5.00). Table 4. In terms of needs assessment, the community pharmacists claim to ask the PCs for the history of aller- gies or other medications 7.00 (1.00)–[Median (inter- quartile range)], respond with empathy to PC’s concerns 8.00 (3.00), and ask about medical, family and social his- tory 7.00 (1.00). However, the PCs claimed that the pro- vision of this counselling was satisfactory, but the use of appropriate counselling aids to support medication counselling by the community pharmacists was poor 2.00 (5.00). As shown in Table 4, the community pharmacists also claim to provide excellent counselling on precau- tions and warnings such as discussion on potential side effects of drugs with PCs 8.00 (2.00) and warning about taking other medications or herbs with the drug 8.00 (2.00). Other satisfactory counsellings provided were what to do with missed doses 7.00 (2.00) and how Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Page 6 of 14 Table 2 Pharmacy customers’ understanding of medication counselling Description Frequency (%) Yes No I don’t know I am aware that I need to be counselled on the use of medications and other drug/ disease-related matters (n = 552) 497 (90.0) 39 (7.1) 16 (2.9) Medication counselling should be provided in community pharmacies (n = 550) 437 (79. Pharmacists’ and pharmacy customers’ opinions on the content and quality of patient medication counsellingh However, in this same study and another in Ethiopia, pharmacists claimed to take medica- tion history during PMC which is similar to our report, but contradicts the report from another study in Nigeria by Osemene et al. [41] where most of the pharmacists did not take patients medication history. Pharmacists can easily identify the error in patients’ self-reported medication history. But when inaccurate and inadequate information on patient medication history and allergies is gathered during PMC, this may hamper the quality of patient care, cause medication errors and affect patient safety. Pharmacists may be unable to promptly prevent inappropriate drug therapy, drug interactions and possi- ble treatment duplications [42].h g p A Mann–Whitney U test revealed a significant differ- ence in the perceived quality of needs assessment coun- selling provided by male pharmacist (mean rank = 68.67, n = 76) and female pharmacist (mean rank = 46.39, n = 44), U = 1051, p = 0.001). Also, the quality of com- munication counselling provided to PCs by community pharmacists differ based on where the pharmacists were trained. The quality of counselling offered by commu- nity pharmacists trained in Nigeria (mean rank = 62.49, n = 116) was higher than those trained outside Nige- ria (mean rank = 26.40, n = 5), U = 228.00, p = 0.024 (Table 6). For the PCs, Table 7 shows that PC’s age, mari- tal status, highest educational qualification and working status were significantly associated with the opinion of PCs on the quality of counselling provided by the com- munity pharmacists (p < 0.05). Discussion There was mutual satisfaction between the community pharmacists and PCs on the provision of PMC on side effects, drug interactions and what to do with leftover medications. There are different reports on the provision of information on medication side effects to patients or Assessing the content and quality of PMC using an estab- lished guideline shows the comprehensiveness of the medication information given to PCs. This is supported by the study among Finnish community pharmacists Page 7 of 14 Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Page 7 of 14 Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Page 7 of 14 Table 3 Demographic characteristics of community pharmacists Age, years 34.54 ± 12.07 Age group (n = 95) ≤ 25 13 (13.7) 26–45 68 (71.6) 46–65 11 (11.6) 66+ 3 (3.2) Gender (n = 120) Male 76 (63.3) Female 44 (36.7) Country of pharmaceutical training (n = 121) Nigeria 116 (95.9) Outside Nigeria 5 (4.1) Cadre of pharmacist (n = 118) Superintendent pharmacist 52 (44.1) NYSC pharmacist 7 (5.9) Post-intern locum pharmacist 9 (7.6) Pharmacy manager 21 (17.8) Staff pharmacist 29 (24.6) Highest educational qualification (n = 122) B.Pharm 94 (77.0) Pharm. Discussion This is essential for the rational use of medicines. consumers and by pharmacists. Some consumers prefer to receive the information on side effects [43–45], some pharmacists prefer not to give it at all [19, 46] while some others seldom provide the information on side effects to consumers [14, 47]. These different reports may be due to the type of population studied, the content of the medi- cation counselling, the use of medication counselling guidelines, the practice setting and whether the medica- tion counselling was for prescription or over-the-counter drugs. Notwithstanding, counselling patients on the side effects of medication is pertinent. Consumers’ or PCs’ knowledge of expected side effects (s) may help them to promptly identify and report such side effects to the pharmacists or other healthcare professionals. This may impel the pharmacist to identify and report adverse drug reactions and identify and resolve drug therapy-related problems. Thus, pharmacists should be able to pass appropriate and balanced information on the beneficial effects and side effects of medications to PCs [48]. Patients or consumers have always been viewed to play a passive role of help seeker in patient medication counselling [52–54] and are seldom encouraged to ask questions during counselling [55, 56]. This tallies with the claim by the PCs that the community pharmacists did not give them enough opportunity to express their concerns or ask questions. Studies showed that patients are interested in receiving personalized information on the use of their medication but their intention to initi- ate consultation or ask questions during counselling is suboptimal [57–59]. Nevertheless, motivating patients’ or consumers’ participation in the counselling process assists in identifying drug therapy-related problems, improving patients’ knowledge of disease and medica- tion, and enhancing patient understanding [10]. Summarily the quality of counselling provided by the pharmacists was satisfactory, but the quality of counsel- ling received by the PCs was unsatisfactory. This find- ing is similar to that conducted in South Korea by Yang et al. [12]. As reported by Yang et al., the major reason why PCs were not satisfied with the PCs’ PMC was insuf- ficient time for counselling [12]. Discussion D 9 (7.4) M.Pharm/M.Sc 13 (10.7) FPCPharm 3 (2.5) MBA 1 (0.8) Ph.D 2 (1.6) Practiced in other pharmacy setting (n = 120) Yes 99 (82.5) No 21 (17.5) Previous area of practice (n = 102) Research/Academia 4 (3.9) Hospital 69 (67.6) Government and NGOs 3 (2.9) Pharmaceutical industry 12 (11.8) Sales representative 13 (12.7) Public health 1 (1.0) Years of post-NYSC community practice Group (n = 108) 6.33 ± 9.34 ≤ 5 74 (68.5) 6–17 24 (22.2) 18–29 1 (0.9) 30+ 9 (8.3) Years of community pharmacy experience Group (n = 121) 6.31 ± 8.69 ≤ 5 84 (69.4) 6–20 29 (24.0) 21–35 5 (4.1) 36+ 3 (2.5) Estimated number of prescriptions filled or seen in the pharmacy per day (n = 113) 36.80 ± 48.93 ≤ 50 94 (83.2) 51–140 16 (14.2) 141–230 1 (0.9) 231+ 2 (1.8) Estimated number of PCs attended to per day (n = 116) 85.16 ± 129.01 ≤ 100 94 (81.0) 101–400 20 (17.2) 701+ 2 (1.7) Highest educational qualification (n = 122) Practiced in other pharmacy setting (n = 120) Previous area of practice (n = 102) Years of post-NYSC community practice Group (n = 108) Years of post-NYSC community practice Group (n = 108) Years of community pharmacy experience Group (n = 121) Page 8 of 14 Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Page 8 of 14 Table 3 (continued) MBA Master in Business Administration, PCs pharmacy customers, B.Pharm Bachelor of Pharmacy, PharmD Doctor of Pharmacy, FPCPharm Fellow Postgraduate College of Pharmacists, M.Sc Master of Science, NYSC National Youth Service Corps, NGO Non-Governmental Organization, M.Pharm Master of Pharmacy * Binned variables based on equal width interval of scanned cases Demographic information Variables Frequency (%) Mean ± SD Estimated time spent with each pharmacy customer (min) Group (n = 120) 8.38 ± 4.87 ≤ 2 5 (4.2) 3–8 59 (49.2) 9–14 37 (30.8) 15+ 19 (15.8) Table 3 (continued) MBA Master in Business Administration, PCs pharmacy customers, B.Pharm Bachelor of Pharmacy, PharmD Doctor of Pharmacy, FPCPharm Fellow Postgraduate College of Pharmacists, M.Sc Master of Science, NYSC National Youth Service Corps, NGO Non-Governmental Organization, M.Pharm Master of Pharmacy Binned variables based on equal width interval of scanned cases siness Administration, PCs pharmacy customers, B.Pharm Bachelor of Pharmacy, PharmD Doctor of Pharmacy, FPCPharm Fellow Postgrad .Sc Master of Science, NYSC National Youth Service Corps, NGO Non-Governmental Organization, M.Pharm Master of Pharmacy b d l id h i l f d MBA Master in Business Administration, PCs pharmacy customers, B.Pharm Bachelor of Pharmacy, PharmD Doctor of Pharmacy, FPCPharm Fellow Postgraduate College of Pharmacists, M.Sc Master of Science, NYSC National Youth Service Corps, NGO Non-Governmental Organization, M.Pharm Master of Pharmacy * Binned variables based on equal width interval of scanned cases * Binned variables based on equal width interval of scanned cases required by the patient or consumer. Discussion Several other reasons have been adduced in the literature and these include PCs forgetting the information provided by the pharma- cist, lack of interest or refusing counselling [60, 61], type of PC (new or old) as new PCs are likely to receive more PMC than old PCs [62], and prescription (new or refill) since pharmacy customers with new prescription are likely to be counselled more than those with refill pre- scription because the pharmacist may assume that the PC on chronic medication(s) has detailed information about the medication(s) [19, 60, 62, 63]. ff Contrary to the report in a study in Vermont USA where 76% of the participants were not informed by the pharmacist on what to do with leftover medications [49], the PCs agree with the pharmacists that this infor- mation was satisfactorily provided. This is important as improperly disposed pharmaceuticals have been reported to have untold consequences on the environment [49, 50]. Patients or PCs equipped with the knowledge of the impact that drugs disposed of improperly could have on the environment are more likely to return leftover medi- cations for proper disposal. Thus, it is paramount that a protocol such as a drug-take-back program is established for the proper disposal of unused and leftover medica- tions in Nigeria and around the world [50].h The PCs however agreed with the pharmacists on the provision of counselling on how to take the medication and when the effect of the drug will be seen. In most studies on PMC, information on the direction for use, dose, name of medicine, and indications, were frequently given [6, 14, 51]. This is probably what most community pharmacists considered the minimum PMC information Contrariwise, pharmacists gave a higher perceived quality of counselling possibly due to over-reporting of their counselling services to fulfill social expectations [64]. But pharmacists tend to provide more PMC to PCs than other pharmacy staff because of their knowledge on drug therapy [60, 63, 65]. Discussion Factors that affect the quality Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Page 9 of 14 Table 4 Pharmacists’ and pharmacy customers’ opinions on the content of pharmacist’s patient medication counselling 1—not done, 2—poor, 3–5—unsatisfactory, 6–7—satisfactory, 8–10—excellent USP-MCBG United States Pharmacopeia Medication Counselling Behaviour Guideline, IQR interquartile range Patient medication counselling content (USP-MCBG) Median (IQR) Pharmacy customers N = 566 Pharmacists N = 122 Component 1: Needs assessment The pharmacist asked me if I had allergies, any other medications taken, my age, etc 6.00 (6.00) 7.00 (1.00) The pharmacist responded with understanding/empathy to my questions and concerns 6.00 (5.00) 8.00 (3.00) The pharmacist asked questions about my illness, family and social history prior to counselling 6.00 (7.00) 7.00 (1.00) The pharmacist explained the purpose of the counselling session to me 6.00 (6.00) 7.00 (2.00) The pharmacist presented facts and concepts concerning the drug and my illness in a logical order 6.00 (6.00) 8.00 (2.00) The pharmacist used appropriate counselling aids such as pictures to support the counselling 2.00 (5.00) 7.00 (2.00) The pharmacist assessed any actual and/or potential concerns or problems of importance 6.00 (6.00) 7.00 (3.00) The pharmacist determined if I have any other medical conditions which could influence the effects of this drug or influence the likelihood of an adverse reaction 6.00 (3.00) 8.00 (2.00) The pharmacist conducted appropriate counselling introduction by identifying him/herself and me 6.00 (6.00) 7.00 (2.00) Component 2: Precautions and warnings The pharmacist explored with me potential problems in taking the medication as prescribed (e.g., cost, access, etc.) 6.00 (2.00) 8.00 (3.00) The pharmacist discussed potential (significant) side effects 6.00 (2.00) 8.00 (2.00) The pharmacist warned me or the patient concerned about taking other medications, including OTCs, herbals/ botanicals and alcohol, which could inhibit or interact with the prescribed medication 6.00 (2.00) 8.00 (2.00) The pharmacist discussed significant interactions that might occur when using my drugs with certain drugs, food and other diseases 7.00 (1.00) 8.00 (3.00) The pharmacist discussed precautions to take (activities to avoid, etc.) 6.00 (2.00) 8.00 (2.00) The pharmacist explained in precise terms what to do if the patient misses a dose 6.00 (6.00) 7.00 (2.00) The pharmacist discussed how to prevent or manage the side effects of the drug if they do occur 6.00 (6.00) 7.00 (2.00) The pharmacist helped me to generate solutions to potential problems 6.00 (6.00) 7.00 (2.00) Component 3: Management of the treatment The pharmacist discussed the storage recommendations, ancillary instructions with respect to the medications (e.g., shake well, refrigerate, etc.) 6.00 (4.00) 8.00 (3.00) The pharmacist explained how long it will take for the drug to show an effect 6.00 (4.00) 7.00 (2.00) The pharmacist told me when I am due for a refill/repurchasing of my medications 6.00 (7.00) 8.00 (3.00) The pharmacist summarized the session by acknowledging and/or emphasizing key points of information 6.00 (6.00) 8.00 (2.00) The pharmacist emphasized the benefits of completing the medication as prescribed 7.00 (3.00) 9.00 (2.00) The pharmacist helped me to plan follow-up and next steps 6.00 (5.00) 8.00 (3.00) The pharmacist provided an opportunity for final concerns or questions 6.00 (6.00) 8.00 (2.00) The pharmacist verified if I understood by requesting feedback or a recap 6.00 (6.00) 8.00 (3.00) The pharmacist maintained control and direction of the counselling session 6.00 (4.00) 8.00 (3.00) The pharmacist assisted me in developing a plan to incorporate the medication regimen into my daily routine 5.00 (6.00) 8.00 (3.00) The pharmacist used open-ended questions that required me giving explanation 6.00 (6.00) 8.00 (3.00) The pharmacist explained to me the way I will take my medications including scheduling and duration of the medication use when appropriate 6.00 (2.00) 8.00 (2.00) The pharmacist probed me for additional information 6.00 (6.00) 8.00 (2.00) Component 4: Communication The pharmacist communicated in a language that I understood 7.00 (2.00) 9.00 (2.00) The pharmacist provided me with accurate information 6.00 (2.00) 9.00 (2.00) The pharmacist discussed with me the name of the medication and what I’m using it for 7.00 (2.00) 9.00 (3.00) The pharmacist displayed effective nonverbal behaviors 7.00 (2.75) 8.38 (2.00) The pharmacist assessed my understanding of the reason(s) for the therapy 6.00 (8.00) 8.00 (2.00) Table 4 Pharmacists’ and pharmacy customers’ opinions on the content of pharmacist’s patient medication counselling Patient medication counselling content (USP-MCBG) Median (IQR) customers’ opinions on the content of pharmacist’s patient medication counselling Component 1: Needs assessment of counselling are the type of prescription (new or refill), pharmacy busyness, pharmacists perceived patients need [66], and the type of consultation with the pharmacist (product request, symptom-based consultation, prescrip- tion counselling, etc.), others include lack of privacy [67], lack of or inadequate remuneration [60], patient relying on the physician recommendation more than the phar- macists counselling [67], patient factors (such as the nature of illness and interest to receive counselling) [60], and time constraint [68]. Pharmacists tend to pay atten- tion to drug more than patients, and pharmacists may assume that patients have prior knowledge on the drug and may feel that the provision of additional information may be construed as patronizing and thus the pharma- cists may fear negative patient reaction. All these affect the content and quality of PMC provided by pharmacists. of counselling are the type of prescription (new or refill), pharmacy busyness, pharmacists perceived patients need [66], and the type of consultation with the pharmacist (product request, symptom-based consultation, prescrip- tion counselling, etc.), others include lack of privacy [67], lack of or inadequate remuneration [60], patient relying on the physician recommendation more than the phar- macists counselling [67], patient factors (such as the nature of illness and interest to receive counselling) [60], and time constraint [68]. Pharmacists tend to pay atten- tion to drug more than patients, and pharmacists may assume that patients have prior knowledge on the drug and may feel that the provision of additional information may be construed as patronizing and thus the pharma- cists may fear negative patient reaction. All these affect the content and quality of PMC provided by pharmacists. Most studies described the rate of counselling rather than the quality of counselling as reported in this study. The rate of counselling was reported as the proportion of consumers who received verbal counselling and ranged from 8 to 56% [7, 35, 38] for consumers and 51 to 100% [69–71] for pharmacists. In comparison, the quality of counselling received by the PCs was 55% and that pro- vided by the community pharmacists was 75%. Though the figures may not be comparable because of the dif- ferences in the definitions of the rate of counselling and quality of counselling, there seems to be a similar trend. Component 1: Needs assessment The pharmacist provided me with accurate information The pharmacist displayed effective nonverbal behaviors The pharmacist assessed my understanding of the reason(s) for the therapy USP-MCBG United States Pharmacopeia Medication Counselling Behaviour Guideline, IQR interquartile range Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Page 10 of 14 Table 5 Differences in the perceived quality of counselling provided by pharmacists and received by pharmacy customers Quality of counselling grading: poor (1–29.9%), unsatisfactory (30–59.9%), satisfactory (60–79.9%); excellent (80–100%) USP-MCBG United States Pharmacopeia Medication Counselling Behaviour Guideline * Independent sample t-test, p < 0.05 is considered significant USP-MCBG scale Description Mean ± SD p-value* Pharmacist N = 122 Pharmacy customers N = 566 Component 1 Needs assessment score 64.05 ± 10.76 43.82 ± 19.73 < 0.001 Mean quality of counselling (%) 71.17 ± 11.96 48.68 ± 21.92 < 0.001 Grading Satisfactory Unsatisfactory Component 2 Precautions and warnings 58.98 ± 11.63 45.61 ± 15.41 < 0.001 Mean quality of counselling (%) 73.73 ± 14.54 57.02 ± 19.26 < 0.001 Grading Satisfactory Unsatisfactory Component 3 Management of the treatment 98.00 ± 20.21 69.22 ± 28.12 < 0.001 Mean quality of counselling (%) 75.39 ± 15.54 53.24 ± 21.63 < 0.001 Grading Satisfactory Unsatisfactory Component 4 Communication 41.47 ± 7.25 33.10 ± 7.81 < 0.001 Mean quality of counselling (%) 82.93 ± 14.50 66.20 ± 15.62 < 0.001 Grading Excellent Satisfactory USP-MCBG Total score 262.50 ± 43.45 191.74 ± 64.53 < 0.001 Mean quality of counselling (%) 75.00 ± 12.41 54.78 ± 18.44 < 0.001 Grading Satisfactory Unsatisfactory rences in the perceived quality of counselling provided by pharmacists and received by pharmacy customers Quality of counselling grading: poor (1–29.9%), unsatisfactory (30–59.9%), satisfactory (60–79.9%); excellent (80–100%) USP-MCBG United States Pharmacopeia Medication Counselling Behaviour Guideline * Independent sample t-test, p < 0.05 is considered significant also for the quality of counselling, pharmacist > phar- macy customers. The type of information considered for the provision of counselling varies across studies. Some studies considered more than 5 items, others focused on 3 items, some studies investigated the type of informa- tion given to consumers [14, 34, 36]. But in this study, 35 items were considered based on the USP-MCBG scale. This affords a comprehensive coverage of PMC. Component 1: Needs assessment For the rate of counselling, pharmacist > consumer, and hf It is obvious from this study that there is the disparity in the quality of counselling purportedly provided by the community pharmacists and that received by PCs. Though from the literature the difference is expected because of the self-report nature of the study. Improvement in the quality of counselling may be enhanced with education, patient-centered communication [72, 73], evaluation of pharmacy practice with feedbacks [62] and the promotion and adoption of the use of counselling guidelines like USP- MCBG. Presently there is no specific patient PMC guide- line used by pharmacists in the country. Thus, there is a need for consensus counselling guidelines. Most studies described the rate of counselling rather than the quality of counselling as reported in this study. The rate of counselling was reported as the proportion of consumers who received verbal counselling and ranged from 8 to 56% [7, 35, 38] for consumers and 51 to 100% [69–71] for pharmacists. In comparison, the quality of counselling received by the PCs was 55% and that pro- vided by the community pharmacists was 75%. Though the figures may not be comparable because of the dif- ferences in the definitions of the rate of counselling and quality of counselling, there seems to be a similar trend. For the rate of counselling, pharmacist > consumer, and Strength and limitations of the studyh This study did not consider written medication counselling in the evaluation of the Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Page 11 of 14 Table 6 Association between pharmacists’ demographic variables and perception of the quality of counselling provided USP-MCBG United States Pharmacopeia Medication Counselling Behaviour Guideline a Mann–Whitney U test Demographics Categories N Quality of patient medication counselling based on the USP-MCBG scale Mean ranks Component 1 Component 2 Component 3 Component 4 USP-MCBG total score Gender Male 76 68.67 63.18 62.72 59.91 64.13 Female 44 46.39 55.88 56.66 61.51 54.23 p-valuea 0.001* 0.267 0.357 0.808 0.133 Age, years ≤ 25 13 44.08 50.88 51.69 58.65 51.73 26–45 68 47.40 47.38 47.84 46.13 47.38 46–65 11 55.45 49.05 46.14 47.18 47.77 66+ 3 51.17 45.67 42.50 47.17 46.67 p-valueb 0.767 0.975 0.939 0.519 0.964 Country of pharmacy training Nigeria 116 61.53 62.24 62.09 62.49 62.19 Outside Nigeria 5 48.60 32.30 35.60 26.40 33.40 p-valuea 0.419 0.062 0.098 0.024* 0.072 Cadre of pharmacists Superintendent pharmacist 52 54.05 54.62 55.83 55.19 54.40 NYSC pharmacist 7 70.50 64.64 61.79 70.57 66.71 Post internship locum phar- macist 9 54.33 65.39 62.33 62.22 60.11 Pharmacy manager 21 74.71 68.33 64.67 63.83 68.90 Staff pharmacist 29 57.21 58.79 60.91 60.57 59.90 p-valueb 0.162 0.574 0.874 0.739 0.548 Years of community pharmacy experience ≤ 5 84 60.43 62.13 61.26 63.70 61.98 6–20 29 63.26 61.90 64.97 58.47 62.97 21–35 5 61.40 52.60 45.80 48.20 46.70 36+ 3 54.50 34.83 40.67 31.17 38.33 p-valueb 0.970 0.559 0.508 0.322 0.524 Estimated time spent with PCs (min) ≤ 2 5 60.80 88.20 90.20 63.90 81.10 3–8 59 56.86 61.97 61.08 62.85 60.80 9–14 37 67.55 57.22 59.24 59.27 60.26 15+ 19 58.00 55.03 53.34 54.71 54.63 p-valueb 0.518 0.253 0.211 0.828 0.512 Table 6 Association between pharmacists’ demographic variables and perception of the quality of counselling provided Table 6 Association between pharmacists’ demographic variables and perception of the quality of counselling provided Demographics Categories N Quality of patient medication counselling based on the USP MCBG sc ween pharmacists’ demographic variables and perception of the quality of counselling provided N Quality of patient medication counselling based on the USP-MCBG scale Mean ranks Component 1 Component 2 Component 3 Component 4 USP-MCBG total score generalization of the result may be limited. The findings presented in this study should thus be considered in light of these limitations. Strength and limitations of the studyh content and quality of counselling, thus the report may be lower compared with when both written and ver- bal medication counselling is considered. Likewise, the provision and receipt of medication counselling con- tent were based on pharmacists’ and PCs’ self-reports, respectively. This is prone to social desirability and recall biases. Another limitation of the study is the employ- ment of a convenient sampling method which may intro- duce selection bias especially in the PCs’ survey where they were sampled only for a day in each pharmacy. The full remit of PCs within the study period was not feasi- ble with this sampling. Also, the study was carried out among community pharmacists and pharmacy custom- ers in a city in one of the southwest states in Nigeria, thus Strength and limitations of the studyh The strength of the study lies in the use of a validated medication counselling guideline to determine the con- tent and quality of PMC offered by community pharma- cists and received by PCs. Acknowledgements The datasets used or analyzed during the current study are available from the corresponding author on reasonable request. The authors thank community pharmacy owners in Ibadan metropolis for permitting the survey in the pharmacy and all the pharmacists and pharmacy clients who took part in the study for the valuable time used in filling the questionnaire. Conclusion Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Page 12 of 14 Table 7 Association between pharmacy customers’ demographic variables and perception of the quality of counselling received USP-MCBG United States Pharmacopeia Medication Counselling Behaviour Guideline Demographics Categories N Quality of patient medication counselling based on the USP-MCBG scale Mean ranks Component 1 Component 2 Component 3 Component 4 USP-MCBG total score Gender Male 278 273.74 277.53 269.89 271.10 271.12 Female 281 286.19 282.45 290.01 288.80 288.79 p-valuea 0.362 0.719 0.141 0.195 0.196 Age, years ≤ 25 192 400.08 405.30 415.02 360.72 417.13 26–44 257 269.04 263.68 263.00 280.17 262.68 45–63 102 133.30 133.62 120.49 150.35 117.40 64+ 15 60.33 83.10 59.83 257.57 59.23 p-valueb < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 Marital status Single 283 355.36 366.09 369.24 334.06 370.25 Married 246 208.87 195.13 191.98 226.45 191.35 Widowed 17 99.59 115.88 99.62 179.24 98.41 Divorced 5 147.90 150.10 165.20 219.30 152.10 Separated 6 170.50 179.42 193.67 169.33 186.17 p-valueb < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 Highest academic qualification No formal education 15 232.60 174.43 219.17 249.43 207.47 Primary education 23 234.13 246.00 261.70 273.98 249.93 Secondary education 180 345.66 342.88 345.15 316.92 350.43 Tertiary education 241 255.51 260.40 253.97 259.53 253.70 Post graduate education 92 210.92 210.06 211.25 243.91 206.49 p-valueb < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 Working status Self-employed 132 204.81 212.12 207.69 227.31 204.51 Employee 116 227.10 225.75 215.63 233.75 220.14 Unemployed 27 250.35 271.87 251.04 291.48 258.67 Civil/public servant 41 189.18 168.96 179.84 231.43 174.84 Artisan 18 220.75 199.94 223.75 210.83 209.22 Student 145 368.17 370.22 380.93 318.03 382.16 Farmer 4 286.63 156.88 244.25 199.63 236.13 Trader 28 157.75 159.04 142.75 201.04 143.16 p-valueb < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 Table 7 Association between pharmacy customers’ demographic variables and perception of the quality of counselling received Table 7 Association between pharmacy customers’ demographic variables and perception of the quality of counselling received Demographics Categories N Quality of patient medication counselling based on the USP-MCBG scale N Quality of patient medication counselling based on the USP-MCBG scale Mean ranks Funding Abbreviations PMC: Patient medication counselling; PC: Pharmacy customer; CP: Clinical pharmacist; USP-MCBG: United States Pharmacopeia Medication Counselling Behaviour Guideline. g This research received no specific grant from any funding agency in the pub- lic, commercial, or not-for-profit sectors. 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Written informed consent was obtained from the owners of the participating pharmacies and pharmacist-participants via a letter where the details of the study were explained. Permission to survey pharmacy customers and the superintendent pharmacists were obtained. Pharmacy customers SJS conceived and designed the study, collected, analyzed and interpreted the data, wrote the draft manuscript, MWL was involved in the design of the study, collected the data, was involved in data analysis and interpretation, read and contributed to the draft manuscript. Both authors read and approved the final manuscript. Page 13 of 14 Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 Showande and Laniyan Journal of Pharmaceutical Policy and Practice (2022) 15:103 gave verbal informed consent. The study was conducted in accordance with the declaration of Helsinki. 20. Kim C. 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https://openalex.org/W1670478309	https://europepmc.org/articles/pmc4561108?pdf=render	English	null	Smoking Status Effect on Inflammatory Markers in a Randomized Trial of Current and Former Heavy Smokers	International journal of inflammation	2,015	cc-by	4,598	Correspondence should be addressed to Janet A. Foote; jfoote@email.arizona.edu Received 30 January 2015; Revised 3 August 2015; Accepted 6 August 2015 Academic Editor: Andrew S. Day Copyright © 2015 Sami Aldaham et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. The level of systemic inflammation as measured by circulating levels of C-reactive protein (CRP) and interleukin- 6 (IL-6) is linked to an increased risk for cardiovascular diseases (CVD) and cancer. Methods. We recruited 154 current and former smokers between 40 and 80 years of age with 25 or more pack-years of smoking history to study the relationship between inflammatory markers (CRP and IL-6) and smoking status. Results. Our results show that male smokers had significantly higher levels of serum IL-6 compared to male former smokers. We did not find any gender specific differences for smoking and CRP levels but the IL-6 levels were slightly lower in females compared to males. Additionally, our results show that CRP is significantly associated with IL-6 regardless of smoking status. Modelling indicates that the significant predictors of CRP levels were biomarkers of the metabolic syndrome while the significant predictors of IL-6 levels were age and plasma triglycerides among former smokers and the numbers of smoked packs of cigarettes per year among smokers. Conclusions. In conclusion, our study showed that CRP levels were not associated with markers of smoking intensity. However, IL-6 levels were significantly associated with smoking especially among current smokers. Hindawi Publishing Corporation International Journal of Inﬂammation Volume 2015, Article ID 439396, 6 pages http://dx.doi.org/10.1155/2015/439396 Hindawi Publishing Corporation International Journal of Inﬂammation Volume 2015, Article ID 439396, 6 pages http://dx.doi.org/10.1155/2015/439396 Hindawi Publishing Corporation International Journal of Inﬂammation Volume 2015, Article ID 439396, 6 pages http://dx.doi.org/10.1155/2015/439396 Research Article Smoking Status Effect on Inflammatory Markers in a Randomized Trial of Current and Former Heavy Smokers Sami Aldaham,1 Janet A. Foote,2 H.-H. Sherry Chow,3 and Iman A. Hakim2,3 1College of Medicine, Al-Imam Mohammad Ibn Saud Islamic University (IMSIU), Riyadh 11432, Saudi Arabia 2Mel & Enid Zuckerman College of Public Health, University of Arizona, Tucson, AZ 85724, USA 3Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA Correspondence should be addressed to Janet A. Foote; jfoote@email.arizona.edu 2. Methods a mean age of 60.3 ± 0.7 years (mean ± SE) were included in the study. Characteristics of the study population by gender and smoking status are presented in Table 1. There were no significant differences between males and females except in reported pack-years among current smokers. Male smokers reported significantly greater pack-years of smoking compared to females (𝑝= 0.004). While the cumulative exposure smoking index of pack-years was not significantly different between current and former smokers among males or females, former smokers reported significantly fewer years of smoking compared to current smokers in both genders (𝑝≤0.001). Current smokers (CS) and former smokers (FS) between 40 and 80 years of age with 25 pack-years or more of smoking history were recruited to a randomized, double-blind, and placebo controlled chemoprevention trial of green tea and black tea. The study protocol received approval by the Univer- sity of Arizona Institutional Review Board. Once informed consent had been obtained, each participant completed a set of smoking, dietary, and health assessment questionnaires and was evaluated for health and respiratory history. A blood sample was collected for a comprehensive metabolic panel. Two additional fasting morning blood samples were collected from which plasma, serum, buffy coat, and erythrocytes were isolated and stored at −80∘C prior to the sample analysis. Body mass index (BMI) for each participant was calculated as weight divided by height2 (kg/m2). 3.2. Inflammation Biomarkers. The differences in the levels of the biomarkers of inflammation and blood lipids between smokers and former smokers are presented in Table 2 for both men and women separately. Male current smokers had significantly higher levels of serum IL-6 compared to male former smokers. Women (smokers and former smokers) had significantly higher levels of HDL compared to men.if 2.1. Human C-Reactive Protein (CRP) Immunoassay and Interleukin-6 (IL-6) Immunoassay in Serum. Aliquots were thawed immediately prior to the analysis. Each immunoassay was completed using a commercially available kit from Quantikine (R & D Systems, Minneapolis, MN, USA). Briefly, specified quantities of the specimen or standard are added to microplates with cells precoated with the CRP antibody or the IL-6 antibody, depending on the assay. The antibodies bind the CRP or IL-6 present in the samples and standards. Any unbound substances are washed away and a monoclonal antibody specific for CRP or a polyclonal antibody specific for IL-6 is added. 2. Methods Again, unbound substances are washed away, substrate solution is added, the plate is incubated, and ampli- fier solution is added. Color develops in proportion to the amount of CRP or IL-6 present in the well and after stopping the color development in the prescribed time, the wells are measured spectrophotometrically for CRP or IL-6 quantifica- tion. Both CRP and IL-6 analyses were completed in duplicate for 154 participants. All data entry and values were checked by additional laboratory personnel for quality control. gi y g p Given the fact that there were no significant differences by gender, the correlations among CRP, IL-6, smoking intensity parameters, and other variables are presented by smoking status in Table 3. Log (CRP) and log (IL-6) were significantly correlated regardless of smoking status (𝑝< 0.001). Log (CRP) was not associated with any marker of smoking inten- sity. Among former smokers, log (CRP) was significantly associated with BMI (𝑟= 0.280, 𝑝= 0.007) and low HDL levels (𝑟= −0.273, 𝑝= 0.011). Among smokers, log (IL-6) was significantly associated with age (𝑟= 0.466, 𝑝< 0.001), years of smoking (𝑟= 0.242, 𝑝= 0.047), pack/years (𝑟= 0.256, 𝑝= 0.033), plasma triglycerides (𝑟= 0.268, 𝑝= 0.028), and low HDL levels (𝑟= −0.029, 𝑝= 0.017). The log (CRP) was only associated with age (𝑟= 0.270, 𝑝= 0.026). 3.3. Models of CRP and IL-6 by Smoking Status. The multi- variate predictors of inflammatory markers by smoking status are presented in Table 4. The best multivariable model for log (CRP) among former smokers included triglycerides (𝑝= 0.009) and the interaction between CRP and IL-6 (𝑝 < 0.001) and the best multivariable model for log (CRP) among current smokers included HDL (𝑝= 0.010), BMI (𝑝= 0.05), and the interaction between CRP and IL-6 (𝑝< 0.001). The best multivariable model for log (IL-6) among former smokers included triglycerides (𝑝= 0.002), age (𝑝= 0.007), and the interaction between CRP and IL-6 (𝑝< 0.001) and the best multivariable model for log (IL-6) among current smokers included pack/year (𝑝= 0.005) and the interaction between CRP and IL-6 (𝑝< 0.001). 2.2. Statistical Analysis. Means and standard errors were cal- culated for descriptive statistics. Because of value skewness, CRP and IL-6 values were log-transformed. Pearson correla- tions were calculated to describe the unadjusted relationships between the smoking parameters, inflammatory markers, and other relevant variables. 4. Discussion In the current study among participants with more than 30 pack-years of smoking exposure, we did not find any signif- icant difference in CRP levels between smokers and former smokers. However, the serum levels of IL-6 were significantly higher among male smokers compared to former smokers (𝑝= 0.04). Exposure to cigarette smoke increases oxidative International Journal of Inflammation International Journal of Inflammation 2 2. Methods Multivariable linear regression models were created to identify independent predictors of log (CRP) and log (IL-6), using variables that had significant correlations (𝑝< 0.10) with these inflammatory markers. A basic model was created for each inflammatory marker that included age, sex, BMI, caloric intake, triglycerides, high-density lipoprotein cholesterol, years of smoking, and cigarette pack-years (cigarettes smoked daily ÷ 20 cigarettes per pack × years of smoking). 1. Introduction effect of cigarette smoking and reflect tissue injury [10, 11]. The potential significance of IL-6 and CRP has been sug- gested in the growth and progression of many malignancies [12–14]. Many large-scale epidemiological studies among apparently healthy men and women have found CRP to be an independent and strong predictor of future cardiovascular risk [15–17].h Inflammation is associated with a number of chronic condi- tions, such as cardiovascular disease and cancer. Reducing inflammation may help prevent or treat these conditions. C-reactive protein (CRP) and interleukin-6 (IL-6) are well- studied proinflammatory cytokines [1, 2]. Inflammation is part of our immune reaction and leads to the release of C- reactive protein (CRP) into the bloodstream [3, 4] and IL- 6 is a major factor driving the chronic elevation of CRP [5]. Elevated levels of IL-6 are predictive of future adverse health events in both healthy and clinical populations, even after controlling for both lifestyle and other clinical risk factors [6– 9]. There is great interest in studying the relationship between inflammatory markers and smoking in an attempt to provide explanations for smoking-mediated morbidity and mortality. The current study is unique with the inclusion and comparison of verified former smokers with a similar smoking history (pack-years and years of smoking) to the current smokers. The profile of inflammation markers is expected to differ following smoking cessation. Therefore, we are reporting here the associations of CRP and IL-6 with smoking status among community-dwelling men and women. Smoking is the major risk factor for lung cancer, the most common cancer worldwide. Cigarette smoking has been associated with increases in CRP and previous investigations have demonstrated that increased CRP levels are a secondary 3. Results Males 𝑛= 71 mean ± SE Females 𝑛= 83 mean ± SE p value (males versus females) Serum C-reactive protein∗ 7.4 ± 0.13 7.4 ± 0.11 0.66 Former smokers 7.2 ± 0.20 7.3 ± 0.16 0.58 Current smokers 7.6 ± 0.17 7.4 ± 0.16 0.27 𝑝value (former versus current) 0.10 0.87 Serum interleukin-6∗ 0.67 ± 0.10 0.43 ± 0.10 0.09 Former smokers 0.44 ± 0.14 0.33 ± 0.13 0.57 Current smokers 0.86 ± 0.13 0.50 ± 0.14 0.08 𝑝value (former versus current) 0.04 0.40 Total cholesterol (mg/dL) 193.2 ± 5.1 206.2 ± 4.0 0.04 Former smokers 189.5 ± 6.7 204.6 ± 5.5 0.08 Current smokers 196.1 ± 7.6 207.3 ± 5.7 0.23 𝑝value (former versus current) 0.52 0.74 High density lipoproteins (mg/dL) 47.0 ± 1.5 61.5 ± 1.6 <0.001 Former smokers 47.1 ± 2.3 59.0 ± 2.7 0.002 Current smokers 47.0 ± 1.9 63.4 ± 1.9 <0.001 𝑝value (former versus current) 0.99 0.17 Low density lipoproteins (mg/dL) 114.0 ± 4.2 119.7 ± 3.6 0.30 Former smokers 111.8 ± 5.3 118.9 ± 4.6 0.32 Current smokers 116.8 ± 1.9 120.4 ± 5.3 0.67 𝑝value (former versus current) 0.99 0.83 Total triglycerides (mg/dL) 175.0 ± 18.5 139.5 ± 7.1 0.06 Former smokers 165.4 ± 18.6 142.5 ± 11.2 0.28 Current smokers 182.9 ± 30.3 137.3 ± 9.2 0.12 𝑝value (former versus current) 0.64 0.71 Table 1: Characteristics of the study population by gender and smoking status (𝑛= 154). Males 𝑛= 71 mean ± SE Females 𝑛= 83 mean ± SE 𝑝value∗ (males versus females) Age (years) 60.8 ± 1.0 59.9 ± 1.0 0.54 Former smokers 62.4 ± 1.5 60.9 ± 1.4 0.45 Current smokers 59.5 ± 1.3 59.2 ± 1.4 0.89 𝑝value (former versus current)∗ 0.14 0.40 Pack-year 45.6 ± 2.6 40.7 ± 1.9 0.12 Former smokers 41.1 ± 3.4 44.6 ± 3.5 0.49 Current smokers 49.2 ± 3.7 37.7 ± 1.9 0.004 𝑝value (former versus current)∗ 0.12 0.07 Years of smoking 34.4 ± 1.1 32.6 ± 1.2 0.28 Former smokers 29.5 ± 1.5 27.6 ± 1.6 0.39 Current smokers 38.4 ± 1.3 36.4 ± 1.5 0.33 𝑝value (former versus current)∗ <0.001 0.001 ∗𝜒2 test. Table 1: Characteristics of the study population by gender and smoking status (𝑛= 154). Table 2: Biomarkers of inflammation and lipid profile by gender and smoking status (𝑛= 154). Table 2: Biomarkers of inflammation and lipid profile by gender and smoking status (𝑛= 154). 3. Results 3.1. Population Characteristics. One hundred and fifty-four current and former smokers (83 women and 71 men) with 3 International Journal of Inflammation 3 International Journal of Inflammation 3 Table 1: Characteristics of the study population by gender and smoking status (𝑛= 154). Males 𝑛= 71 mean ± SE Females 𝑛= 83 mean ± SE 𝑝value∗ (males versus females) Age (years) 60.8 ± 1.0 59.9 ± 1.0 0.54 Former smokers 62.4 ± 1.5 60.9 ± 1.4 0.45 Current smokers 59.5 ± 1.3 59.2 ± 1.4 0.89 𝑝value (former versus current)∗ 0.14 0.40 Pack-year 45.6 ± 2.6 40.7 ± 1.9 0.12 Former smokers 41.1 ± 3.4 44.6 ± 3.5 0.49 Current smokers 49.2 ± 3.7 37.7 ± 1.9 0.004 𝑝value (former versus current)∗ 0.12 0.07 Years of smoking 34.4 ± 1.1 32.6 ± 1.2 0.28 Former smokers 29.5 ± 1.5 27.6 ± 1.6 0.39 Current smokers 38.4 ± 1.3 36.4 ± 1.5 0.33 𝑝value (former versus current)∗ <0.001 0.001 ∗𝜒2 test. Table 2: Biomarkers of inflammation and lipid profile by gender and smoking status (𝑛= 154). ∗Log transformed data for C-reactive protein and interleukin-6. 3. Results Variables CRP IL-6 𝑟 𝑝 𝑟 𝑝 Interleukin-6 (pg/mL) Former smoker 0.387 0.000 1.000 Current smoker 0.589 0.000 1.000 Age (years) Former smoker 0.161 0.140 0.198 0.067 Current smoker 0.270 0.026‡ 0.446 0.000‡ Years of smoking Former smoker 0.081 0.461 0.202 0.062 Current smoker 0.017 0.889 0.242 0.047‡ Pack/year Former smoker 0.136 0.212 0.313 0.003‡ Current smoker 0.028 0.823 0.256 0.033‡ Caloric intake/day Former smoker 0.206 0.065 0.014 0.904 Current smoker 0.092 0.474 0.054 0.677 Total fat intake (g/day) Former smoker 0.173 0.126 0.007 0.951 Current smoker 0.090 0.488 0.124 0.338 Total cholesterol (mg/dL) Former smoker 0.024 0.825 0.024 0.829 Current smoker 0.134 0.275 0.086 0.484 HDL-C (mg/dL) Former smoker −0.273 0.011‡ −0.207 0.056 Current smoker −0.131 0.287 −0.290 0.017‡ LDL-C (mg/dL) Former smoker 0.028 0.799 0.072 0.517 Current smoker 0.182 0.141 0.061 0.623 Total triglycerides (mg/dL) Former smoker 0.168 0.123 0.134 0.220 Current smoker 0.196 0.110 0.268 0.028‡ BMI (kg/m2) Former smoker 0.287 0.007‡ 0.129 0.236 Current smoker 0.203 0.100 0.130 0.299 CRP: C-reactive protein; HDL-C = high density lipoproteins; LDL-C: low density lipoproteins; BMI: body mass index. ∗Log transformed data for CRP (ng/mL) and IL-6 (pg/mL). ‡Significant correlation. that mean CRP levels were significantly lower in never- smokers (𝑝< 0.0001) compared to current smokers [20]. One study conducted in people of Japanese ethnicity did not find any significant relationship between serum CRP levels and the number of cigarettes smoked per day [4].h g p y The MONICA study from Germany examined gender specific differences for smoking and CRP levels, finding that serum CRP concentrations were significantly higher in male regular smokers than male never-smokers but no significant differences were observed in women [21]. In our study we did not find any gender specific differences for smoking and CRP levels but the IL-6 levels were slightly lower in females compared to males. Also, women had significantly higher levels of HDL compared to men. In addition, our results showed that among smokers, CRP was associated with age while IL-6 was significantly associated with age, smoking variables as measured by years of smoking and number of pack-years, and dyslipidemia as measured by low HDL and high triglycerides levels.h There is strong evidence that IL-6 serum concentration increases with age [22, 23] and it is associated with an increased risk of cardiovascular disease (CVD) mortality, independent of CRP level [24]. 3. Results Males 𝑛= 71 mean ± SE Females 𝑛= 83 mean ± SE p value (males versus females) Serum C-reactive protein∗ 7.4 ± 0.13 7.4 ± 0.11 0.66 Former smokers 7.2 ± 0.20 7.3 ± 0.16 0.58 Current smokers 7.6 ± 0.17 7.4 ± 0.16 0.27 𝑝value (former versus current) 0.10 0.87 Serum interleukin-6∗ 0.67 ± 0.10 0.43 ± 0.10 0.09 Former smokers 0.44 ± 0.14 0.33 ± 0.13 0.57 Current smokers 0.86 ± 0.13 0.50 ± 0.14 0.08 𝑝value (former versus current) 0.04 0.40 Total cholesterol (mg/dL) 193.2 ± 5.1 206.2 ± 4.0 0.04 Former smokers 189.5 ± 6.7 204.6 ± 5.5 0.08 Current smokers 196.1 ± 7.6 207.3 ± 5.7 0.23 𝑝value (former versus current) 0.52 0.74 High density lipoproteins (mg/dL) 47.0 ± 1.5 61.5 ± 1.6 <0.001 Former smokers 47.1 ± 2.3 59.0 ± 2.7 0.002 Current smokers 47.0 ± 1.9 63.4 ± 1.9 <0.001 𝑝value (former versus current) 0.99 0.17 Low density lipoproteins (mg/dL) 114.0 ± 4.2 119.7 ± 3.6 0.30 Former smokers 111.8 ± 5.3 118.9 ± 4.6 0.32 Current smokers 116.8 ± 1.9 120.4 ± 5.3 0.67 𝑝value (former versus current) 0.99 0.83 Total triglycerides (mg/dL) 175.0 ± 18.5 139.5 ± 7.1 0.06 Former smokers 165.4 ± 18.6 142.5 ± 11.2 0.28 Current smokers 182.9 ± 30.3 137.3 ± 9.2 0.12 𝑝value (former versus current) 0.64 0.71 ∗Log transformed data for C-reactive protein and interleukin-6. International Journal of Inflammation International Journal of Inflammation 4 Table 4: Stepwise regression analysis for CRP and IL-6 by smoking status. Subjects Explanatory variable 𝛽 𝑝 Adjusted 𝑅2 CRP (mg/L) Former smokers Total triglycerides 0.496 0.009 0.455 CRP ∗IL-6 <0.001 0.000 Smokers HDL-C (mg/dL) −0.017 0.010 0.369 BMI (kg/m2) 0.028 0.052 CRP ∗IL-6 <0.001 0.000 IL-6 (pg/mL) Former smokers Total triglycerides 0.442 0.002 0.547 Age 0.027 0.007 CRP ∗IL-6 <0.001 0.000 Smokers Pack/year 0.012 0.005 0.436 CRP ∗IL-6 <0.001 0.000 CRP: C-reactive protein; IL-6: interleukin 6; HDL-C: high density lipopro- tein; BMI: body mass index, and CRP ∗IL-6: interaction between CRP and IL-6. Variables included in the model are gender, age, BMI, total triglycerides, HDL-C, years of smoking, pack/year, caloric intake/day, and interaction between CRP and IL-6. Table 3: Pearson’s correlation of CRP∗and IL-6∗with each parameter by smoking status (𝑛= 154). Table 4: Stepwise regression analysis for CRP and IL-6 by smoking status. parameter by smoking status (𝑛= 154). International Journal of Inflammation [3] B. G. Nordestgaard and J. Zacho, “Lipids, atherosclerosis and CVD risk: is CRP an innocent bystander?” Nutrition, Metabolism and Cardiovascular Diseases, vol. 19, no. 8, pp. 521– 524, 2009. between former and current smokers. Additionally, former smoker IL-6 levels were significantly lower than the level among current smokers only among the males. These results suggest that CRP levels and IL-6 levels may not be the primary modulators of health improvements following cessation. Alternatively, intense smoking (high frequency over multiple decades) may have more longstanding effects on some types of inflammation. Our CRP results are consistent with results of Asthana et al. among smokers with a similar three-decade history of smoking but with half the pack-years [26]. [4] M. Ohsawa, A. Okayama, M. Nakamura et al., “CRP levels are elevated in smokers but unrelated to the number of cigarettes and are decreased by long-term smoking cessation in male smokers,” Preventive Medicine, vol. 41, no. 2, pp. 651–656, 2005. [5] R. Roubenoff, T. B. Harris, L. W. Abad, P. W. E. Wilson, G. E. Dallal, and C. A. Dinarello, “Monocyte cytokine production in an elderly population: effect of age and inflammation,” Journals of Gerontology—Series A Biological Sciences and Medical Sci- ences, vol. 53, no. 1, pp. M20–M26, 1998. Numerous studies have now confirmed that CRP levels are elevated in patients with the metabolic syndrome. CRP levels were shown to be strongly associated with BMI, low HDL, triglycerides, and levels of the proinflammatory cytokine, IL-6 [6, 27, 28]. Our results that showed that CRP levels were significantly influenced by HDL, triglycerides, and IL-6 levels support a potential relationship between CRP levels and the metabolic syndrome. [6] S. Cheng, A. Lyass, J. M. Massaro, G. T. O’Connor, J. F. Keaney Jr., and R. S. Vasan, “Exhaled carbon monoxide and risk of metabolic syndrome and cardiovascular disease in the community,” Circulation, vol. 122, no. 15, pp. 1470–1477, 2010. [7] W. B. Ershler and E. T. Keller, “Age-associated increased interleukin-6 gene expression, late-life diseases, and frailty,” Annual Review of Medicine, vol. 51, pp. 245–270, 2000. In conclusion, our data supports previous studies that showed that serum CRP is associated with biomarkers of the metabolic syndrome but not with smoking. However, serum IL-6 levels are mainly affected not only by the metabolic syndrome but also by age and smoking status. [8] J. A. Nettleton, L. M. Steffen, E. J. Conflict of Interests [13] M. Hara, Y. Matsuzaki, T. Shimuzu et al., “Preoperative serum C-reactive protein level in non-small cell lung cancer,” Anti- cancer Research, vol. 27, no. 4, pp. 3001–3004, 2008. The authors declare no conflict of interests. Acknowledgment [14] D.-K. Kim, S. Y. Oh, H.-C. Kwon et al., “Clinical significances of preoperative serum interleukin-6 and C-reactive protein level in operable gastric cancer,” BMC Cancer, vol. 9, article 155, 2009. This work was partially supported by a grant (DOD PR 023104) from the Department of Defense. This work was partially supported by a grant (DOD PR 023104) from the Department of Defense. [15] J. Danesh, P. Whincup, M. Walker et al., “Low grade inflamma- tion and coronary heart disease: prospective study and updated meta-analyses,” British Medical Journal, vol. 321, no. 7255, pp. 199–204, 2000. Highlights (i) We conducted a cross-sectional study on a sample of 154 current and former smokers. (i) We conducted a cross-sectional study on a sample of 154 current and former smokers. [10] J. Danesh, R. Collins, P. Appleby, and R. Peto, “Association of fibrinogen, C-reactive protein, albumin, or leukocyte count with coronary heart disease: meta-analyses of prospective studies,” Journal of the American Medical Association, vol. 279, no. 18, pp. 1477–1482, 1998. (ii) We investigated the relationship between inflamma- tory biomarkers and smoking status. (ii) We investigated the relationship between inflamma- tory biomarkers and smoking status. (iii) CRP levels were significantly associated with IL-6 levels regardless of smoking status. (iii) CRP levels were significantly associated with IL-6 levels regardless of smoking status. [11] M. Miller, M. Zhan, and S. Havas, “High attributable risk of elevated C-reactive protein level to conventional coronary heart disease risk factors: the third National Health and Nutrition Examination Survey,” Archives of Internal Medicine, vol. 165, no. 18, pp. 2063–2068, 2005. (iv) The significant predictors of CRP levels were biomarkers of the metabolic syndrome. (iv) The significant predictors of CRP levels were biomarkers of the metabolic syndrome. (v) IL-6 levels were significantly associated with smoking especially among current smokers. (v) IL-6 levels were significantly associated with smoking especially among current smokers. (v) IL-6 levels were significantly associated with smoking especially among current smokers. [12] M. Groblewska, B. Mroczko, U. Wereszczy´nska-Siemia¸tkowska et al., “Serum interleukin 6 (IL-6) and C-reactive protein (CRP) levels in colorectal adenoma and cancer patients,” Clinical Chemistry and Laboratory Medicine, vol. 46, no. 10, pp. 1423– 1428, 2008. International Journal of Inflammation Mayer-Davis et al., “Dietary patterns are associated with biochemical markers of inflamma- tion and endothelial activation in the Multi-Ethnic Study of Atherosclerosis (MESA),” American Journal of Clinical Nutri- tion, vol. 83, no. 6, pp. 1369–1379, 2006. [9] P. M. Ridker, N. Rifai, M. J. Stampfer, and C. H. 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https://openalex.org/W4382490827	https://zenodo.org/record/8095991/files/8123ijhas01%20%281%29.pdf	English	null	ASSESSMENT OF NUTRITIONAL STATUS BY MIDUPPER-ARM-CIRCUMFERENCE (MUAC) AMONG THE UNIVERSITY FEMALE BOARDERS OF MIDNAPORE TOWN, WEST BENGAL	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	3,384	International Journal of Humanities, Art and Social Studies (IJHAS), Vol. 8, No.1, February 2023 International Journal of Humanities, Art and Social Studies (IJHAS), Vol. 8, No.1, February 2023 ABSTRACT A cross sectional study has been conducted among 270 university female boarders of Vidyasagar University, Paschim Medinipur district, West Bengal. All of the participants belong to 20-26 years of age. The objective of the study is to find out the nutritional status of female boarders of Vidyasagar University and the facors influencing the nutritional status of the participants. Body Mass Index (BMI), Waist Circumference (WC), Waist Hip Ratio (WHR), Mid Upper Arm Circumference (MUAC) and Waist Height Ratio (WHTR) have been used to evaluate nutritional status of the participants. Central obesity is found more among the participants according to WC (5.6%), WHR (8.9%) and WHTR (13%). In this study, MUAC shows significant association with occupation and educational status of father of the participants. No other associated factor has been found to influence the nutritional status of the participants. ASSESSMENT OF NUTRITIONAL STATUS BY MID- UPPER-ARM-CIRCUMFERENCE (MUAC) AMONG THE UNIVERSITY FEMALE BOARDERS OF MIDNAPORE TOWN, WEST BENGAL Arunima Kundu h.D. Scholar, Department of Anthropology, Vidyasagar University,Midnapore-721102 KEYWORDS Nutritional status, University students, Female students, Boarders, MUAC 1. INTRODUCTION Nutritional status assessment is very important to understand the standard of living of any individual in any population. Nutritional status is influenced by food consumption [3][14][19]. In the era of globalization, the changes in the socio economical factor also influence the nutritional status of individuals. This results in an increase in illnesses either by overnutrition or undernutrition [3]. Globally it seems to be a serious health risk among the 20–39 years old population [21]. Proper nutrition, adequate sleep, and physical activity help to increase immunity power, combat disease, develop physical and mental growth, and increased productivity [3][12]. World Health Organization (WHO) report said that, globally the frequency of underweight adults are 462 million while overweight or obese are 1.9 billion. In India, the prevalence of being underweight declined between 2006-2016, from 36% to 23% among women and 34% to 23% in the case of men. According to WHO, the overall prevalence of underweight, overweight and obesity in male university students were 14.2%, 11.5%, and 2.5%, respectively, in female university students were 27.5%, 2.4%, and 0.3%, respectively. In the context of globalization and industrialization, both developing and developed countries have faced the problem of malnutrition. The mushrooming of shopping malls, fast food outlets, different social and cultural pressure, peer group influences, availability of oily food in university campuses, and adequacy of funds have compelled young adults to adopt unhealthy food habits [7][17][2][10][6]. University students, usually within the age group of 17-25 years, cover a large number of populations [1]WHO 2002,. Living away from home influences the nutritional status which leads to under and over-nutrition [17][6]. Most university students did not consume recommended macronutrients/micronutrients [22][7][15][4][19][20]. A report has shown that the underweight prevalence of university students is increasing day by day, especially in females .Female 1 International Journal of Humanities, Art and Social Studies (IJHAS), Vol. 8, No.1, February 2023 university students are more akin to losing weight than male students [9]. A women’s nutritional status is very important for her child’s good health [12]. A small mother will have small babies who will grow to become small mothers. There is an association between maternal and child nutrition[23][8][11]. In developing country like India women faces serious health concern related to physical growth and nutritional status which starts at an early age[12][5]. Anthropometry is affordable and easily accessible technique for evaluating nutritional status of different populations [18]. (a) To assess the nutritional status of the female boarders of Vidyasagar University. (b) To find the factors affecting the nutritional status of female boarders of Vidyasagar University. (a) To assess the nutritional status of the female boarders of Vidyasagar University. (b) To find the factors affecting the nutritional status of female boarders of Vidyasagar University. (b) To find the factors affecting the nutritional status of female boarders of Vidyasagar University. 1. INTRODUCTION Hence, I have used different anthropometric measurements to acess the nutritional status of the university female students who used to live in the hostels affiliated with Vidyasagar University. 2. OBJECTIVE (a) To assess the nutritional status of the female boarders of Vidyasagar University. 3. MATERIALS AND METHOD A cross-sectional study was conducted in the two affiliated girls’ hostels of Vidyasagar University, Midnapore, West Bengal named Pritilata Chhatrinibas and Matangini Chhatrinibas among female students. Among 300 female students, only 270 students have been chosen to conduct the study based on their willingness to participate as a subject. They are pursuing postgraduate courses under Vidyasagar University and of 20-26 years of age. Purposive sampling was used to select the participants of the study. A pre-structured schedule was made to collect data. A quantitative technique was applied here. Anthropometric measurements were obtained by using Martins Anthropometer, a weighing machine, and a calibrated cloth tape. Socioeconomic variables and anthropometric variables are taken for the study. They are as follows, Socio-economic variable: Socio-economic variables are family type, education of father, education of mother, Occupation of father, occupation of mother, family income, Anthropometric variable: The anthropometric variables which have been used in this study are as follows, height (cm), weight (kg), Mid Upper Arm Circumference (mm), Waist Circumference (mm), Hip Circumference (mm). The derived variables which I have found are Body Mass Index (BMI), Waist Hip ratio (WHR) and Weight Height Ratio (WTHR) accordingly. SPSS (IBM Statistical Package for the Social Sciences, version18) software has been used for statistical analysis. To find the associated factors which influence the nutritional status of the study participants, a chi-square test has been performed. 2 2 International Journal of Humanities, Art and Social Studies (IJHAS), Vol. 8, No.1, February 2023 4. RESULTS Table 1- Anthropometric characters of the study participants Anthropometric variable Mean SD Prevelence of BMI Height 154.10 5.16 Category n % Weight 49.44 4.50 CED grade III 12 4.4 MUAC 24.18 2.39 CED grade II 8 3.0 WC 30.0 1.89 CED grade I 38 14.1 HC 69.47 7.33 Normal 188 69.6 Overweight 21 7.8 Derived Variable Mean SD Obese 3 1.1 BMI 20.81 3.12 Prevelence of MUAC WHR 0.77 0.05 Undernutrition 62 23.0 WHTR 1.13 0.07 Normal 208 77.0 Prevelence of WHR Prevelence of WHTR n % Normal 246 91.10 Normal 235 87.0 Central obese 24 8.9 Central obese 35 13.0 Prevelence of WC Normal 255 94.4 obese 15 5.6 4. RESULTS Table 1 shows that the mean and standard deviation of the anthropometric variables viz. height, weight, MUAC, WC, and HC are 154.10 ±5.16, 49.44 ±4.50, 24.18 ±2.39, 30.0 ±1.89, 69.47 ±7.33 respectively. The derived variables are Body Mass Index(BMI), Waist Hip Ratio(WHR), and Waist Height Ratio(WHTR). The mean and standard deviation of the BMI, WHR, and WHTR are 20.81± 3.12, 0.77 ±0.05 and 1.13± 0.07 respectively. The BMI is an attempt to quantify the amount of tissue mass (muscle, fat, and bone) in an individual, and then categorize that person as Chronic Energy Deficiency (CED), normal weight, overweight, or obese based on that value. According to the table, the prevalence of nutritional status based on BMI according to WHO (1995) it is revealed that among 270 individuals, 12 females (4.4%) are under CED III, 8 females (3%) are under CED II, 38 females (14.1%) are under CED I. while 188 females (69.6%)are normal, 21 females (7.8%) are overweight and 3 females (1.1%) are obese. Hence, 58 females (CED grade III+CED grade II+CED grade I) have BMI less than 18.5, 188 females are normal and lie in between the BMI range 18.5-24.9 and the rest 24 have BMI more than 25. Waist Circumference (WC) is another measurement of central obesity. According to the table, the prevalence of nutritional status based on WC shows that among 270 individuals, 255 individuals (94.4) are normal and 15 individuals (5.6%) are centrally obese. Waist hip Ratio (WHR) is the measure to calculate central obesity. Table 5 reveals that among 270 individuals, 246 individuals (91.1%) are normal and 24 individuals (8.9%) are centrally obese. Waist height ratio (WHTR) is another measurement of central obesity. This table shows that among 270 individuals, 235 (87%) individuals are normal and 35 (13%) are centrally obese. MUAC has been used to detect short- term changes in the nutritional status of a population. It is revealed that among 270 individuals(females) 208 individuals (77%) are normal and 62 individuals (23%) are in the undernutrition category. 3 3 International Journal of Humanities, Art and Social Studies (IJHAS), Vol. 4. RESULTS 8, No.1, February 2023 Table 2: Socio demographic and economic profile Education (%) Father Mother Father occupation % Mother occupation % Non- literate 10 18.5 Agricultural labour 37.8 Housewife 85.6 Primary 30.4 34.1 Daily labour 8.5 Dialy labour 4.4 Secondary 34.1 28.1 Business 35.2 Service 8.1 HS 11.9 11.5 Service 15.6 Non manual labour 0.4 Graduate and more 10.4 5.6 Income category Family type % Income group Income range n % Nuclear 69.3 Lower middle 1036-4045 5 1.8 Joint 11.1 Upper middle 4046-12535 122 45.2 Extended 19.6 high >12535 143 53.0 Table 2 illustrates the socio-demographic profile and economic profile. Three types of families are found here. These are the nuclear family, joint family, and extended family. Observance of Nuclear family is found to be more (69.3%) than joint (11.1%) and extended (19.6%) family. Father’s and mother’s education has been categorised into five divisions. They are Non- literate/can sign (10%, 18.5%), primary (30.4%, 34.1%), secondary (34.1%, 28.1%), higher secondary (11.9%, 11.5%), and Graduate (10.4%, 5.6%) respectively. It is seen that the prevalence of literacy among mothers is more than that of the father. Primary education among mothers is more than that of the father Table 2 illustrates the socio-demographic profile and economic profile. Three types of families are found here. These are the nuclear family, joint family, and extended family. Observance of Nuclear family is found to be more (69.3%) than joint (11.1%) and extended (19.6%) family. Father’s and mother’s education has been categorised into five divisions. They are Non- literate/can sign (10%, 18.5%), primary (30.4%, 34.1%), secondary (34.1%, 28.1%), higher secondary (11.9%, 11.5%), and Graduate (10.4%, 5.6%) respectively. It is seen that the prevalence of literacy among mothers is more than that of the father. Primary education among Table 2 illustrates the socio-demographic profile and economic profile. Three types of families are found here. These are the nuclear family, joint family, and extended family. Observance of Nuclear family is found to be more (69.3%) than joint (11.1%) and extended (19.6%) family. Father’s and mother’s education has been categorised into five divisions. They are Non- literate/can sign (10%, 18.5%), primary (30.4%, 34.1%), secondary (34.1%, 28.1%), higher The monthly income of the participants has been categorized according to World Bank classification which shows that total family income less than 1036 rs per month belongs to the low-income category. 4. RESULTS The lower middle-class category range is from 1036 -4045 rs and the upper-middle-class category range is from 4046-12535. Monthly family Income more than 12535 rs belongs to high-income category. The table shows that, majority of the families belong to agriculture-oriented economy. They are agriculture (37.8%), daily labor (8.5%), business (35.2%), and service (15.6) respectively. Also, the major frequency is observed among the housewife category (85.6%). Then Labour (4.4%), service (8.1%), and non-manual labor (0.4%) are the remaining three categories of mother occupation. 4 International Journal of Humanities, Art and Social Studies (IJHAS), Vol. 8, No.1, February 2023 Table 3: Association between father’s education category and MUAC Table 4: Association between father’s occupation category and MUAC Association between Father education category and MUAC category χ2 = 10.024 ;df=4 ; p=0.040 Association between Father occupation category and MUAC category χ2 = 17.833; df=3 ; p=0.000 Father education Under nutrition Normal Father occupation Under nutrition Normal Non- literate 12 (44.4%) 15 (55.6%) Agriculture 27 (26.5%) 75 (73.5%) Primary 21 (25.6%) 61 (74.4%) Manual labour 11 (47.8%) 12 (52.2%) Secondary 15 (16.3%) 77 (83.7%) Business 10 (10.5%) 85 (89.5%) HS 6 (18.8%) 26 (81.3%) Service 12 (28.6%) 30 (71.4%) Graduate and more 6 (21.4%) 22 (78.6) The table 3 shows that, 44.4% participants fall under nutritional category whose fathers are non- literate followed by primary education category which shows 25.6% individuals are in undernutrition category. Hence, an association has been found between father education category and MUAC category. Father education category and MUAC category are significantly associated (χ2 = 10.024 ;df=4 ; p=0.040). The table 4 shows that, 47.8% indivials are in undernutritional category according to MUAC whose fathers are used to do manual labour jobs and 89.5% participants are normal whose father are businessman. Hence, an association has been found between father occupation category and MUAC category. Father occupation category and MUAC category is significantly associated (χ2 = 17.833; df=3 ; p=0.000). 5. DISCUSSION I have found that, the mean height and weight of the participants are 154.10 cm and 49.44 kg respectively. Rasoul stated that, mean height is 154.43 cm and 48.9 kg for girlssimilar to my study [17]. In my study, I have found that, 12 females (4.4%) are under CED III, 8 females (3%) are under CED II, 38 females (14.1%) are under CED I. while 188 females (69.6%)are normal, 21 females (7.8%) are overweight and 3 females (1.1%) are obese .Another study showed that, prevalence of overweight and obesity is 17.35% and 14.33% which is high than my study [12]. In my study, The prevalence of central obeseity is among 15 individuals (5.6%) according to WC category,24 individuals (8.9%) according to WHR category and 35 (13%) according to WHTR category. Another scholars used MUAC as best indicator to detect short term changes in the case of nutritional status. It is revealed that among 270 individuals(females) 208 individuals (77%) are normal and 62 individuals (23%) are in the undernutrition category. Another study showed that, the prevalence of undernutrition is 21.9% which is less than my study [17]. In this study, MUAC shows significant association with occupation and educational status of father of the participants. But in another study the scholars had found that only father occupation is significantly associated with obesity and overweight [6]. 5 5 International Journal of Humanities, Art and Social Studies (IJHAS), Vol. 8, No.1, February 2023 International Journal of Humanities, Art and Social Studies (IJHAS), Vol. 8, No.1, February 2023 International Journal of Humanities, Art and Social Studies (IJHAS), Vol. 8, No.1, February 202 6. CONCLUSION This paper has tried find out the nutritional status of the female boarders of Vidyasagar University by evaluating nutritional status through BMI, HC, WC, WHR, WHTR and MUAC. Which fulfils the first objective of the study. Also socio demograpgic and economic distributuion been shown which suggests that, MUAC strongest association with father education and father occupation. Hence, among all the associated factors, only father education and father occupation can influence the nutritional status of the study participants. It can be concluded that, MUAC is the best indicator to evaluate nutritional status of the study participants. The prevalence of overweight and obesity according to BMI category and undernutriton according to MUAC category have been found high if compared to another studies. Hence, I will suggest that, Proper intervention is needed in the hostels. Also nutritional awareness is necessary among the participants for the good health and better quality of life of the individuals. ACKNOWLEDGEMENT I am very thankful to the hostel superintendent of Vidyasagar University, Midnapore to permit me for data collection. I also thankful to all of the participants of my study. REFERENCES 1. 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Huda, N., & Ahmad, Rajia., (2010) Preliminary Survey 0n Nutritional Status among Univers Students at Malaysia, Pakistan Journal of Nutrition, vol 9, issue 2, pp 125-127 9. Desai, S. (1994) Gender Inequalities and Demographic Behavior, India, New York Population Council 10. Achinihu, G. REFERENCES (2010) Nutritional Status of University Students in South-Eastern States of Nigeria, Journal of Research in National Development, vol 7, issue 2 f p 11. Sogari, G., Argumedo, C.V., Gomez, M.I., & Mora, C., (2018). College Students and Eating Habits: A Study Using an Ecological Model for Healthy Behaviour, Nutrients, vol 10. 6 6 International Journal of Humanities, Art and Social Studies (IJHAS), Vol. 8, No.1, February 2023 12. Mushonga , N.G.T. , Chagwena, D. , Benhura, C., & Nyanga, L. K., (2013) An Assessment of the Nutritional Status of University of Zimbabwe Students, Pakistan Journal of Nutrition, vol 12, issue 1, pp 30-33 pp 13. Chin, Y.S., & Mohd, N.M. 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Rasoul, G., (2011) Health and Nutritional Status of Students Residing at University of Agricultural Sciences Hostel, Bengaluru, Karnataka, India, Department of Food Science and Nutrition University of Agricultural Sciences Bengaluru BIOGRAPHY Author Name: Arunima Kundu Profile: I am a regular Ph.D. scholar of Department of Anthropology, Vidyasagar University, Midnapore-721102, West Bengal. I have done specialization in Biological Anthropology from the same department in 2019. 7 7
W3205867107.txt	https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/ejhf.1963	fr		Supplement Article	European journal of heart failure	2,020	cc-by	3,350	European Journal of Heart Failure (2020) 22 (Suppl. S1) 2–415 doi:10.1002/ejhf.1963 Basic Science – Vascular Diseases: Biomarkers 22 Development of reactive pulmonary hypertension induced by left heart failure can be predicted by the assessment of the level of new biomarkers - results on experimental rat model M Milan Chovanec1 ; F Malek2 ; Z Jiraskova-Zakostelska3 ; J Durisova4 ; B Kaftanova4 ; T Andreasova2 ; J Herget4 ; 1 Charles University of Prague and Na Homolce Hospital, Prague, Czechia; 2 Na Homolce Hospital, Cardiology department, Prague, Czechia; 3 Academy of Sciences of the Czech Republic, Laboratory of Cellular nad Molecular Immunology, Institute of Microbiology , Prague, Czechia; 4 Charles University Prague, 2nd Faculty of Medicine, Department of Physiology, Prague, Czechia; Funding Acknowledgements: Grant Agency of the Czech Republic (GACR) 17-11223S and Institutional Research Grant MH-CZ DRO NNH 00023884 IG160502 and IG190501 Introduction: The most common cause of pulmonary hypertension (PH) in patients is due to left heart failure (HF). Diagnosis of early stages of HF or PH have not been clearly obvious, recently. Elevated filling pressures in the heart and pulmonary vascular remodelling is associated with expression changes of the various plasma levels of biomarkers. Purpose: To assess the plasma levels of HF biomarkers on a new rodent model of HF induced by the left ventricle pressure-overload which leads to the development of reactive PH consist of pulmonary vascular remodelling. Methods: The left heart failure was induced by pressure overload in adult male Wistar rats by inserting a polyethylene tubing into aorta trough the right carotid artery. Three weeks later experimental animals were studied (the group E, n=6) and compared to the controls (n=6). Serial venous blood samples were taken from both experimental groups to determine levels of biomarkers involved in pathophysiology cardiac and vascular remodelling: Troponin I, N-proBNP, Copeptin, Apelin, Endostatin, Asymmetric dimethylarginine (ADMA), Growth/differentiation factor 15 (GDF-15), Ceruloplasmin and Cystatin-C. The biomarker levels were assessed by ELISA method. Results: The left ventricle end-diastolic pressure was elevated in the group E (1.34±0.07 mmHg vs. 0.41±0.13 mmHg in the controls; p<0.0001). Mean pulmonary arterial blood pressure measured by catheterization was increased 22.9±0.7 mmHg compared to the controls 16.9±1.0 mmHg; p<0.05. Weight of the right ventricle relative to body weight was 5.5±0.3.10-4 compared to the controls 4.6±0.2.10-4; p<0.05. In lung histology, 74% of small pulmonary vessels had muscularized media (24% in controls; p<0.01). Elevated blood plasma levels of biomarkers in the group E compared to the controls were found in: NT-proBNP (671.8±61.2 pg.mL-1 vs. 450.3±47.5 pg.mL-1; p<0.05; respectively) and Copeptin (251.9±41 pg.mL-1 vs. 141.3±10.1 pg.mL-1; p<0.05; respectively). Significantly decreased blood plasma levels of biomarkers in the group E compared to the controls were found in the values of Apelin (4.0±0.09 ng.mL-1 vs. 4.3±0.05 ng.mL-1; p<0.05; respectively) and ADMA (12.1±0.5 mg.mL-1 vs. 15.3±0.8 mg.mL-1; p<0.05; respectively). We found no significant changes in the blood plasma levels compared to the controls in the values of TnI, GDF-15, Endostatin, Cystatin-C and Ceruloplasmin. Conclusion: We develop a brand new rodent model of PH accompanied with pulmonary vascular remodelling induced by left HF. Presented experimental model was associated with increased concentration of biomarker of cardiac remodelling: NT-proBNP and Copeptin and with decreased level of biomarkers that have protective effect against vascular remodelling: Apelin and ADMA. Atherosclerosis, Cerebrovascular Diseases, Aneurysm, Restenosis 23 Microwave spectrometry for coronary stent monitoring S Susana Amoros Garcia De Valdecasas1 ; GGL Gonzalez-Lopez1 ; IJC Jimenez2 ; LLJ Jofre1 ; ABG Bayes-Genis3 ; JT Tejada2 ; JOC O’callaghan Castella1 ; ORL Rodriguez-Leor4 ; CGM Galvez-Monton3 ; 1 Polytechnic University of Catalonia, Barcelona, Spain; 2 University of Barcelona, Grup de Magnetisme, Departament de Física de la Matèria Condensada, Barcelona, Spain; 3 Fundació Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, ICREC Research Program, Barcelona, Spain; 4 Germans Trias i Pujol University Hospital, Servei de Cardiologia, Barcelona, Spain; Funding Acknowledgements: 20153530/31, 2017 SGR 00483, RD16/0011/0006, CB16/11/00403, MDM-2016-0600, PI18/00256, PI18/00256, Sociedad Española de Cardilogía Background: Coronary artery disease (CAD) is the leading cause of death worldwide, and percutaneous coronary intervention with stenting the most widely performed procedure to treat CAD. However, current stent monitoring techniques are invasive and/or ionizing. Microwave spectrometry (MWS) may provide a non-invasive, non-ionizing and cost-effective alternative capable of detecting stent-related pathologies before they cause fatal heart failure. MWS response to monitor stent status Purpose: To develop a new MWS-based technology to monitor coronary stents in an in vivo swine model. Methodology: First, using a new MWS device, an in vitro experiment was carried out to demonstrate: (1) the ability of detecting the presence of a stent and (2) stent fractures (SF). To that end, an intact stent was distanced 3, 7, 11 and 15 mm from a MWS near-field probe in open-air conditions. Afterwards, three identical stents were piecemeal cut to emulate type I, II and III SF at different fractions of the stent’s length (l): l/5, l/3 or l/2. Additionally, the stent was measured in a phantom substance, to simulate in vivo conditions: it was distanced from 0 to 40 mm in steps of 5 mm. Likewise, a pair of MWS far-field antennas measured the stent at 10, 20, 30 and 40 mm. © 2020 The Authors European Journal of Heart Failure © 2020 European Society of Cardiology, 22 (Suppl. S1), 2–415 108 Resynchronization Therapy P569 Myocardial dyssynchrony and a different response to cardiac resynchronization therapy in patients with chronic heart failure and atrial ﬁbrillation T Troyanova-Shchutskaia1 ; A Kurlianskaya1 ; T Denisevich1 ; D Goncharik1 ; A Chasnoyt1 ; T Asmalouskaia1 ; O Shatova1 ; 1 Republican Scientiﬁc and Practical Centre of Cardiology, Minsk, Belarus; Background: The search for ideal responders to cardiac resynchronization therapy (CRT) remains relevant and is widely discussed nowadays. Purpose: To assess the echocardiographic response to CRT depending on the myocardial dyssynchrony in patients with chronic heart failure (CHF) and permanent atrial fibrillation (AF). Methods: The study included 101 patients with CHF NYHA II – III, left ventricular ejection fraction <35% and QRS≥130 ms. All patients underwent CRT. Then atrioventricular node ablation was performed to 83 patients, but 18 patients refused it. The criterion for the effectiveness of CRT was a decrease in the end-systolic volume ≥15%. The survey was conducted initially, after 3 and 12 months from the date of inclusion in the study. According to the results of the prospective observation groups of patients with CHF and AF were formed in dependence on the duration and sustainability of the response to CRT: 1a - early response (3 month) and preservation of effect by 12 months, 1b - ‘elusive’ early effect, 2a - delayed response (12 month), 2b – non-responders. Results: The study revealed the correlation between the increase in the presystolic delay on the aortic valve and the delayed response to CRT that was confirmed by the results of the frequency analysis of this interventricular delay. In groups 1a, 1b and 2b the frequency of cases of presystolic delay on the aortic valve varied from 67% to 73%, but in group 2a it was 100%. We obtained similar results analyzing dispersion of intraventricular contraction. In group 2a a variance of intraventricular contraction of more than 100 ms was found in 90.5% of patients (no more than 70.0% of patients in the remaining groups). Responders and non-responders significantly differed in the incidence of moderately extended and wide QRS. Picture 1 demonstrates the incidence of QRS wide (>150 ms) in patients with a different response to CRT. The results indicated the dependence of the positive effect of CRT on the width of the QRS complex. Conclusions: The early sustained response to CRT was more commonly detected in NYHA II patients. There were more non-responders among those with NYHA III. Patients with an early and sustained response to CRT were characterized by higher median values of the 6-minute walk test. Diabetes mellitus was more often detected in individuals with the ’escape’ of the early positive effect. A smaller proportion of atrioventricular node ablation was found in the subgroup with an elusive effect, which allows us to conclude that ablation of the AV compound increases the effectiveness of CRT in individuals with AF. QRS wide incidence and CRT response Ventricular Assist Devices P571 Effect of levosimendan infusion prior to left ventricular assist device implantation A Amitai Segev1 ; J Lavee1 ; Y Kassif1 ; Y Shemesh1 ; D Lotan1 ; D Freimark1 ; A Morgan1 ; A Kogan1 ; A Grupper1 ; 1 Sheba Medical Center, Ramat Gan, Israel; Funding Acknowledgements: None Introduction: Data on the safety and efficacy of preoperative levosimendan infusion in patients undergoing left ventricular assist device (LVAD) implantation is scarce. Purpose: To investigate the clinical utility of preoperative levosimendan administration in patients prior to LVAD implantation. Our hypothesis was that preoperative levosimendan will be associated with improved post-operative outcomes due to extended cardiovascular therapeutic effects from its long-acting active metabolites. Methods: A retrospective analysis of all consecutive patients who received continuous-flow LVADs (HeartMate 2, 3, and HVAD) between August 2012 and May 2018 in a single tertiary center. INTERMACS profile 1 patients were excluded. Patients who received preoperative levosimendan were compared to patients who did not receive levosimendan preoperatively. Results: The study cohort consisted of 62 patients (40 [65%] in the levosimendan group and 22 [35%] in the no-levosimendan group) with a median follow-up of 21 months. Preoperative laboratory parameters were similar in both groups except for lower median albumin levels among patients in the levosimendan group (3.7 g/dL vs. 4 g/dL; P=0.024). Preoperative echocardiographic parameters, including left ventricular ejection fraction, the presence of enlarged right ventricle (RV), moderate to severe reduction in RV function and above moderate tricuspid regurgitation were similar between the levosimendan and no-levosimendan groups (median of 15% vs. 17.5%; 18% vs. 7%; 52.5% vs. 40.9%; and 10% vs. 13.6%, respectively; p>0.1 for all). Preoperative cardiac output, mean pulmonary artery and right atrium pressures and pulmonary vascular resistance were also similar between the groups (median of 2.93 L/min vs. 3.19 L/min; 41.5 mmHg vs. 33 mmHg; 10 mmHg vs. 8 mmHg; and 3.17 wood units vs. 4.02 wood units, respectively; p>0.1 for all). Preoperative median pre-capillary wedge pressure was higher in the levosimendan group (28 mmHg vs. 23.5 mmHg; P=0.047). No patient in either group required right ventricular assist device support. There was no difference in post-operative inotropes and ventilation support time between the levosimendan and no-levosimendan groups (median of 51 hours vs. 72 hours; P=0.41 and 24 hours vs. 27 hours; P=0.19, respectively). Length of hospitalization, both total and in the intensive care unit was similar (median of 13 days vs. 16 days; P=0.34 and 3 days vs. 4 days; P=0.44, respectively). Post-operative laboratory and echocardiographic parameters did not differ between the groups. The in-hospital complication rate including bleeding, surgical re-exploration and cardiac arrhythmias was also similar. There was no significant difference in the in-hospital and long term mortality throughout the follow-up period between the groups (2.5% vs. 4.5%; P>0.99 and 10% vs. 27.3% respectively; P=0.64). Conclusion: Levosimendan infusion prior to LVAD implantation was not associated with improved post-operative outcomes. Chronic Heart Failure: Rehabilitation P572 Characteristics of good response to aerobic axercise training in decompensated heart failure patients BM Bruno Miguel Delgado1 ; I Lopes1 ; B Gomes2 ; A Novo3 ; 1 Hospital Center of Porto, Porto, Portugal; 2 University of Porto, Porto, Portugal; 3 Escola Superior de Saude do IPB, Bragança, Portugal; Introduction: Exercise training is an excellent tool to promote functional capacity in chronic heart failure (HF) patients. Although its benefits in this population, it needs to be demonstrated in decompensated HF patients. A way to optimize an intervention is to evaluate who are the good responders and understand the causes of no response. Knowing the factors of good response is also important to emphasize the feasibility of an intervention and to deliver it to the ones who get the most benefit of it. Purpose: To identify the characteristics that lead a patient to have a better response to an aerobic exercise training program for decompensated HF inpatients – ERIC-HF (early rehabilitation in cardiology – heart failure) Methods: 50 patients who performed ERIC-HF program during the phase of stabilization were evaluated in terms of their sociodemographic, functional and physiological characteristics and performance during the program. The main variable used to understand the performance of the patients was the variation of the distance walked in the 6-minute walking test (6MWT), performed as soon as the patient were able to do it (6MWTinicial) and at discharge (6MWTdischarge). A multiple linear regression was made in order to determine which variables are related to a better variation on the 6MWT, namely: age, LCADL and Barthel index (BI) scores at admission and discharge, number of days of hospitalization, number of cardiovascular risk factors, NYHA class, etiology of HF and ventricular function. Durbin-Watson test was used to analyze the existence of independence of residual random variables. It was assumed a significance level at p<0.05. Results: Patient’s average age was 71 (±11) years old, 34 are male, 80% are in NYHA class III and 73% have severe left ventricular depression. Patients present a median of 76 points in BI at admission (minimum of 45 and maximum of 97) and a median of 32 at LCADL (minimum of 24 and maximum of 45 points). The mean distance walked in the 6MWTinicial performed by the patients was 199.9 (±115.9) meters and 287.6 (±128.9) meters at 6MWTdischarge, representing a 87.7 (±170.6) meters variation. © 2020 The Authors European Journal of Heart Failure © 2020 European Society of Cardiology, 22 (Suppl. S1), 2–415 109 According to the linear regression, an equation was obtained: Difference of the 6MWT = 454.694-16MWTinitial+2.981Barthelinitial-5.554*age. This equation explains 65% of the variation of the model in this sample of patients. Using this variables it’s possible to know how much distance a patient can walk, and understand if he is going to have a good performance in the program. Conclusions: Patients with the worst results in the initial 6MWT, higher initial Barthel and younger ages, will get the most gains in terms of difference walked between the initial and final 6MWT and have the most benefit from the intervention program. Chronic Heart Failure: Multidisciplinary Interventions P574 The impact of percutaneous mitral valve repair with mitraclip in cardiopulmonary performance JP Joao Pedro Dias Ferreira Reis1 ; J Viegas1 ; A Goncalves1 ; A Galrinho1 ; L Branco1 ; A Fiarresga1 ; D Cacela1 ; R Cruz Ferreira1 ; 1 Hospital de Santa Marta, Lisbon, Portugal; Aim: The aim of this study was to evaluate changes in Ney York Heart Association (NYHA) functional class, cardiopulmonary exercise test (CPET) variables, 6-min walk test (6MWT) in patients (pts) submitted to percutaneous mitral valve repair with MitraClip (MC). Methods: Prospective analysis of 46 pts with moderate to severe mitral regurgitation - MR - (grades III-IV) submitted to MC between 2013 and 2019 in our institution. The Wilcoxon sign test was used to assess the procedure’s effect on NYHA Class, CPET and 6MWT, by comparing several variables at baseline and at 1 year postprocedure follow-up. P-values<0.05 were considered significant. Results: 46pts, 61% male, with a mean age of 65±14 years (Y) and mean follow-up time of 17±14 months, of which 58.7% presented with grade IV MR (mean regurgitant volume - 49.6±21 ml; mean EROA - 35.1±13 mm2 ) and 75% with functional MR. Mean LVEF of 36%±11.8, with 55% presenting a LVEF <35%. Mean Euroscore II of 6.0±7. 27.5% had already undergone a previous cardiac surgery, in most cases CABG (63.6%). 34.8% had already suffered an acute coronary syndrome and 63% had atrial fibrillation. Mean pre-procedural peak oxygen uptake (pVO2) of 14.3mL/kg/min and mean distance in the 6 minute walk test (6MWT) of 322±96m. Device implantation was successful in 45 pts with a device success rate of 87% (successful implantation and reduction in MR to grade 2 or less), with 54% of pts presenting mild MR before discharge. There were immediate complications related to the procedure in 17.5% of pts, with 4pts experiencing tendinous cord rupture and 2pts leaflet tear. There were no cases of pericardial tamponade or embolic complications. Follow-up mortality of 34.8% (16pts), with 9 deaths in the first-year post-procedure, 1 pt referred to cardiac surgery due leaflet tear and 1pt to heart transplantation. Successful MC was associated with an improvement in pVO2 (14.3 vs 18.6 mL/kg/min, p=0.043), 6MWT distance (322.2m vs 373.2m, p=0.001) at 1 year follow-up, irrespective of MR aetiology and left ventricular systolic function. It also led to a an improvement in NYHA class both at 6 months (mean NYHA class: 2.9 vs 2.1, p=0.009) and 1 year post-procedure (mean NYHA class: 2.9 vs 2.0, p=0.001). However, these improvements weren’t verified at the 1 and 6 months follow-up. MC wasn’t associated with a reduction of neither the natriuretic peptide value (854.6 vs 450.1 pg/ml, p=0.241) nor the VE/VCO2 slope value (36.2 vs 34.1, p=0.144) at 1 year follow-up. Conclusion: Percutaneous mitral valve repair with MitraClip was associated with an enhancement in cardiopulmonary performance and reduction in the symptomatic burden after the first year post-procedure. Chronic Heart Failure: Pharmacotherapy P575 Prognostic impact of digoxin use in a heart failure population JP Joao Pedro Dias Ferreira Reis1 ; A Goncalves1 ; P Bras1 ; R Ilhao Moreira1 ; T Pereira Silva1 ; A Timoteo1 ; R Soares1 ; R Cruz Ferreira1 ; 1 Hospital de Santa Marta, Lisbon, Portugal; Aim: Digoxin (D) may be considered in patients (pts) in sinus rhythm with symptomatic HFrEF to reduce the risk of hospitalization and in pts with HFrEF and atrial fibrillation (AF) for rate control. There are some controversies regarding its safety in this population, with some studies suggesting a higher risk of events, while others showed no deleterious effect on mortality. Methods: Prospective evaluation of adult pts with HFrEF submitted to CPET in a tertiary centre. Pts were followed up for at least 1 year for the primary endpoints of cardiac death, urgent heart transplantation/ ventricular assist device implantation in the first year of follow-up (MH1) and sudden cardiac death (SCD). Univariate followed by Cox multivariate regression analysis were performed to evaluate the impact of D use in the study’s endpoints. Survival analysis was performed using Kaplan-Meier plots. Results: CPET was performed in 487 HRrEF pts, with a mean age of 56.3 ± 12.5 years, of which 79.1% were male, 46.3% of ischemic aetiology (IA), with a mean LVEF of 30.4 ± 7.6%, a mean heart failure survival score (HFss) of 8.6±1.1. At baseline, 134 (29.3%) pts were receiving D. These pts presented lower LVEF (26.7% vs 30.9%, p<0.001), HFss (8.3 vs 8.7, p<0.001) and sodium values (137.0 vs 138.3, p<0.001), a lower prevalence of coronary artery disease (38.8% vs 49.2%, p=0.042), but a higher prevalence of AF (38.8% vs 19.8%, p<0.001). There was no difference regarding patient’s age, prevalence of chronic kidney disease (CKD), peak oxygen uptake (pVO2) or VE/VCO2 slope values. Baseline D use was independently associated with an increased risk of SCD in our population (HR: 3.45; 95%CI 1.28-9.27, 0.014), aswell as in pts of IA (HR: 4.45, 95%CI 1.25-15.83, p=0.014) and with CKD (HR: 15.57, 95%CI 1.97-123.02, p=0.009). There was no association with SCD in pts of non-ischemic aetiology, preserved renal function and AF. Pts taking D presented a significantly higher incidence of SCD (log rank p<0.001). D use was not independently associated with MH1 in the general population (p=0.122 in multivariate analysis), but it was in pts of IA (HR: 4.94, 95%CI 1.32-18.39, p=0.017). Conclusion: In our HF population, D use was an independent predictor of SCD, particularly in pts with coronary artery disease and CKD. Resynchronization Therapy P576 Prognostic value of submaximal cpet variables in heart failure patients with biventricular pacing JP Joao Pedro Dias Ferreira Reis1 ; A Goncalves1 ; P Bras1 ; R Ilhao Moreira1 ; T Pereira Silva1 ; A Timoteo1 ; R Soares1 ; R Cruz Ferreira1 ; 1 Hospital de Santa Marta, Lisbon, Portugal; Aims: The 2016 International Society for Heart Lung Transplantation listing criteria for heart transplantation (HT) states that the peak volume of oxygen consumption © 2020 The Authors European Journal of Heart Failure © 2020 European Society of Cardiology, 22 (Suppl. S1), 2–415
https://openalex.org/W2953629909	https://ieeexplore.ieee.org/ielx7/6287639/8600701/08752345.pdf	English	null	An Online Correction Predictive EMS for a Hybrid Electric Tracked Vehicle Based on Dynamic Programming and Reinforcement Learning	IEEE access	2,019	cc-by	11,129	An Online Correction Predictive EMS for a Hybrid Electric Tracked Vehicle Based on Dynamic Programming and Reinforcement Learning This work was supported in part by the Beijing Institute of Technology Research Fund Program for Young Scholars and in part by the National Natural Science Foundation of China under Grant 51775039 and Grant 51861135301. ABSTRACT Energy management strategy is critical in the development of hybrid electric vehicles. It is used to improve fuel economy and sustain battery state of charge by splitting the power demand to different power sources while satisfying various physical constraints and vehicle performance simultaneously. However, it is challenging to achieve an optimal control performance due to the complexity of the hybrid powertrain, the time-varying constraints, and stochastic of the load power. Focusing on these problems, this paper presents an online correction predictive energy management (OCPEM) strategy for a hybrid electric tracked vehicle based on dynamic programming (DP) and reinforcement learning (RL). First, a multi-time-scale prediction method is proposed to realize the short-period future driving cycle prediction. Then, the DP algorithm is applied to obtain the local control policy based on the short-period future driving cycle. The RL algorithm is combined with the fuzzy logic controller to optimize the control policy by eliminating the inﬂuence of imprecise prediction. Finally, the simulations are conducted in Matlab/Simulink to evaluate the control effectiveness and adaptability of the proposed method. The results indicate that the fuel economy of the proposed OCPEM is improved by 4% compared with the original predictive energy management and achieve 90.51% of that of the DP benchmark. INDEX TERMS Predictive energy management, hybrid electric tracked vehicle, online correction, rein- forcement learning, fuzzy logic controller. NOMENCLATURE A. ABBREVIATIONS EV Electric vehicle HEV Hybrid electric vehicle FCHEV Fuel cell hybrid electric vehicle PHEG Plug-in hybrid electric vehicle EMS Energy management strategy DP Dynamic programming PSO Particle swarm optimization IPSO Improved particle swarm optimization ECMS equivalent consumption minimization strategy A-ECMS Adaptive equivalent consumption minimization strategy BP Back propagation The associate editor coordinating the review of this manuscript and approving it for publication was Muhammad Imran Tariq. MPC Model predictive control GA genetic algorithm RL Reinforcement learning GT Game theory KL Kullback-Leibler Divergence DNN Deep neural network HETV Hybrid electric tracked vehicle PEM Predictive energy management EGS Engine generator set SOC State of charge RMSE root mean square error OCPEM online correction predict energy management SD standard deviation B. SYMBOLS Pdem Power demand Fa Accelerating resistance 98252 This work is licensed under a Creative Commons Attribution 4.0 License. Received May 7, 2019, accepted June 24, 2019, date of publication July 1, 2019, date of current version August 7, 2019. Digital Object Identifier 10.1109/ACCESS.2019.2926203 An Online Correction Predictive EMS for a Hybrid Electric Tracked Vehicle Based on Dynamic Programming and Reinforcement Learning For more information, see http://creativecommons.org/licenses/by/4.0/ VOLUME 7, 2019 J. Wu et al.: Online Correction Predictive EMS for a Hybrid Electric Tracked Vehicle Fr Rolling resistance Fw Aerodynamic resistance vave Average speed of two tracks v1, v2 Speed of two tracks M Resistance yaw moment ω Rotational speed of the vehicle B Track gauge m Vehicle mass g Acceleration of gravity f Coefﬁcient of rolling resistance CD Coefﬁcient of aerodynamic resistance A Fronted area of the vehicle a Longitudinal acceleration µmax Maximum value of the lateral resis- tance coefﬁcient µt Lateral resistance coefﬁcient R Turning radius of the vehicle Ug Output voltage of the generator Ig Output current of the generator Tg Electromagnetic torque of the generator Ke Coefﬁcient of the electromotive force Kx Equivalent impedance coefﬁcient P Pole-pairs number of the generator Lg Synchronous inductance of the armature thr Throttle of the engine neng The speed of the engine neng,min, neng,max Minimum and maximum value of neng ng speed of the generator Teng output torque of the engine Je moment of inertia for the engine Jg moment of inertia for the generator SOC The state of charge SOCmin, SOCmax Allowed minimize and maximum of SOC Ib The battery current VOC Open-circuit voltage Ubat Output voltage of the battery Rint Internal resistance Ib,min, Ib,max minimum and maximum value of Ib .Pg. power of the generator Pb Power of the battery Pb,min, Pb,max Minimum and maximum value of Pb Udc Bus voltage PL Load power of the vehicle J Cost function J∗ Optimal cost function · fuel Fuel consumption ε Penalty factor pij (n) One-step transition probability Nij Transition number from i to j Ni Total transition number initiated from i x (n + 1)p One-step velocity prediction value VOLUME 7, 2019 tp Prediction horizon pt ij(n) Multi-step transition probability x(n + t)p Multi-step velocity prediction value Error(k) Root mean square error of prediction horizon at k moment Q(s, a) Action-value function in terms of s, a λ Learning rate of RL γ Discount factor r Instant reward of the selected action a π∗(s) Optimal policy α Adjustment factor I. INTRODUCTION Nowadays, electric vehicles (EVs), hybrid EVs (HEVs), and fuel cell HEVs (FCHEVs) have been widely considered to be the most promising solutions to environmental pollution and energy crisis [1]. Although EVs have gained tremen- dous momentum in addressing issues faced by automotive industry worldwide on energy and environment, they still face signiﬁcant challenges including driving distance anxi- ety, high initial cost, energy storage reliability, and degra- dation [2]. HEVs are becoming a promising solution to the environmental issues by improving fuel economy through rational distribution of power demand between multi-power sources, especially for the long-distance vehicle, construction vehicle and military vehicle. Existing energy management strategies can be divided into two main categories: rule-based energy management strategies [3], [4] and optimization- based energy management. So far, rule-based energy strategy is one of the most popular control strategies in the auto- motive industry, for its reliability and simplicity in develop- ment [4], [5]. Generally, it is implemented by an ‘‘if-else’’ structure, whose initial threshold values are designed by the expert experience. However, tuning these values requires a lot of repeated simulations and experiments on the vehicle. To address this issue, a state-of-the-art approach is proposed to extract the rules from optimization-based algorithms [4], but the obtained rules have a great dependence on the original driving cycles, and the cycle adaptability is relatively poor. Furthermore, comparative researches indicate that the rule- based energy management strategies generally do not per- form as well as those optimization-based approaches [2], [6], so that researches on optimization-based strategies are popu- lar in recent years. The main objective of the optimization-based EMS (EMS) can be described as an optimization of a cost function includ- ing the fuel consumption, state of charge (SOC) variation of the battery, and emissions. Various optimization algorithms are applied to exploit the global optimal control strategy, including dynamic programming (DP) [7]–[9], Pontryagin’s minimum principle [10], [11], Particle swarm optimization (PSO) [12], [13] and so on. Xie et al. used DP to develop a global optimization strategy for a plug-in serial-parallel hybrid electric bus [7]. Reference [8] employed DP to develop 98253 98253 J. Wu et al.: Online Correction Predictive EMS for a Hybrid Electric Tracked Vehicle model and applied a back propagation (BP) neural network to compensate the prediction error. Then MPC is adopted to obtain a prediction EMS with DP as an optimal solver. I. INTRODUCTION [15] proposed adaptive-ECMS (A-ECMS) method with an estimation of equivalence factor λ∗according to the driving cycle. Yang et al. [16] proposed an A-ECMS based EMS for a plug-in hybrid electric bus by adjusting the equivalence factor to different route segment. Zeng et al. [17] applied the PSO algorithm to optimize the equivalent factor and got a map of the factor under several variables to improve the performance of A-ECMS strategy. Rezaei et al. [18] use two soft bounds of battery SOC and the feedback of the SOC to calculate equivalence factor at each moment to build a new A-ECMS. Li et al. [19] proposed an A-ECMS EMS considering the lateral dynamics of the vehicle. A-ECMS methods transform the original global optimization problems to instantaneous ones by minimizing objective function at each step. Although they have great potential in being imple- mented online, the improvement of fuel economy would be limited without due consideration of the future driving cycle. With the development of machine learning and artiﬁcial intelligence, some inspiring innovative methods are also applied to deal with the energy management issues of HEVs, such as reinforcement learning (RL) [32], [33] and game theory (GT). As one branch of machine learning, RL can achieve a model-free and adaptive control for the sequence decision problem. Zou et al. [33] proposed a Markov chain- based power recursive algorithm and applied RL to quan- titatively compare the effects of different forgetting factors and KL divergence rates on reducing fuel consumption for a hybrid electric tracked vehicle. Reference. [34] applied Markov chain-based power transition prediction and RL to the hybrid energy storage system in a plug-in hybrid elec- tric vehicle. Although these researches can yield a close-to- optimal solution by solving the optimal control problem for hundreds of thousands of times, they still face the ‘‘curse of dimensionality’’ due to the discrete of the state variables. On the other hand, the issue that the Q-learning algorithm may overestimate action value in some conditions is not con- sidered. To address this issue, Reference [30] put forward a continuous reinforcement learning EMS by applying the deep Q network to the hybrid electric bus. Unlike the discretization matrix, continuous change in state variables can be reﬂected in a DNN-based system, which is insensitive to the increase of state dimension. I. INTRODUCTION Since BP is used to ﬁt the relationship between actual veloc- ity and prediction error under a speciﬁc cycle, the trained deterministic BP network lack generalization. The Markov Chain Monte-Carlo method is applied to predict the veloc- ity sequences in the short future, and MPC-based energy management has been put forward [24]. Xie et al. [25] also used Markov chain to predict the short period future div- ing cycle and proposed a stochastic MPC EMS based on PMP, and analyzed the inﬂuence of the prediction horizon on computational time and fuel economy. Then they considered the depth of battery discharge to optimize the EMS [26]. Another method called radial basis function neural network was also used for velocity prediction and embedded into MPC in literature [27], [28]. Guo et al. [29] optimized the velocity trajectory with the onboard navigation system and proposed a bi-level MPC-based EMS. Although it is a good idea, the assumption about the trafﬁc information is a little idealistic. To reduce online computation burden, an explicit MPC-based EMS was proposed by Li et al. [30]. Apart from the abovementioned methods, other prediction energy man- agement or combination methods are also used for improving the performance of optimization-based energy management. An intelligent EMS based on hierarchical approximate global optimization for plug-in hybrid electric vehicle was pro- posed [31], with a combination of long-term and short-term average speed prediction. Although these predictive energy management methods have relatively good performance yet are limited by prediction error. the optimal energy allocation for a plug-in hybrid electric vehicle with an engine-generator, battery, and ultra-capacitor. Hou et al. [11] combined a piecewise linear approximation strategy with PMP to establish optimal energy management for a Plug-in HEV (PHEV). Additionally, Chen et al. [13] developed an EMS by using improved PSO (IPSO) and compared the effectiveness of PSO and IPSO. These energy management strategies can realize the global optimization of the cost function with the complete knowledge of the global vehicle-speed trajectory in advance. However, for a practical trip path, it is difﬁcult to gain the whole future driving cycle in advance, so these strategies are usually used to extract rules for online implementation or as a benchmark to evaluate other energy management strategies. I. INTRODUCTION In addition to the above global optimal methods, equivalent consumption minimization strategy (ECMS), which is based on PMP and ﬁrst introduced in [14], is also a sound technology for real- time optimal energy management, but the ECMS optimal equivalent factor λ∗can be determined only with the pre- knowledge of demanded power. To overcome this problem, Musardo et al. [15] proposed adaptive-ECMS (A-ECMS) method with an estimation of equivalence factor λ∗according to the driving cycle. Yang et al. [16] proposed an A-ECMS based EMS for a plug-in hybrid electric bus by adjusting the equivalence factor to different route segment. Zeng et al. [17] applied the PSO algorithm to optimize the equivalent factor and got a map of the factor under several variables to improve the performance of A-ECMS strategy. Rezaei et al. [18] use two soft bounds of battery SOC and the feedback of the SOC to calculate equivalence factor at each moment to build a new A-ECMS. Li et al. [19] proposed an A-ECMS EMS considering the lateral dynamics of the vehicle. A-ECMS methods transform the original global optimization problems to instantaneous ones by minimizing objective function at each step. Although they have great potential in being imple- mented online, the improvement of fuel economy would be limited without due consideration of the future driving cycle. the optimal energy allocation for a plug-in hybrid electric vehicle with an engine-generator, battery, and ultra-capacitor. Hou et al. [11] combined a piecewise linear approximation strategy with PMP to establish optimal energy management for a Plug-in HEV (PHEV). Additionally, Chen et al. [13] developed an EMS by using improved PSO (IPSO) and compared the effectiveness of PSO and IPSO. These energy management strategies can realize the global optimization of the cost function with the complete knowledge of the global vehicle-speed trajectory in advance. However, for a practical trip path, it is difﬁcult to gain the whole future driving cycle in advance, so these strategies are usually used to extract rules for online implementation or as a benchmark to evaluate other energy management strategies. In addition to the above global optimal methods, equivalent consumption minimization strategy (ECMS), which is based on PMP and ﬁrst introduced in [14], is also a sound technology for real- time optimal energy management, but the ECMS optimal equivalent factor λ∗can be determined only with the pre- knowledge of demanded power. To overcome this problem, Musardo et al. I. INTRODUCTION g y In order to improve the online implementation of optimization-based method, prediction energy management (PEM) strategy is proposed to get a sub-optimal fuel economy based on velocity prediction. The velocity prediction based on Markov chain is one of the most common methods for PEM. Liu et al. [20], proposed a look-ahead EMS based on a ﬁnite-state Markov chain velocity prediction and rein- forcement learning to reduce fuel consumption and compu- tational time. They then rephrased the prediction algorithm and proposed an online Markov Chain-based energy man- agement [21], but the inﬂuence of the prediction error was not discussed. Liu et al. [22] established a trip condition prediction model by using BP neural network to obtain a real- time vehicle-speed trajectory and applied both the genetic algorithm (GA) and PSO to improve the prediction accu- racy of the trip condition prediction model. Based on the velocity prediction methods model predict control (MPC) is a commonly used framework to explore EMS. Reference [23] formulated a velocity prediction method based on Markov 98254 VOLUME 7, 2019 VOLUME 7, 2019 J. Wu et al.: Online Correction Predictive EMS for a Hybrid Electric Tracked Vehicle FIGURE 2 Th h t f th HETV From the above analysis, it can be concluded that predictive EMS can realize online implementation for hybrid electric vehicles with better driving cycles adaptability. However, these strategies cannot guarantee the robustness especially when the actual driving cycles are signiﬁcantly different from the training driving cycles, which means the performance of the predictive EMS highly depends on the prediction accu- racy of the future driving cycle. Although many authors focus on improving the prediction accuracy of the driving cycles, few people study how to deal with the inaccurate prediction case. This paper proposes a novel online correction predic- tive energy management (OCPEM) strategy to solve this issue, which integrates the reinforcement learning algorithm to achieve online correction based on a fuzzy logic controller for a hybrid electric tracked vehicle (HETV). First, a multi- time-scale prediction algorithm based on Markov Chain is proposed to realize the prediction of the future driving cycles with better prediction accuracy. Then the DP algorithm is applied to obtain the local control policy based on the future short-period predicted driving cycle. II. POWERTRAIN MODELING OF HYBRID ELECTRIC TRACKED VEHICLE A. STRUCTURE OF THE HETV II. POWERTRAIN MODELING OF HYBRID ELECTRIC TRACKED VEHICLE A. STRUCTURE OF THE HETV The power train of the HETV is illustrated in Fig. 1, and the photo of the HETV is shown in Fig. 2. The tracked vehicle is driven by two traction motors with 50 kW rated power on each side. Engine-generator set (EGS) and battery pack are the two power sources of the vehicle. The 3-cylinder gasoline engine can provide 93 Nm maximum torque at 2500∼3500rpm, The power train of the HETV is illustrated in Fig. 1, and the photo of the HETV is shown in Fig. 2. The tracked vehicle is driven by two traction motors with 50 kW rated power on each side. Engine-generator set (EGS) and battery pack are the two power sources of the vehicle. The 3-cylinder gasoline engine can provide 93 Nm maximum torque at 2500∼3500rpm, I. INTRODUCTION Finally, our previous study about reinforcement learning [33], regarded as an online correction algorithm, are dynamically integrated to the ﬁnal control policy by a fuzzy logic controller according to the real-time calculated prediction accuracy. This character- istic of the OCPEM makes it has a better driving cycle adapt- ability compared with the original RL or traditional PEM. FIGURE 2. The photo of the HETV. FIGURE 2. The photo of the HETV. TABLE 1. Essential parameters of the vehicle. TABLE 1. Essential parameters of the vehicle. TABLE 1. Essential parameters of the vehicle. The remainder of this paper is organized as follows: the mathematical model of the series HETV is described in Section II. The multi-time-scale prediction method for realiz- ing the prediction of the future driving cycle and the OCPEM are proposed in Section III. Section IV shows the simulation and control performance validation results. Section V con- cludes the paper. FIGURE 1. The driving system structure of the HETV. 52kW maximum power at 6200rpm. The generator’s rated power is 40 kW at 3500 rpm. The battery pack is composed of LiFePO4 batteries, with 307 V rated voltage and 37.6 Ah capacity, which can store 11.5kWh electric power. The EGS, battery pack and two driving motors are all electrically con- nected to the integrated power electric module, which inte- grates power distribution unit, AC/DC, and DC/AC. In the integrated power electronic module, the EGS feeds the main electric bus through the AC/DC converter, and the battery directly feeds the electric bus. The bus voltage provides power to the two traction motors to drive the HETV. Table 1 shows the parameters of the tracked vehicle. 52kW maximum power at 6200rpm. The generator’s rated power is 40 kW at 3500 rpm. The battery pack is composed of LiFePO4 batteries, with 307 V rated voltage and 37.6 Ah capacity, which can store 11.5kWh electric power. The EGS, battery pack and two driving motors are all electrically con- nected to the integrated power electric module, which inte- grates power distribution unit, AC/DC, and DC/AC. In the integrated power electronic module, the EGS feeds the main electric bus through the AC/DC converter, and the battery directly feeds the electric bus. The bus voltage provides power to the two traction motors to drive the HETV. Table 1 shows the parameters of the tracked vehicle. FIGURE 1. The driving system structure of the HETV. B. MODELING OF THE HETV In order to solve the problem of energy management of the HETV, it is necessary to build models for components of the VOLUME 7, 2019 VOLUME 7, 2019 J. Wu et al.: Online Correction Predictive EMS for a Hybrid Electric Tracked Vehicle FIGURE 4. The simplified hybrid powertrain model. powertrain. The models are detailed as follows, and they are veriﬁed against experimental data as attached in the appendix. powertrain. The models are detailed as follows, and they are veriﬁed against experimental data as attached in the appendix. 1) MODELING OF THE POWER DEMAND The power demand of the vehicle consists of heading power and steering power, shown in the following equations: The power demand of the vehicle consists of heading power and steering power, shown in the following equations: FIGURE 4. The simplified hybrid powertrain model.    Pdem = (Fa + Fr + Fw)vave + Mω vave = (v1 + v2)/2 ω = (v1 −v2)/B (1) (1) where Tg means the electromagnetic torque of the generator, Keωg is the electromotive force and Ke is the coefﬁcient of the force. Kx means the equivalent impedance coefﬁcient, and Kx = 3PLg/π, Lg is the synchronous inductance of the armature, P is the pole-pairs number. where Fa means the accelerating resistance, Fr is the rolling resistance, Fw denotes the aerodynamic resistance, vave means the average speed of two tracks, v1 and v2 denote the speed of two tracks respectively, M means resistance yaw moment, ω is yaw angular velocity of the vehicle, B means the track gauge. For the series hybrid system, the engine and generator are directly connected, so the rotating speed of engine and gen- erator is the same. According to the torque balance, the fol- lowing relationship should be satisﬁed: The resistances of the vehicle are calculated by:    Fr = mgf Fw = CDAv2 ave/21.15 Fa = ma (2)    neng = ng Teng −Tg = π 30(Je + Jg)dng dt (6) (2) (6) where m means the vehicle mass. g means the acceleration of the gravity, and g = 9.8N/kg, f is the coefﬁcient of rolling resistance, CD is the coefﬁcient of aerodynamic resistance, A means the fronted area of the vehicle, a is the longitudinal acceleration. where neng and ng represent the speed of engine and generator respectively, and it is chosen as one state variable in the optimization algorithm, Teng is engine output torque, Je and Jg are the moment of inertia of the engine and generator. The resistance yaw moment M is computed by: 4) MODELING OF ELECTRIC POWER COUPLING FIGURE 3. The equivalent electric circuit of the EGS. The electric power coupling of a hybrid electric vehicle refers to the power balance relationship between the front and rear power chains. The front power chain means the power pro- vided by the EGS and the power battery pack, while the rear power chain refers to the power consumed by the two driving motors, i.e. the power demand Pdem. The power demand may be supplied by the two power sources of the HETV. Since the EGS, battery pack and the driving motor controllers are all electrically connected to the main electric bus, the simpliﬁed electric powertrain model is expressed in Fig. 4. 3) MODELING OF THE BATTERY M = utmgL/4 (3) (3) The internal resistance model of the battery pack is shown as follow [13]: The internal resistance model of the battery pack is shown as follow [13]: where L means the track contacting length, ut is the lat- eral resistance coefﬁcient and calculated by the empirical formula:    dSOC(t) dt = −Ib/Qb Ib = (VOC − q V 2 OC −4RintPb) /(2Rint) Ubat = Voc −IbRint (7) (7) ut = umax/(0.925 + 0.15R/B) (4) (4) where umax means the maximum value of the lateral resis- tance coefﬁcient,R denotes the turning radius of the vehicle. where umax means the maximum value of the lateral resis- tance coefﬁcient,R denotes the turning radius of the vehicle. where Qb is the total battery capacity. Ib means the battery current, VOC means the open-circuit voltage, and Ubat means the output voltage of the battery, Rint means the internal resistance of the battery. Besides, the battery current and SOC should satisfy the following constraints: where Qb is the total battery capacity. Ib means the battery current, VOC means the open-circuit voltage, and Ubat means the output voltage of the battery, Rint means the internal resistance of the battery. Besides, the battery current and SOC should satisfy the following constraints: FIGURE 3. The equivalent electric circuit of the EGS. ( Ib,min ≤Ib(t) ≤Ib,max SOCmin ≤SOC(t) ≤SOCmax (8) (8) A. DRIVING CYCLE PREDICTION METHOD Using the singe-time-scale prediction method, the driving cycle prediction process is conducted for the driving cycle shown in Fig. 5. The prediction results are illustrated in Fig. 6 with the prediction horizon tp = 5s, 10s, 15s, and 20s. The prediction result of the next moment is determined by the prediction value at the last moment, so there will be error accumulation in the singe-time-scale prediction method. 1) SINGLE-TIME-SCALE PREDICTION METHOD The velocity of the vehicle is modeled as a Markov chain. The transition probability of the velocity is calculated based on the maximum likelihood estimator and the nearest neighbor approach, and the equation is as follows:    pij(n) = P(xn+1 = j\|xn = i) = Nij/Ni Ni = m X j=1 Nij (12) (12) 5) ENERGY MANAGEMENT MODEL FIGURE 6. Driving cycle prediction of the singe-time-scale prediction method. The cost function J is a combination of fuel consumption and battery SOC deviation, which can be expressed as follow: FIGURE 6 Driving cycle prediction of the singe time scale prediction J = T Z 0 [ · fuel(t) + ε(SOC(t) −SOCref )2] (10) (10) where · fuel is the fuel consumption rate. ε is a penalty factor, which is used to satisfy the charge-sustainability constraint. Optimization target is to minimize cost function J. The dynamic system also has to meet the following constraints because of physical limitations.    neng,min ≤neng(t) ≤neng,max 0 ≤Teng(t) ≤Teng,max Pb,min ≤Pb(t) ≤Pb,max Ig,min ≤Ig ≤Ig,max (11) (11) FIGURE 6. Driving cycle prediction of the singe-time-scale prediction method. 2) MODELING OF THE EGS The equivalent electric circuit of the engine-generator set (EGS) is illustrated in Fig. 3 [21]. The rectiﬁed output voltage and current of the generator are Ug and Ig respectively, which are calculated by: ( Ug = Keωg −KxωgIg Tg = KeIg −KxI2 g (5) (5) 98256 VOLUME 7, 2019 VOLUME 7, 2019 J. Wu et al.: Online Correction Predictive EMS for a Hybrid Electric Tracked Vehicle FIGURE 5. Driving schedule and power demand for the HETV. FIGURE 6. Driving cycle prediction of the singe-time-scale prediction method. FIGURE 5. Driving schedule and power demand for the HETV. FIGURE 5. Driving schedule and power demand for the HETV. According to the circuit principle, the power balance rela- tionship is shown as follow:    Pg = UdcIg Pb = UdcIb Udc = Voc −IbRint Udc = Keωg −KxωgIg PL = Pdem = Pg + Pb (9) (9) where Udc means the bus voltage. PL means the load power of the vehicle. Pb means the power of the battery pack. Pg is the power of the generator. FIGURE 5. Driving schedule and power demand for the HETV. 2) MULTI-TIME-SCALE PREDICTION METHOD To avoid the inﬂuence of the accumulative error on predic- tion accuracy, a multi-time-scale prediction method based on the multi-step transition probability matrix is developed as follows: where pij (n) means the one-step transition probability as the velocity transfers from i at time n to j at next time n + 1, Nij is transition number from i to j, Ni denotes total transition number initiated from i.    pt ij(n) = P(xn+t = j\|xn = i) = Nij/Ni Ni = m X j=1 Nij (14) Based on the one-step transition probability matrix, the future driving cycle is predicted as follows: (14) x(n + 1)p = M X j=1 pij(n) · xi (13) (13) where pt ij (n) means the transition probability as the velocity transfers from i at time n to j at time n + t. Fig.7 shows the transition probability matrix for different time scale. where x (n + 1)p is the velocity prediction value. Based on the prediction value, the singe-time-scale prediction is achieved by repeating the above process. The velocity predictions in the prediction horizon are acquired according to the corresponding transition VOLUME 7, 2019 98257 J. Wu et al.: Online Correction Predictive EMS for a Hybrid Electric Tracked Vehicle FIGURE 7. Transition probability matrix for different time scale. probability matrix as follows: x(n + t)p = M X j=1 pt ij(n) · xi (t = 1, 2 · · · , tp) (15) Fig.8 shows the processes of both the single-time-scale and multi-time-scale prediction with 5s prediction. In the process of multi-time-scale prediction, prediction at each step with different time scale is based on the corresponding time scale transition probability matrix and the true value of the current moment, so there are no accumulate errors caused by iteration, which is different to the single-time-scale predic- i Th f h i ll h l i i l di i FIGURE 9. Driving cycle prediction of the multi-time-scale prediction method. FIGURE 9. Driving cycle prediction of the multi-time-scale prediction method. FIGURE 10. The RMSE of driving cycle prediction. FIGURE 7. Transition probability matrix for different time scale. FIGURE 9. Driving cycle prediction of the multi-time-scale prediction h d FIGURE 9. Driving cycle prediction of the multi-time-scale prediction method. FIGURE 7. Transition probability matrix for different time scale. FIGURE 7. Transition probability matrix for different time scale. FIGURE 7. 1) DYNAMIC PROGRAMMING ALGORITHM Based on the future short-period driving cycle obtained by the multi-time-scale prediction method, the local control policy is achieved. The whole driving cycle is divided into several stages at regular intervals. For each stage, the driving cycle in the future interval is predicted, and DP is applied to solve the optimal control policy for the future stage. The local optimal process is shown in Fig. 12. TABLE 2. RMse of Two new driving cycles. TABLE 2. RMse of Two new driving cycles. In the reverse solution, the cost function is calculated by: N-1 stage [37]: N-1 stage [37]: J∗(x(N −1)) = min u(N−1)[L(x(N −1), u(N −1))] (17) k stage (0≤k ≤N-2):    J∗(x(k)) = min u(N−1)[L(x(k), u(k)) + J∗(x(k + 1))] x(k + 1) = f (x(k), u(k)) (18) J∗(x(N −1)) = min u(N−1)[L(x(N −1), u(N −1))] (17) (17) B. ENERGY MANAGEMENT STRATEGY The proposed OCPEM strategy developed in this paper con- sists of two parts. In the ﬁrst part, the MPC framework is applied to develop a PEM strategy on the velocity predic- tion horizon with the DP algorithm as optimization solver, assuming that all of the predictions are precise. Although the prediction accuracy is improved by using multi-time-scale prediction method, the prediction error still exists, which will reduce the effect of the optimization. Therefore, in the sec- ond part, a reinforcement learning framework working as an online correction algorithm is adopted to reduce the inﬂuence of prediction error on control performance according to the fuzzy logic controller. These two parts are discussed in detail in the following sections. k stage (0≤k ≤N-2): (18) where J∗(x(k)) is the optimal cost function when the sys- tem applies optimal control, transferring x(k) to x(N). L(x(k), u(k)) is the instantaneous cost at k stage. In the energy management problem, there are a set of state variables st ∈S = {0.4 < SOC(t) < 0.9, 1200 < neng (t) < 6000} and a set of actions at ∈A = {0 < thr(t) < 1}. thr is the throttle of the engine. The discretization of the variables and the computation burden on different prediction horizon are list in Table 3. The time step of all the computation is 0.1s. In order to implement in real time, both the state and control variables are discretized into 50 grids. And the prediction horizon is set to 5s for better prediction accuracy. 2) MULTI-TIME-SCALE PREDICTION METHOD Transition probability matrix for different time scale. FIGURE 9. Driving cycle prediction of the multi-time-scale prediction method. probability matrix as follows: probability matrix as follows: FIGURE 10. The RMSE of driving cycle prediction. FIGURE 10. The RMSE of driving cycle prediction. x(n + t)p = M X j=1 pt ij(n) · xi (t = 1, 2 · · · , tp) (15) (15) Fig.8 shows the processes of both the single-time-scale and multi-time-scale prediction with 5s prediction. In the process of multi-time-scale prediction, prediction at each step with different time scale is based on the corresponding time scale transition probability matrix and the true value of the current moment, so there are no accumulate errors caused by iteration, which is different to the single-time-scale predic- tion. Therefore, theoretically, the multi-time-scale prediction method has better prediction accuracy. FIGURE 10. The RMSE of driving cycle prediction. FIGURE 8. Schematic of the prediction processes with 5s prediction. to evaluate the accuracy of the two driving cycle prediction methods, which can be calculated as follows [36]:    error = nP k=1 error(k)/n error(k) = s tpP i=1 (vp(k + i) −v(k + i))2/tp (16) (16) The RMSEs of the two methods are shown in Fig. 10. The results indicate that the prediction accuracy of both single- time-scale and multi-time-scale prediction decrease with the increasing of the prediction horizon. The multi-time-scale prediction method has higher prediction accuracy compared with the singe-time-scale prediction method at different pre- diction horizon. The RMSE of the multi-time-scale method with the prediction horizon of 5s is 1.545km/h. FIGURE 8. Schematic of the prediction processes with 5s prediction. To further verify the prediction performance, the two meth- ods are applied to another two diving cycles collected in ﬁeld test. The prediction results at 10s prediction horizon are plotted in Fig. 11, and the similar result at other prediction horizon are no longer presented. The RMSE of the two The multi-time-scale prediction method is applied to the driving cycle shown in Fig. 5, and the results are shown in Fig. 9. The root mean square error (RMSE) is investigated 98258 The multi-time-scale prediction method is applied to the driving cycle shown in Fig. 5, and the results are shown in Fig. 9. The root mean square error (RMSE) is investigated VOLUME 7, 2019 VOLUME 7, 2019 98258 98258 J. 2) MULTI-TIME-SCALE PREDICTION METHOD Wu et al.: Online Correction Predictive EMS for a Hybrid Electric Tracked Vehicle methods is listed in Table 2, which indicate that the multi- prediction methods have better performance than the single- time-scale method regardless of the driving cycles. FIGURE 12. The process of DP-based predictive energy management. FIGURE 12. The process of DP-based predictive energy management. FIGURE 11. Prediction of the two methods on different driving cycles. FIGURE 12. The process of DP-based predictive energy management. TABLE 3. Computation consuming of DP. TABLE 3. Computation consuming of DP. FIGURE 11. Prediction of the two methods on different driving cycles. By using the DP algorithm, the optimal control sequence is achieved after the reverse solution to obtain the optimal cost function and the optimal control sequence. Then according to the initial state of the system, the positive solution is carried out. The optimal control policy is imposed on the state to obtain the optimal trajectory. TABLE 2. RMse of Two new driving cycles. 2) ONLINE CORRECTION PREDICTIVE ENERGY MANAGEMENT In the Q-learning algorithm, the action-value function Q, which is the function of the pair (s, a), is obtained by recursive form as follows [32]: Q(s, a) := Q(s, a) + λ r + γ min a′ Q s′, a′ −Q(s, a) (19) FIGURE 14. The membership functions of inputs and output. FIGURE 13. Flowchart of OCPEM. FIGURE 14. The membership functions of inputs and output. FIGURE 14. The membership functions of inputs and output. FIGURE 13. Flowchart of OCPEM. FIGURE 13. Flowchart of OCPEM. FIGURE 13. Flowchart of OCPEM. FIGURE 14. The membership functions of inputs and output. FIGURE 14. The membership functions of inputs and output. TABLE 4. Fuzzy control rules. FIGURE 15. The value of the adjustment factor α. TABLE 4. Fuzzy control rules. to maximize the accumulated reward. For an energy man- agement problem, RL can map the powertrain situations to actions with a brilliant optimal performance by training the RL agent in various driving cycles. In this paper, Q-learning, which is a famous RL algorithm, is applied to train a RL EMS. Then the obtained RL EMS, which is a control map from state and action, is used as an online correction to the PEM through a fuzzy logic controller, aiming to decrease the sensitivity of the EMS to the prediction accuracy. The operational process of the OCPEM, as shown in Fig. 13, is based on the reinforcement learning algorithm and fuzzy logic controller. The process of the OCPEM is described in detail as follows: Step 1: DP is applied to obtain an optimal control policy U ′(t) based on the future predicted driving cycle. For the OCPEM, only the ﬁrst element of U ′(t) is applied as one part of the ﬁnal control policy. FIGURE 15. The value of the adjustment factor α. p y Step 2: Given that the prediction accuracy of adjacent historical prediction is similar. we use the prediction accuracy of the previous stage to reﬂect the current one. Then a fuzzy logic controller [38] is adopted to evaluate the prediction accuracy of the previous stage, outputting an adjustment factor, α ∈[0, 1]. The adjustment factor is used to adjust the conﬁdence level of the predictive EMS algorithm. The smaller the adjustment factor is, the higher the conﬁdence level of the predictive energy management is, and the greater the proportion of the result in the ﬁnal control is. 2) ONLINE CORRECTION PREDICTIVE ENERGY MANAGEMENT RL algorithm can make optimal decisions through trial-and- error based on observations and analysis of system behavior VOLUME 7, 2019 J. Wu et al.: Online Correction Predictive EMS for a Hybrid Electric Tracked Vehicle FIGURE 13. Flowchart of OCPEM. to maximize the accumulated reward. For an energy man- agement problem, RL can map the powertrain situations to actions with a brilliant optimal performance by training the RL agent in various driving cycles. In this paper, Q-learning, which is a famous RL algorithm, is applied to train a RL EMS. Then the obtained RL EMS, which is a control map from state and action, is used as an online correction to the PEM through a fuzzy logic controller, aiming to decrease the sensitivity of the EMS to the prediction accuracy. The operational process of the OCPEM, as shown in Fig. 13, is based on the reinforcement learning algorithm and fuzzy logic controller. The process of the OCPEM is described in detail as follows: Step 1: DP is applied to obtain an optimal control policy U ′(t) based on the future predicted driving cycle. For the OCPEM, only the ﬁrst element of U ′(t) is applied as one part of the ﬁnal control policy. Step 2: Given that the prediction accuracy of adjacent historical prediction is similar. we use the prediction accuracy of the previous stage to reﬂect the current one. Then a fuzzy logic controller [38] is adopted to evaluate the prediction accuracy of the previous stage, outputting an adjustment factor, α ∈[0, 1]. The adjustment factor is used to adjust the conﬁdence level of the predictive EMS algorithm. The smaller the adjustment factor is, the higher the conﬁdence level of the predictive energy management is, and the greater the proportion of the result in the ﬁnal control is. Since the actual driving cycle in the current interval is recorded, the root mean square error (RMSE) and error standard deviation (SD) are used as inputs for the fuzzy logic controller The fuzzy FIGURE 14. The membership functions of inputs and output. TABLE 4. Fuzzy control rules. FIGURE 15. The value of the adjustment factor α. Step 3: The Q-learning, which is a famous RL algorithm, is applied as an online correction algorithm for the PEM if the future driving cycle prediction is inaccurate. 2) ONLINE CORRECTION PREDICTIVE ENERGY MANAGEMENT Since the actual driving cycle in the current interval is recorded, the root mean square error (RMSE) and error standard deviation (SD) are used as inputs for the fuzzy logic controller. The fuzzy logic controller can math the RSME, SD, and α to small, medium and big. The membership functions of the inputs and outputs are plotted and approximated using the trimf and trapmf distributions respectively as shown in Fig. 14. And the parameters of the membership functions are optimized to map the RMSE and SD to proper α. The fuzzy control rules are listed in Table 4. Then, we get the adjustment factor map as shown in Fig. 15. Step 3: The Q-learning, which is a famous RL algorithm, is applied as an online correction algorithm for the PEM if the future driving cycle prediction is inaccurate. In the Q-learning algorithm, the action-value function Q, which is the function of the pair (s, a), is obtained by recursive form as follows [32]: Q(s, a) := Q(s, a) + λ r + γ min a′ Q s′, a′ −Q(s, a) (19) (19) where Q(s, a) is the accumulative reward at state s, action a taken at time t. γ is the discount factor with a value between 0 and 1. λ means the learning rate, and λ = 1/(k + 1). r is the reward obtained by selecting action a, and can be expressed as follow: r = − fuel(t) + ε(SOC(t) −SOCref )2 (20) (20) where fuel(t) is the gasoline cost at step t. 98260 98260 VOLUME 7, 2019 VOLUME 7, 2019 J. Wu et al.: Online Correction Predictive EMS for a Hybrid Electric Tracked Vehicle FIGURE 17. The trajectories of the battery SOC and engine throttle. (a) The battery SOC corresponding to the four algorithms. (b) Engine throttle of the four algorithms. The optimal policy is obtained as follow: π∗(s) = arg min a Q∗(s, a) (21) (21) After training convergence, we get the control map of RL EMS. Based on the system real-time feedback state, the real- time control U∗(t) of the correction algorithm is obtained by looking up the control map, and a part of the control maps is shown as Fig. 16. FIGURE 16. Part of the control map calculated by RL. FIGURE 17. The trajectories of the battery SOC and engine throttle. 2) ONLINE CORRECTION PREDICTIVE ENERGY MANAGEMENT (a) The battery SOC corresponding to the four algorithms. (b) Engine throttle of the four algorithms. which is not conducive to the battery life. Besides, the battery is not fully utilized to improve fuel economy. However, the proposed OCPEM can reduce the ﬂuctuation in battery SOC. Speciﬁcally, during 500s-700s, the power demand of the vehicle is large, so the battery and engine jointly provide electricity for the motor, and SOC decreases to about 0.73. During 700s-900s, the power demand of vehicle is small, so the engine individually increases the throttle, as shown in Fig.17 (b), to provides the electric power to drive the vehicle and recharges the battery at the same time to ensure the engine working in the region of the fuel economy. The battery SOC eventually approaches to the initial value. FIGURE 16. Part of the control map calculated by RL. Step 4: The OCPEM is a combination of the predictive EMS and reinforcement learning strategy with an adjustment factor α, which can be expressed as follow: U (t) = αU∗(t) + (1 −α) U′ (t) (22) (22) FIGURE 18. The trajectories of the adjustment factor α, RSME, and SD. where U (t) is the OCPEM control policy. The adjustment factor α is used to determine the proportion between pre- dictive energy management and online correction algorithm. It has to be pointed out that the OPCEM policy can be implemented on real-time because both of the two parts of the policy can obviously meet the real-time requirements. TABLE 6. Fuel consumption of the four algorithms. FIGURE 20. The engine working points of the four algorithms. trajectory, the adjustment factor is set to be 1.0, which means the control policy only contains the RL part in this period. During about 200s-300s, the adjustment factor is larger than other periods, which means the proportion of the PEM is smaller during this period. It is caused by the large ﬂuctuation of the driving cycle, as shown in Fig. 5, which inﬂuence the prediction accuracy of this period. IV. SIMULATION AND VALIDATION A. CONTROL PERFORMANCE VALIDATION To validate the control performance of the proposed method, PEM without online correction method, RL algorithm and DP are introduced to make a comparison with OCPEM. The driving cycle for simulation is shown in Fig. 5. The initial value of the engine speed neng is set as 1200 r/min, and the initial value of the battery SOC is set as 0.75. Fig.17 shows the trajectories of the battery SOC and the engine throttle obtained from the four methods. The results show that in PEM without online correction algorithm, the battery SOC has little change with ﬂuctuating around the initial value because SOC variation is constrained in each prediction horizon to solve the optimal control. The ﬂuctuation will lead to frequent battery charge and discharge, FIGURE 18. The trajectories of the adjustment factor α, RSME, and SD. The adjustment factor trajectory is shown in Fig.18. It takes several seconds to record the actual driving cycle and calcu- late the RMSE and SD. Therefore, at the beginning of the VOLUME 7, 2019 J. Wu et al.: Online Correction Predictive EMS for a Hybrid Electric Tracked Vehicle FIGURE 19. The power distribution of the four algorithms. FIGURE 20. The engine working points of the four algorithms. TABLE 5. Fuel consumption of the four algorithms. FIGURE 21. Driving cycle and power demand for verifying OCPEM. FIGURE 22. The trajectories of the battery SOC. FIGURE 19. The power distribution of the four algorithms. FIGURE 20. The engine working points of the four algorithms. TABLE 5. Fuel consumption of the four algorithms. FIGURE 21. Driving cycle and power demand for verifying OCPEM. FIGURE 22. The trajectories of the battery SOC. TABLE 6. Fuel consumption of the four algorithms. TABLE 5. Fuel consumption of the four algorithms. FIGURE 21. Driving cycle and power demand for verifying OCPEM. TABLE 5. Fuel consumption of the four algorithms. FIGURE 21. Driving cycle and power demand for verifying OCPEM. FIGURE 19. The power distribution of the four algorithms. TABLE 5. Fuel consumption of the four algorithms. TABLE 5. Fuel consumption of the four algorithms. FIGURE 21. Driving cycle and power demand for verifying OCPEM. FIGURE 22. The trajectories of the battery SOC. FIGURE 19. The power distribution of the four algorithms. FIGURE 20. The engine working points of the four algorithms. FIGURE 20. The engine working points of the four algorithms. FIGURE 22. The trajectories of the battery SOC. TABLE 6. IV. SIMULATION AND VALIDATION A. CONTROL PERFORMANCE VALIDATION Fuel consumption of the four algorithms. TABLE 6. Fuel consumption of the four algorithms. B. ADAPTABILITY VALIDATION To validate the effectiveness and adaptability of the OCPEM, we apply another new completely different driving cycle, as shown in Fig. 21, to the OCPEM and make another simulation. Fig. 22 shows that all these four methods have good performance in maintaining the SOC even for the new driving cycle. It should be pointed out that the SOC of OCPEM increase at the beginning of the process where the power demand is low, which means the engine work in high- power area with high fuel economy to charge the battery and drive the HETV simultaneously. DP, with an assumption of knowing the driving cycle in advance, stops the engine during 0-300s and 500-700s as shown in Fig. 23. The engine working points are plotted in Fig. 24, and it can be seen that the OCPEM method have more engine working points The power distributions between the engine and battery corresponding to the four methods are illustrated in Fig. 19. Fig. 20 shows the engine working points in the fuel con- sumption map. Compared with PEM and RL algorithms, the OCPEM has more working points in the lower fuel con- sumption area. It can be seen from Table 5 that the fuel econ- omy of the proposed method is increased by 4% compared with the previous predictive EMS and reaches 90.51% of that of the DP benchmark. The results show that applying the reinforcement learning algorithm to correct the control pol- icy of predictive EMS through fuzzy control can effectively improve the fuel economy of the vehicle. 98262 98262 VOLUME 7, 2019 VOLUME 7, 2019 J. Wu et al.: Online Correction Predictive EMS for a Hybrid Electric Tracked Vehicle FIGURE 23. The power distribution of the four algorithms. FIGURE 24. The engine working points of the four algorithms. G h di ib i f h f l i h FIGURE 23. The power distribution of the four algorithms. Results indicate that the fuel economy of the proposed OCPEM strategy is reduced by 4%, compared with the pre- vious predictive energy management, and achieve 90.51% of that of DP benchmark. In addition, the present method is conducive to the battery life because it can reduce the frequent charge and discharge of the battery, compared with the original predictive energy management. Since solving the optimization problems in the internal prediction horizon, the proposed method can be implemented online. V. CONCLUSION This paper develops an OCPEM strategy for series HETV, which integrate an online correction algorithm to the PEM by a fuzzy logic controller. To reduce the accumulative error of singe-time-scale prediction method, the multi-time-scale prediction method is developed to realize the prediction of the future short-period driving cycle with a better prediction precision. To realize real-time control, DP is adopted to opti- mize the power-split during the prediction horizon. Moreover, through dynamically adjusting the coefﬁcient α by a fuzzy logic controller, the reinforcement learning ofﬂine policy is employed as an online correction algorithm to solve the ﬁnal optimal control policy. APPENDIX In order to verify the accuracy of the model built in this paper, the main components and the entire HETV has been validated against the experimental data. First, we drive the HETV in a real way for a period of time to collect the experiment data. Then, divide the experiment data into input data and output data of different components. Finally, make a computer simulation with the established model based on the input data, and compare the simulation output of the models with the corresponding experiment output to see the accuracy of the models. FIGURE 23. The power distribution of the four algorithms. FIGURE 24. The engine working points of the four algorithms. FIGURE 25. The experimental real driving trajectory. FIGURE 25. The experimental real driving trajectory. FIGURE 24. The engine working points of the four algorithms. A. THE VALIDATION OF EGS MODEL located in the high economy area compared with PEM and RL. Table 6 shows that the fuel economy of the OCPEM increases 4.1% compared with the PEM and achieves 88.58% of that of DP benchmark, which means the OCPEM can adapt well to a new driving cycle. The experimental driving trajectory is shown in Fig. 25, and Fig. 26 shows the corresponding trajectory of the cur- rent, rotational speed, and the voltage of the EGS. Take the rotational speed and output current as the input of computer simulation of the EGS model, the simulation output voltage of the EGS model is achieved. The comparison result of the experiment voltage and simulation voltage is shown is Fig. 27. The root square mean error of the EGS model, which can be used to reﬂect the accuracy of the built model, is 0.074V. B. ADAPTABILITY VALIDATION In future work, the application of the proposed method will be con- ducted on a real hybrid electric tracked vehicle to prove the performance. B. THE VALIDATION OF BATTERY MODEL should have a great performance on tracing the demand power of the vehicle and simulating the power distribution. For the validation of the powertrain model, we conduct the simulation with the collected real driving cycle as shown in Fig. 25. In addition, both EMS and initial state of the simulation are set as the same as the real experiment. The simulation result demonstrates that the powertrain model has a good perfor- mance in tracing the demand power of the HETV, as shown in Fig. 30. The experimental power value is calculated by multiplying the value from the voltage sensor and current sensor with some ﬁltering. The root mean square error of the demand power, battery power, and EGS power are 0.0359 kW, 0.347kW, and 0.0228kW, respectively. FIGURE 28. The experimental data of the battery. error of the battery voltage and SOC are about 0.06V and 0.007, respectively. B. THE VALIDATION OF BATTERY MODEL The collected experiment data of the battery is shown in Fig. 28. Since the battery and the EGS are connected to the bus of the vehicle, the collected battery voltage is the same as the EGS voltage. Take the battery current Ib as the input of simulation based on the battery model. Result Demonstrates that the battery model can reﬂect the battery characteristics, as shown in Fig. 29. And the root square mean VOLUME 7, 2019 98263 J. Wu et al.: Online Correction Predictive EMS for a Hybrid Electric Tracked Vehicle y FIGURE 26. The experimental data of the EGS. FIGURE 27. The comparison of experiment and simulation output voltage of the EGS. FIGURE 28. The experimental data of the battery. error of the battery voltage and SOC are about 0.06V and 0.007, respectively. C THE VALIDATION OF POWERTRAIN MODEL FIGURE 29. The Comparison of experiment and simulation result of the battery model. FIGURE 30. The comparison of experiment and simulation result of the entire HETV model. should have a great performance on tracing the demand power of the vehicle and simulating the power distribution. For the validation of the powertrain model, we conduct the simulation with the collected real driving cycle as shown in Fig. 25. In addition, both EMS and initial state of the simulation are set as the same as the real experiment. The simulation result demonstrates that the powertrain model has a good perfor- mance in tracing the demand power of the HETV, as shown in Fig. 30. The experimental power value is calculated by multiplying the value from the voltage sensor and current sensor with some ﬁltering. The root mean square error of the demand power, battery power, and EGS power are 0.0359 kW, 0.347kW, and 0.0228kW, respectively. ACKNOWLEDGMENT FIGURE 29. The Comparison of experiment and simulation result of the battery model FIGURE 29. The Comparison of experiment and simulation result of the battery model. FIGURE 26. The experimental data of the EGS. FIGURE 27. The comparison of experiment and simulation output voltage of the EGS. FIGURE 27. The comparison of experiment and simulation output voltage of the EGS. FIGURE 28. The experimental data of the battery. FIGURE 28. The experimental data of the battery. 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He is currently pursuing the Ph.D. degree with the Beijing Collab- orative and Innovative Center for Electric Vehicle, School of Mechanical Engineering, Beijing insti- tute of Technology. His research interests include the vehicle dynamic control and energy manage- ment of hybrid propulsions. JINLONG WU received the B.S. and M.S. degrees from Yanshan University, Hebei, China, in 2012 and 2015, respectively. He is currently pursuing the Ph.D. degree with the Beijing Collab- orative and Innovative Center for Electric Vehicle, School of Mechanical Engineering, Beijing insti- tute of Technology. His research interests include the vehicle dynamic control and energy manage- ment of hybrid propulsions. [19] L. Li, S. Coskun, F. Zhang, R. Langari, and J. Xi, ‘‘Energy management of hybrid electric vehicle using vehicle lateral dynamic in velocity predic- tion,’’ IEEE Trans. Veh. Technol., vol. 68, no. 4, pp. 3279–3293, Apr. 2019. [20] T. Liu, X. Hu, S. E. Li, and D. 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YUAN ZOU received the Ph.D. degree from the Beijing Institute of Technology, in 2005, where he is currently a Professor with the Beijing Collab- orative and Innovative Center for Electric Vehi- cles, School of Mechanical Engineering. He also serves as the Co-Director of the ETHZ-BIT Joint Research Center for New Energy Vehicle Dynamic System and Control. He conducted research about ground vehicle propulsion modeling and optimal control at the University of Michigan Ann Arbor and ETH Zürich. XUDONG ZHANG, p XUDONG ZHANG, photograph and biography not available at the time of publication. REFERENCES His research interest includes modeling and control for electriﬁed vehicle and transportation systems. XUDONG ZHANG, photograph and biography not available at the time of publication. TENG LIU (M’18) received the B.S. degree in mathematics and the Ph.D. degree in automotive engineering from the Beijing Institute of Tech- nology (BIT), Beijing, China, in 2011 and 2017, respectively. His Ph.D. dissertation, under the supervision of Prof. F. Sun, was Reinforcement learning-based energy management for hybrid electric vehicles. He is currently a Postdoc- toral Fellow with the Department of Mechani- cal and Mechatronics Engineering, University of Waterloo, ON, Canada. He has more than 8 years’ research and working experience in new-energy vehicle and connected autonomous vehicle. His current research interests include parallel driving, parallel reinforcement learning, automated driving, and the energy management of hybrid electric vehicles. He has published more than 30 papers in these areas. DINGBO HE received the B.S. degree in mecha- tronics from Central South University, Changsha, China, in 2016. He is currently pursuing the mas- ter’s degree with the Beijing Collaborative and Innovative Center for Electric Vehicles, School of Mechanical Engineering, Beijing Institute of Tech- nology. His research interest includes the model- ing, control, and optimization of vehicle systems. DINGBO HE received the B.S. degree in mecha- tronics from Central South University, Changsha, China, in 2016. He is currently pursuing the mas- ter’s degree with the Beijing Collaborative and Innovative Center for Electric Vehicles, School of Mechanical Engineering, Beijing Institute of Tech- nology. His research interest includes the model- ing, control, and optimization of vehicle systems. 98266 VOLUME 7, 2019
https://openalex.org/W2091385681	https://europepmc.org/articles/pmc3637097?pdf=render	English	null	Discontinuous microduplications at chromosome 10q24.31 identified in a Chinese family with split hand and foot malformation	BMC medical genetics	2,013	cc-by	4,374	Li Dai1,2,3,5, Ying Deng1,2, Nana Li1,2, Liang Xie2,4,5, Meng Mao5 and Jun Zhu1,2,3,5 Li Dai1,2,3,5, Ying Deng1,2, Nana Li1,2, Liang Xie2,4,5, Meng Mao5 and Jun Zhu1,2,3,5 Li Dai1,2,3,5, Ying Deng1,2, Nana Li1,2, Liang Xie2,4,5, Meng Mao5 and Jun Zhu1,2,3,5 Li Dai1,2,3,5, Ying Deng1,2, Nana Li1,2, Liang Xie2,4,5, Meng Mao5 and Jun Zhu1,2,3,5 Dai et al. BMC Medical Genetics 2013, 14:45 http://www.biomedcentral.com/1471-2350/14/45 Dai et al. BMC Medical Genetics 2013, 14:45 http://www.biomedcentral.com/1471-2350/14/45 Open Access Abstract Background: Split hand/foot malformation (SHFM) is a congenital disorder characterized by a cleft of the hands and/or feet due to dificiency of central rays. Genomic rearrangement at 10q24 has been found to cause nonsyndromic SHFM (SHFM3). Methods: Four patients and fourteen unaffected individuals from a four-generation Chinese pedigree with typical SHFM3 phenotypes were recruited for this study. After informed consent was obtained, genome-wide copy number analysis was performed on all patients and two normal family members using the Affymetrix Cytogenetics Whole-Genome 2.7M Array. The results were then confirmed by real-time quantitative polymerase chain reaction in all available individuals of this pedigree. Candidate genes were further screened for mutation through sequence analyses. Results: Copy number analysis showed a microduplication at chromosome 10q24.31-q24.32 co-segregating with the SHFM phenotype. Compared to other known genomic duplications for SHFM3, the duplication described here contains two discontinuous DNA fragments. The minimal centromeric duplicated segment of 259 kb involves LBX1, POLL and a disrupted BTRC. The minimal telomeric duplication of 114 kb encompasses DPCD and one part of FBXW4. No coding and splice-site mutations of candidate genes in the region were found. Conclusions: Genomic duplications at chromosome 10q24.3, which were identified in the current study, provide further evidence for limb-specific cis-regulatory sequences in this region, highlighting the importance of chromosome 10q24.31-q24.32 in limb development and SHFM pathogenesis. Keywords: SHFM, Microduplication, Han Chinese © 2013 Dai 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. * Correspondence: daili@scu.edu.cn 1National Center for Birth Defects Monitoring, Chengdu, China 2Lab of Molecular Epidemiology of Birth Defects, Chengdu, China Full list of author information is available at the end of the article Background can occur as an isolated entity or a part of other anoma- lies, with an estimated incidence of 1/8,500 ~1/25,000 [2]. Split hand/foot malformation (SHFM) is a congenital dis- order characterized by variable degrees of median clefts of hands and/or feet due to absence of the central ray digits. It also presents syndactyly, and aplasia/hypoplasia of the phalanges, metacarpals and metatarsals. SHFM shows highly variability in phenotypes between and/or within families, even between limbs of a single patient, ranging from mild syndactyly to severe central clefting of the autopods, oligodactyly or monodactyly [1]. The condition Currently, six loci for nonsyndromic SHFM phenotype have been identified: SHFM1 on 7q21 (OMIM 183600) [3], SHFM2 on Xq26 (OMIM 313350) [4-6], SHFM3 on 10q24 (OMIM 246560) [7-9], SHFM4 on 3q27 (OMIM 605289) [10], SHFM5 on 2q31 (OMIM 606708) [11,12] and SHFM6 on 12q13 (OMIM 225300) [13]. The auto- somal dominant inherited SHFM1, 3 and 4 have been associated with deletions and translocations on 7q, ge- nomic duplications on 10q24 and mutations in TP63, respectively. The only autosomal recessive SHFM6 have been found to be caused by mutations in WNT10B [13-15]. However, genetic causes for SHFM2 and SHFM5 are still unknown. * Correspondence: daili@scu.edu.cn 1National Center for Birth Defects Monitoring, Chengdu, China 2Lab of Molecular Epidemiology of Birth Defects, Chengdu, China Full list of author information is available at the end of the article Page 2 of 6 Page 2 of 6 Dai et al. BMC Medical Genetics 2013, 14:45 http://www.biomedcentral.com/1471-2350/14/45 Dai et al. BMC Medical Genetics 2013, 14:45 http://www.biomedcentral.com/1471-2350/14/45 Mutation screening The SHFM3 locus was initially mapped to chromo- some 10q24 by linkage analysis [7-9,16], then narrowed to a 0.5-Mb region through the identified genomic du- plications involving LBX1, BTRC and FBXW4 [17]. No intragenic mutations to date have been detected in can- didate genes within or flanking duplicated regions. The relationship has been well established between SHFM3 and genomic duplications in 10q24 [17-20], a few were documented in Chinese patients [21] though a relative high birth prevalence rate of 0.64/10000 for nonsyndro- mic SHFM was reported [22]. In this study, we conduc- ted an array-based genome-wide copy number variation (CNV) analysis on a Chinese family with SHFM and identified a discontinuous microduplication on 10q24.31- 24.32 as the likely cause. Genomic DNA was isolated from blood samples using a commercially available kit (Bio Teke, Beijing, China). As TP63 is the only disease-causing gene for autosomal dominant SHFM, we first searched for possible muta- tions in the gene in family members as outlined previ- ously [23]. We also screened candidate genes within the regions defined by array-based copy number variation analysis. Exons and their flanking intronic sequences of the LBX1, BTRC, DPCD and FBXW4 were amplified by polymerase chain reaction (PCR). All the resulting pro- ducts were purified, and sequenced bi-directionally using an ABI 3730xl sequencer. All primer sequences are avai- lable on request. Identification of the duplication by quantitative PCR 5 9 3 10 Figure 2 Clinical and X-ray photos of selected patients from this SHFM family. 5 Identification of the duplication by quantitative PCR To confirm duplications at 10q24.31-q24.32 mapped by CNV analysis, six EvaGreen assays were designed to de- termine the relative copy number (RCN) for all family members. Primers for amplifying selected fragments in duplicated regions were described previously [20]. PCR conditions and locations of assays are presented in Additional file 1: Table S1. All reactions used 500nM of each primer, 10 ng of genomic DNA, and SsoFast EvaGreen Supermix quantitative PCR (qPCR) buffer (Bio-Rad, California, USA). PCRs were performed by using a Bio-Rad C1000™Thermal Cycler (Bio-Rad, USA) in a 10-μl volume in 96-well plates with four replicates per sample. Reactions were run in a Bio-Rad CFX96™Real- Time System with the following conditions: 98°C for 2 min, 98°C for 1 sec, annealing for 5 sec, and 40 cycles of 98°C 1 sec. The quantification of the target sequences was normalized to one assay from chromosome 11 [18], the RCN was determined on the basis of the comparative 2-ΔΔCt method with a normal control DNA as the cali- brator [24]. The experiments were repeated three times. A ~1.5-fold RCN was used for duplication. Relative DNA copy number was obtained by pair-wise comparisons of test and control DNAs. 5 9 10 10 Mutation screening in candidate genes but not in the two unaffected family members. The du- plicated genomic contents contain two segments. The centromeric duplications, ranging from 247 kb to 260 kb, share a common overlapping segment containing LBX1, POLL and a disrupted BTRC. The telomeric duplica- tions of 114–125 kb comprise DPCD and one part of FBXW4 gene (Additional file 3: Table S3, Additional file 4: Figure S1). With use of qPCR assay, we confirmed the duplication co-segregating with SHFM phenotypes in Before genome-wide genotyping, we did not identify any mutation in exons of TP63 gene using PCR and direct sequencing analysis. Also we sequenced four known genes in the critical regions at 10q24, and found no pa- thogenic mutation in the coding region of these genes. Clinical report p Five individuals in this family were affected by SHFM with autosomal dominant transmission, but only four of them were available for this study (Figure 1). The pro- band (IV:3), one three-year-old boy, was affected by severe distal deficiency affecting all 4 limbs. Other three patients exhibited similar phenotypes including ectrodactyly of central digits, bilateral median cleft in feet, 3/4 finger or toe syndactly, hypoplasia of metacarpals and meta- tarsals. Representative clinical observations from photo- graphs and X-ray data are summarized in Figure 2 and Additional file 2: Table S2. Notably, phenotypic variations were observed between affected family members. Triphala- ngeal thumb was only identified in patient II:5, and complete 1/2 toe syndactyly in individual IV:3. Similar hy- poplasia/agenesis of 1st ray existed in III:9, III:10 and IV:3. No non-limb malformations were identified in any patient. 3 Figure 2 Clinical and X-ray photos of selected patients from this SHFM family. 3 Figure 2 Clinical and X-ray photos of selected patients from this SHFM family. Figure 2 Clinical and X-ray photos of selected patients from this SHFM family. Methods Subjects Using the Affymetrix Cytogenetics Whole-Genome 2.7M array (Affymetrix Inc, USA), four affected and two unaffected individuals (II:6, III:8) in this family were selected to undertake CNV analysis. Experiments were carried out by Peking CapitalBio Company (Beijing, China) according to the manufacturer's protocol. The Affymetrix Chromosome Analysis Suite Software (ChAS) was used for genotype calling, quality control and CNV identification. A SHFM family with four-generation-span was recruited after informed consent (Figure 1). Four affected (II:5, III:9, III:10 and IV:3) and 14 unaffected family members received full clinical evaluation and blood samples were collected for further studies. Clinical records and radio- graphic images were published under the patients’ written permission. This study was approved by the Research Ethics Committee of Sichuan University. Figure 1 Pedigree chart of the Chinese SHFM family. Figure 1 Pedigree chart of the Chinese SHFM family. Figure 1 Pedigree chart of the Chinese SHFM family Dai et al. BMC Medical Genetics 2013, 14:45 http://www.biomedcentral.com/1471-2350/14/45 Dai et al. BMC Medical Genetics 2013, 14:45 http://www.biomedcentral.com/1471-2350/14/45 Page 3 of 6 Discussion In this study, the duplicated segments contain three entire genes (LBX1, DPCD and POLL) and one part of two genes (BTRC and FBXW4). The duplicated portion of BTRC gene contains its promoter and exon 1, while the duplicated segment of FBXW4 includes 30 end of the gene. Since BTRC and FBXW4 are only partially dupli- cated in our SHFM cases, it is less likely that overex- pression of these two gene causes SHFM. Comparing our findings with those of previous studies, BTRC is the most frequently identified gene involved in the genomic re- arrangements. A duplication affecting BTRC and POLL only has been reported to be responsible for SHFM3 [20]. We speculate that limb-specific regulatory elements around or in BTRC gene may play an important role in SHFM pathogenesis, by controlling the expression of genes at 10q24 or the related downstream genes. SHFM is a complicated limb malformation that shows highly phenotypic variability and genetic heterogeneity. Even though six SHFM loci have been mapped to date, causal genes for a large number of cases are still unknown. At the gene level, mutations in TP63 and WNT10B can lead to autosomal dominant SHFM4 and autosomal re- cessive SHFM6, respectively. Recently a point mutation of DLX5 gene was reported to be responsible for auto- somal recessive SHFM [25]. Genomic rearrangements at 10q23-q24 have been identified in many familial or spor- adic SHFM cases [17-20], the mechanisms remain to be elucidated. In current study, we identified a previously unreported copy number variation at 10q24.31-q24.32, segregating with SHFM in a four-generation Chinese fa- mily. The minimal duplicated genomic contents include a 247-kb fragment comprising LBX1 and part of BTRC, and another fragment of 114 Kb containing POLL, DPCD, and disrupted FBXW4. Our findings expand the knowledge of genomic rearrangements leading to SHFM, and highlight the important role of chromosomal 10q24 region in regu- lating limb development. It is unclear how genomic rearrangement at this critical region causes the SHFM phenotype. The elevated expres- sion of BTRC and SUFU in lymphoblastoid cells detected in SHFM3 patients suggested that the gene dosage (BTRC) or long-range control mechanisms (SUFU) may underlie overexpression [20]. In the Dactylaplasia mouse model, changes in the expression levels of Lbx1, Btrc, Poll, and Dpcd were not observed except for reduced levels of nor- mal Fbxw4 transcript [19], which implies duplications may disrupt normal expression of related genes. Discussion An in- creasing number of studies have shown that copy number variations are a common cause of human genetic disor- ders [29]. Duplications of regulatory elements have been described for human limb malformations. Brachydactyly type A2 is caused by a tandem duplication of a 5.5 kb region 3’ of BMP2 [30]. Triphalangeal thumb-polysyndac- tyly syndrome is associated with duplications of ZPA regu- latory sequence, a long range cis-regulator for SSH gene [31]. Based on current understanding of the molecular mechanisms for limb anomalies associated with duplica- tions, the duplications described here may alter the dosage of a regulatory element involved in limb development or disrupt the co-expression domains, consequently leading to SHFM phenotypes. A genotype-phenotype study showed that preaxial in- volvement of the upper limbs can be locus discrimi- nators for SHFM3 [26]. In the current study, various preaxial upper limb anomalies were found in the affected individuals including proximal placed thumb, triphala- ngeal thumb, preaxial polydactyly, and absence of par- axial rays. In addition, more severe affected feet than hands, and worsening phenotype in patients of younger generations, were observed in the family. As noted in lit- erature [9,19], the observed phenotypes strongly sug- gested that current disorder should be mapped to SHFM3 locus, chromosomal 10q24. SHFM is considered a failure to maintain medial apical ectodermal ridge (AER) [1], so genes expressed in AER are reasonable candidates for SHFM3, such as the three known SHFM genes TP63 ,WNT10B and DLX5. Micro- duplications involving LBX1, BTRC, POLL, DPCD and one part of FBXW4 (human dactylin gene) were shown for SHFM3 in two studies [17,19]. Another study re- ported that a 325-kb duplication containing BTRC and POLL leads to SHFM3, which is the smallest one identi- fied to date [20]. A small number of genes at 10q24 like Copy number variation analysis Using array-based genome-wide CNV, we identified simi- lar duplications at 10q24.31-q24.32 in the four patients, Page 4 of 6 Dai et al. BMC Medical Genetics 2013, 14:45 http://www.biomedcentral.com/1471-2350/14/45 Page 4 of 6 BTRC, FBXW4, SUFU and FGF8 are also expressed in the developing limbs [1]. Among them, FBXW4 has been considered as the best candidate gene due to its role in the SHFM3 ‘like’ Dactylaplasia mouse model [27,28]. BTRC is another promising candidate, because its pro- duct gets involved in essential pathways for limb develop- ment, such as the canonical WNT/β-catenin and NKFα signaling pathways. Despite the frequent observations of genomic rearrangements at 10q24 in SHFM patients, no exon mutations were found in these candidate genes. the family. As predicted by ENCODE (http://www. genome.gov/10005107), this genomic region contains a variety of regulatory elements including enhancers, pro- moters and multiple transcription factor-binding sites. Par- ticularly, the centromeric duplications involve two negative enhancers, hs324 and hs939, while the region between two duplications harbors a human limb-development-related positive enhancer, hs326 (chr10:103,266,649-103,267,972, http://enhancer.lbl.gov). BTRC, FBXW4, SUFU and FGF8 are also expressed in the developing limbs [1]. Among them, FBXW4 has been considered as the best candidate gene due to its role in the SHFM3 ‘like’ Dactylaplasia mouse model [27,28]. BTRC is another promising candidate, because its pro- duct gets involved in essential pathways for limb develop- ment, such as the canonical WNT/β-catenin and NKFα signaling pathways. Despite the frequent observations of genomic rearrangements at 10q24 in SHFM patients, no exon mutations were found in these candidate genes. Author details 1 1National Center for Birth Defects Monitoring, Chengdu, China. 2Lab of Molecular Epidemiology of Birth Defects, Chengdu, China. 3National office for Maternal and Child Health Surveillance, Chengdu, China. 4Pulmonary Vascular Remodeling Research Unit, Chengdu, China. 5Key Laboratory of Obstetric & Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu, China. 1National Center for Birth Defects Monitoring, Chengdu, China. 2Lab of Molecular Epidemiology of Birth Defects, Chengdu, China. 3National office for Maternal and Child Health Surveillance, Chengdu, China. 4Pulmonary Vascular Remodeling Research Unit Chengdu China 5Key Laboratory of Obstetric & 18. Everman DB, Morgan CT, Lyle R, Laughridge ME, Bamshad MJ, Clarkson KB, Colby R, Gurrieri F, Innes AM, Roberson J, et al: Frequency of genomic rearrangements involving the SHFM3 locus at chromosome 10q24 in syndromic and non-syndromic split-hand/foot malformation. Am J Med Genet A 2006, 140(13):1375–1383. Education, West China Second University Hospital, Sichuan University, Chengdu, China. 19. Kano H, Kurosawa K, Horii E, Ikegawa S, Yoshikawa H, Kurahashi H, Toda T: Genomic rearrangement at 10q24 in non-syndromic split-hand/split-foot malformation. Hum Genet 2005, 118(3–4):477–483. Received: 10 November 2012 Accepted: 8 April 2013 Published: 18 April 2013 20. Lyle R, Radhakrishna U, Blouin JL, Gagos S, Everman DB, Gehrig C, Delozier-Blanchet C, Solanki JV, Patel UC, Nath SK, et al: Split-hand/split- foot malformation 3 (SHFM3) at 10q24, development of rapid diagnostic methods and gene expression from the region. Am J Med Genet A 2006, 140(13):1384–1395. Conclusion In summary, this report describes a discontinuous du- plication at 10q24.3 responsible for a typical familial Page 5 of 6 Page 5 of 6 Dai et al. BMC Medical Genetics 2013, 14:45 http://www.biomedcentral.com/1471-2350/14/45 SHFM3. Our data suggest that chromosome 10q24.3 may contain key elements for regulating the coordinate expres- sion of many genes for limb development. Future studies on the developmental effects of genomic rearrangements at this region in animal models would thus complement the human data presented here. Our findings could be of value to molecular diagnosis and deeper understanding of pathogenesis of this disorder. dominant split hand/split foot locus on chromosome 7, band q21.3-q22.1. Am J Hum Genet 1994, 55(1):12–20. 5. Faiyaz U, Haque M, Uhlhaas S, Knapp M, Schuler H, Friedl W, Ahmad M, Propping P: Mapping of the gene for X-chromosomal split-hand/split- foot anomaly to Xq26-q26.1. Hum Genet 1993, 91:17–19. 6. Ahmad M, Abbas H, Haque S, Flatz G: X-chromosomally inherited split-hand/split-foot anomaly in a Pakistani kindred. Hum Genet 1987, 75(2):169–173. The authors declare that they have no competing interests. The authors declare that they have no competing interests. 14. Khan S, Basit S, Zimri F, Ali N, Ali G, Ansar M, Ahmad W: A novel homozygous missense mutation in WNT10B in familial split-hand/foot malformation. Clin Genet 2012, 82(1):48–55. Acknowledgements W ld lik h We would like to thank the family for their participation. This work was supported by the Natural Science Foundation grant 30700679 and the Program for Changjiang Scholars and Innovative Research Team in University “PCSIRT” (IRT0935). 17. de Mollerat XJ, Gurrieri F, Morgan CT, Sangiorgi E, Everman DB, Gaspari P, Amiel J, Bamshad MJ, Lyle R, Blouin JL, et al: A genomic rearrangement resulting in a tandem duplication is associated with split hand-split foot malformation 3 (SHFM3) at 10q24. Hum Mol Genet 2003, 12(16):1959–1971. Program for Changjiang Scholars and Innovative Research Team in University “PCSIRT” (IRT0935). Authors’ contributions LD studied the family, designed research plan and prepared manuscript. YD, NL and LX performed the molecular genetic studies. MM participated in clinical evaluation. JZ participated in collecting specimens. All authors reviewed the final manuscript and approved the publication of the clinical images. All authors read and approved the final manuscript. 15. Blattner A, Huber AR, Rothlisberger B: Homozygous nonsense mutation in WNT10B and sporadic split-hand/foot malformation (SHFM) with autosomal recessive inheritance. Am J Med Genet A 2010, 152A(8):2053–2056. 16. Raas-Rothschild A, Manouvrier S, Gonzales M, Farriaux JP, Lyonnet S, Munnich A: Refined mapping of a gene for split hand-split foot malformation (SHFM3) on chromosome 10q25. J Med Genet 1996, 33(12):996–1001. Additional file 7. Gurrieri F, Prinos P, Tackels D, Kilpatrick MW, Allanson J, Genuardi M, Vuckov A, Nanni L, Sangiorgi E, Garofalo G, et al: A split hand-split foot (SHFM3) gene is located at 10q24– > 25. Am J Med Genet 1996, 62(4):427–436. Additional file 1: Table S1. Quantitatibe PCR Primers used in the study. Additional file 2: Table S2. Clinical features of four patients in the SHFM family. Additional file 3: Table S3. Genomic duplications at 10q21.31-q21.32 identified in SHFM patients. Additional file 4: Figure S1. Schematic overview of microduplications at chromosome 10q24.3 associated with SHFM. This graph was generated by the Affymetrix ChAS software. Additional file 1: Table S1. Quantitatibe PCR Primers used in the study. Additional file 2: Table S2. Clinical features of four patients in the SHFM family. 8. Nunes ME, Schutt G, Kapur RP, Luthardt F, Kukolich M, Byers P, Evans JP: A second autosomal split hand/split foot locus maps to chromosome 10q24-q25. Hum Mol Genet 1995, 4(11):2165–2170. Additional file 3: Table S3. Genomic duplications at 10q21.31-q21.32 identified in SHFM patients. 9. Ozen RS, Baysal BE, Devlin B, Farr JE, Gorry M, Ehrlich GD, Richard CW: Fine mapping of the split-hand/split-foot locus (SHFM3) at 10q24: evidence for anticipation and segregation distortion. Am J Hum Genet 1999, 64(6):1646–1654. Additional file 4: Figure S1. Schematic overview of microduplications at chromosome 10q24.3 associated with SHFM. This graph was generated by the Affymetrix ChAS software. 10. Celli J, Duijf P, Hamel BC, Bamshad M, Kramer B, Smits AP, Newbury-Ecob R, Hennekam RC, Van Buggenhout G, van Haeringen A, et al: Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome. Cell 1999, 99(2):143–153. 10. Celli J, Duijf P, Hamel BC, Bamshad M, Kramer B, Smits AP, Newbury-Ecob R, Hennekam RC, Van Buggenhout G, van Haeringen A, et al: Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome. Cell 1999, 99(2):143–153. Abbreviations SHFM S li h 11. Boles RG, Pober BR, Gibson LH, Willis CR, McGrath J, Roberts DJ, Yang-Feng TL: Deletion of chromosome 2q24-q31 causes characteristic digital anomalies: case report and review. Am J Med Genet 1995, 55(2):155–160. SHFM: Split hand/foot malformation; BTRC: Beta-transducin repeat containing E3 ubiquitin protein ligase; LBX1: Ladybird homeobox 1; DPCD: Deleted in primary ciliary dyskinesia homolog (mouse); POLL: Polymerase (DNA directed), lambda; FBXW4: F-box and WD repeat domain containing 4; SUFU: Suppressor of fused homolog (Drosophila); BMP2: Bone morphogenetic protein 2. 12. Goodman FR, Majewski F, Collins AL, Scambler PJ: A 117-kb microdeletion removing HOXD9-HOXD13 and EVX2 causes synpolydactyly. Am J Hum Genet 2002, 70(2):547–555. 13. Ugur SA, Tolun A: Homozygous WNT10b mutation and complex inheritance in Split-Hand/Foot Malformation. Hum Mol Genet 2008, 17(17):2644–2653. 3. Scherer SW, Poorkaj P, Allen T, Kim J, Geshuri D, Nunes M, Soder S, Stephens K, Pagon RA, Patton MA, et al: Fine mapping of the autosomal 2. Elliott AM, Reed MH, Chudley AE, Chodirker BN, Evans JA: Clinical and epidemiological findings in patients with central ray deficiency: split hand foot malformation (SHFM) in Manitoba. Canada Am J Med Genet A 2006, 140(13):1428–1439. References 1. Duijf PH, van Bokhoven H, Brunner HG: Pathogenesis of split-hand/ split-foot malformation. Hum Mol Genet 2003, 12:R51–R60. Spec No 1. 21. Yang W, Hu ZJ, Yu XF, Li QH, Zhang AJ, Deng X, Zhang AY, Gao CS, Liu Y, Ao Y, et al: [A DNA duplication at chromosome 10q24.3 is associated with split-hand split-foot malformation in a Chinese family]. Zhonghua Yi Xue Za Zhi 2006, 86(10):652–658. 2. Elliott AM, Reed MH, Chudley AE, Chodirker BN, Evans JA: Clinical and epidemiological findings in patients with central ray deficiency: split hand foot malformation (SHFM) in Manitoba. Canada Am J Med Genet A 2006, 140(13):1428–1439. 22. Dai L, Li YH, Deng Y, Zhu J, Wang YP, Liang J, Zhang YW, Liu ZY: [Prevalence of congenital split hand/split foot malformation in Chinese population]. Sichuan Da Xue Xue Bao Yi Xue Ban 2010, 41(2):320–323. 3. Scherer SW, Poorkaj P, Allen T, Kim J, Geshuri D, Nunes M, Soder S, Stephens K, Pagon RA, Patton MA, et al: Fine mapping of the autosomal Page 6 of 6 Page 6 of 6 Dai et al. BMC Medical Genetics 2013, 14:45 http://www.biomedcentral.com/1471-2350/14/45 23. van Bokhoven H, Hamel BC, Bamshad M, Sangiorgi E, Gurrieri F, Duijf PH, Vanmolkot KR, van Beusekom E, van Beersum SE, Celli J, et al: p63 Gene mutations in eec syndrome, limb-mammary syndrome, and isolated split hand-split foot malformation suggest a genotype-phenotype correlation. Am J Hum Genet 2001, 69(3):481–492. 24. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 2001, 25(4):402–408. 25. Shamseldin HE, Faden MA, Alashram W, Alkuraya FS: Identification of a novel DLX5 mutation in a family with autosomal recessive split hand and foot malformation. J Med Genet 2012, 49(1):16–20. 26. Elliott AM, Evans JA: Genotype-phenotype correlations in mapped split hand foot malformation (SHFM) patients. Am J Med Genet A 2006, 140(13):1419–1427. 27. Sidow A, Bulotsky MS, Kerrebrock AW, Birren BW, Altshuler D, Jaenisch R, Johnson KR, Lander ES: A novel member of the F-box/WD40 gene family, encoding dactylin, is disrupted in the mouse dactylaplasia mutant. Nat Genet 1999, 23(1):104–107. 28. Ianakiev P, Kilpatrick MW, Dealy C, Kosher R, Korenberg JR, Chen XN, Tsipouras P: A novel human gene encoding an F-box/WD40 containing protein maps in the SHFM3 critical region on 10q24. Biochem Biophys Res Commun 1999, 261(1):64–70. 29. doi:10.1186/1471-2350-14-45 Cite this article as: Dai et al.: Discontinuous microduplications at chromosome 10q24.31 identified in a Chinese family with split hand and foot malformation. BMC Medical Genetics 2013 14:45. References Zhang F, Gu W, Hurles ME, Lupski JR: Copy number variation in human health, disease, and evolution. Annu Rev Genomics Hum Genet 2009, 10:451–481. 30. Dathe K, Kjaer KW, Brehm A, Meinecke P, Nurnberg P, Neto JC, Brunoni D, Tommerup N, Ott CE, Klopocki E, et al: Duplications involving a conserved regulatory element downstream of BMP2 are associated with brachydactyly type A2. Am J Hum Genet 2009, 84(4):483–492. 31. Lettice LA, Heaney SJ, Purdie LA, Li L, de Beer P, Oostra BA, Goode D, Elgar G, Hill RE, de Graaff E: A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly. Hum Mol Genet 2003, 12(14):1725–1735. doi:10.1186/1471-2350-14-45 Cite this article as: Dai et al.: Discontinuous microduplications at chromosome 10q24.31 identified in a Chinese family with split hand and foot malformation. BMC Medical Genetics 2013 14:45. 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https://openalex.org/W3210260466	https://zenodo.org/record/5810937/files/First%20Genomic%20Evidence%20of%20Dual%20African%20Swine%20Fever.pdf	English	null	First Genomic Evidence of Dual African Swine Fever Virus Infection: Case Report from Recent and Historical Outbreaks in Sardinia	Viruses	2,021	cc-by	9,708	Citation: Fiori, M.S.; Ferretti, L.; Floris, M.; Loi, F.; Nardo, A.D.; Sechi, A.M.; Ladu, A.; Puggioni, G.; Sanna, D.; Scarpa, F.; et al. First Genomic Evidence of Dual African Swine Fever Virus Infection: Case Report from Recent and Historical Outbreaks in Sardinia. Viruses 2021, 13, 2145. https://doi.org/10.3390/v13112145 Academic Editors: Alejandro Núñez and David T. Williams Received: 19 September 2021 Accepted: 19 October 2021 Published: 25 October 2021 Citation: Fiori, M.S.; Ferretti, L.; Floris, M.; Loi, F.; Nardo, A.D.; Sechi, A.M.; Ladu, A.; Puggioni, G.; Sanna, D.; Scarpa, F.; et al. First Genomic Evidence of Dual African Swine Fever Virus Infection: Case Report from Recent and Historical Outbreaks in Sardinia. Viruses 2021, 13, 2145. https://doi.org/10.3390/v13112145 * Correspondence: federica.loi@izs-sardegna.it † These authors contributed equally to this work. Abstract: African swine fever virus (ASFV) is one of the pathogens of highest concern worldwide. Despite different virus lineages co-circulating in several areas, dual infections in the same animal have been rarely observed, suggesting that ASF superinfections are infrequent events. Here we present the ﬁrst genome-wide detection and analysis of two intragenotype dual ASFV infections. The dual infections have been detected in a hunted wild boar and in a pig carcass, both infected by ASFV genotype I in Sardinia in 1984 and 2018, respectively. We characterize the genetic differences between the two sequences, their intra-host frequency, and their phylogenetic relationship among fully sequenced ASFV strains from Sardinia. Both dual infections involve pairs of closely related but different viruses that were circulating in Sardinia in the same period. The results imply that dual ASFV infections or similar ASFV strains are more common than expected, especially in ASF endemic areas, albeit difﬁcult to detect. Received: 19 September 2021 Accepted: 19 October 2021 Published: 25 October 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Keywords: African swine fever; dual infection; phylogenetic analysis; whole genome sequence; Sardinia Article First Genomic Evidence of Dual African Swine Fever Virus Infection: Case Report from Recent and Historical Outbreaks in Sardinia Mariangela Stefania Fiori 1,† , Luca Ferretti 2,† , Matteo Floris 3,† , Federica Loi 4,* , Antonello Di Nardo 5 , Anna Maria Sechi 1, Anna Ladu 1, Graziella Puggioni 1, Daria Sanna 3 , Fabio Scarpa 6 , Maria Luisa Sanna 1, Maria Paola Madrau 1, Claudia Torresi 7 , Roberto Sirica 8, Eloisa Evangelista 8, Annalisa Oggiano 1 and Silvia Dei Giudici 1 1 Department of Animal Health, Istituto Zooproﬁlattico Sperimentale della Sardegna, 07100 Sassari, Italy; mariangela.ﬁori@izs-sardegna.it (M.S.F.); annamaria.sechi@izs-sardegna.it (A.M.S.); anna.ladu@izs-sardegna.it (A.L.); Graziella.puggioni@izs-sardegna.it (G.P.); marialuisa.sanna@izs-sardegna.it (M.L.S.); paola.madrau@izs-sardegna.it (M.P.M.); annalisa.oggiano@izs-sardegna.it (A.O.); silvia.deigiudici@izs-sardegna.it (S.D.G.) 1 Department of Animal Health, Istituto Zooproﬁlattico Sperimentale della Sardegna, 07100 Sassari, Italy; mariangela.ﬁori@izs-sardegna.it (M.S.F.); annamaria.sechi@izs-sardegna.it (A.M.S.); anna.ladu@izs-sardegna.it (A.L.); Graziella.puggioni@izs-sardegna.it (G.P.); marialuisa.sanna@izs-sardegna.it (M.L.S.); paola.madrau@izs-sardegna.it (M.P.M.); annalisa.oggiano@izs-sardegna.it (A.O.); silvia.deigiudici@izs-sardegna.it (S.D.G.) 2 Big Data Institute, Nufﬁeld Department of Medicine, University of Oxford, Oxford OX1 4BH, UK; 2 Big Data Institute, Nufﬁeld Department of Medicine, University of Oxford, Oxford OX1 4BH, UK; luca.ferretti@gmail.com g 3 Department of Biomedical Sciences, University of Sassari, 07100 Sassari, Italy; matteo.ﬂoris@gmail.com (M.F.); darsanna@uniss.it (D.S.) 4 Osservatorio Epidemiologico Veterinario Regionale, Istituto Zooproﬁlattico Sperimentale della Sardegna, 09125 Cagliari, Italy g y 5 The Pirbright Institute, Ash Road, Pirbright, Woking GU24 0NF, UK; antonello.dinardo@pirbright.ac.uk 6 f d f S S l f 5 The Pirbright Institute, Ash Road, Pirbright, Woking GU24 0NF, UK; antonello.dinardo@pirbright.ac.uk 6 Department of Veterinary Medicine, University of Sassari, 07100 Sassari, Italy; fscarpa@uniss.it 7 Istituto Zooproﬁlattico Sperimentale dell’Umbria e delle Marche, 06126 Perugia, Italy; c.torresi@izsum.it 8 Ames Polydiagnostic Group Center SRL, 80013 Napoli, Italy; roberto.sirica@centroames.it (R.S.); elo.evangelista@gmail.com (E.E.) viruses viruses viruses viruses viruses 1. Introduction African swine fever (ASF) is an infectious, often lethal disease affecting suid species caused by the African swine fever virus (ASFV) [1]. The virus is the only member of the family Asfarviridae, characterized by a large, double-stranded DNA genome of around 180–190 kb that encodes for over 150 open reading frames [1]. The virus mainly infects monocyte and macrophages but is also able to infect dendritic cells [1,2]. ASF is considered the most serious animal disease [3], given the lack of licensed vaccines [4], its capacity of affecting different target populations [5] with consequent high morbidity and mortality, and its transboundary and transcontinental spread [6]. The ﬁrst ASF cases were reported Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/viruses Viruses 2021, 13, 2145. https://doi.org/10.3390/v13112145 Viruses 2021, 13, 2145 2 of 13 in Kenya in 1914 [7], from where it spread to the Iberian Peninsula [8] and subsequently to Eastern Europe [9]. Since 2018, the ASFV has overrun the Asian continent [10]. in Kenya in 1914 [7], from where it spread to the Iberian Peninsula [8] and subsequently to Eastern Europe [9]. Since 2018, the ASFV has overrun the Asian continent [10]. p Dual infections (i.e., infections by multiple variants of the same pathogen) are not uncommon in viral infections and represent a necessary condition for recombination [11]. Speciﬁc disease conditions are mandatory for the occurrence of such events; in particular, the prevalence of the disease should be high enough that it is not unlikely for a host to be repeatedly infected by two different strains that co-circulate in the same area at the same time [12]. It is not surprising that this event seems to be very rare for ASF. In Mozambique between 1960 and 1994, the presence of two genetically distinct viruses circulating simultaneously during the same outbreak has been conﬁrmed, but no evidence of coinfection was detected [13]. Despite extensive evidence of co-circulation of different genotypes of ASFV in the same group of animals, dual infections were not detected, possibly because of technical limitations during those years. 1. Introduction To date, the only evidence of infection with different strains was reported by Mulumba-Mfumu [14] and occurred in the Democratic Republic of the Congo during 2010, where two different variants of the central variable region (CVR) of the B602L gene associated with two strains both belonging to genotype I were sequenced from two different tissues of the same pig. However, such evidence relies on a single hypervariable region in a single sample; hence, alternative explanations such as intra-host mutations or mislabeling of one of the samples cannot be excluded. Some authors also suggested recombination processes during coinfections in ticks [15,16]. Considering the speciﬁc epidemiological context that may prompt dual infections (i.e., co-circulation of different strains in the same area within the same host population and high disease prevalence), the Mediterranean island of Sardinia (Italy) could be considered a favorable land for such rare events to occur. Molecular studies revealed that Sardinian ASFV belongs to genotype I (vp72) [13,17]. Additional studies focusing on other regions of the genome (p54) have classiﬁed Sardinian isolates within genotype Ia [18]. Differences were observed in the B602L gene, which is involved in viral morphogenesis [19,20] allowing the differentiation of Sardinian isolates in two temporally related subgroups (X and III). Almost all of the strains isolated from 1990 onwards (subgroup X) showed the deletion of 12–13 tetramers [21] with respect to those isolated before 1990 (subgroup III). Likewise, Sanna in 2017 [22] reported an identical temporal subdivision of Sardinian ASF viruses into two subgroups. These groups, differing from the deletion of a six-amino-acid, repeat at the C-terminus of the CD2v protein encoded by the EP402R (CD2 homolog). From the characteristic of the strains isolated after 1990, the gene was detected as responsible for adsorption of erythrocytes around infected cells (haemadsorption) facilitating virus spread in the host [19]. Almost all of the Sardinian ASF viruses isolated after 1990 (modern strains) showed deletions in both the B602L and EP402R genes if compared to viruses isolated before 1990 (historical strains). The ﬁrst two Whole Genome Sequences (WGS) of Sardinian ASFV were obtained in 2016 [23,24]. Three more recent studies [17,25,26] analyzed 73 ASFV Sardinian full genomes showing a remarkable genetic stability of the strains. 1. Introduction In this paper, we present the ﬁrst genome-wide analysis of dual infection with viruses belonging to the same ASFV genotype I, detected in both a wild boar and a domestic pig in 1984 and 2018, respectively, at the time of the ﬁrst and the last epidemic peaks of ASF in Sardinia. We characterized the nucleotide differences between the different sequences in the viral population, their frequency within each sample, and their phylogenetic origin among Sardinian sequences and presented these as case report. 1.1. Dual Infections, Superinfections, and Coinfections 1.1. Dual Infections, Superinfections, and Coinfections We borrow the nomenclature from studies of other viruses, such as Human Immunod- eﬁciency Virus (HIV) and herpesviruses, where multiple infections have been observed and analyzed in great detail [27–31]. Dual infections are characterized by the presence of multiple strains (usually two) infecting the same individual. They can be conceptually cate- Viruses 2021, 13, 2145 3 of 13 gorized into coinfections, if all the strains were transmitted during the same exposure event, and superinfections, if they correspond to different exposure possibly from different sources. 1.2. Sardinian Epidemiological Context The virus was introduced to Sardinia in 1978, probably through infected food waste com- ing from the Iberian Peninsula [32,33]. Although the first Sardinian eradication plan started during the 1980s, several risk factors (i.e., epidemiological, environmental, and sociocultural) allowed the persistence of the disease in the island until 2019 [34–38]. After only six months from its ﬁrst detection, ASFV recorded more than 12,000 dead pigs [32,39]. The difﬁculties in the implementation of disease control measures (i.e., killing of all animals infected or suspect of infection, movement and export ban) led to ~200 domestic pig outbreaks recorded during the ﬁrst 5 years of the epidemic [33]. Consequently, the disease spread all over the island, mainly in the Nuoro province where ASFV infected the full spectrum of its target populations (i.e., domestic pigs, wild boar (WB), and free-ranging pigs). The initial epidemic peaks were recorded in 1979–1984 in domestic pigs and in 1985 in wild boar, ﬁnding the suitable conditions for endemicity for more than 42 years [21,36,38–41]. During the last ASF Eradication Program 2015–2018, a signiﬁcant decrease in disease prevalence was observed, and some areas demonstrated being free from ASFV [42]. The main role of illegal free-ranging pigs in disease persistence and the secondary one of WB have been recently demonstrated [17,41]. Several municipalities of the Nuoro province have been historically deﬁned as the main endemic area for ASFV circulation, where the virus has been maintained not only by the high animal density and contact rate [41] but more im- portantly by the lack of high biosecurity measures that afford the interactions between the three susceptible populations [36]. 2.2. DNA Extraction, Quantiﬁcation, and Sequencing Viral DNA was extracted from cell culture supernatant to perform the sequencing through the Illumina platform using a QIAmp UltraSens Virus Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA quantification was performed using an Epoch microplate spectrophotometer (BioTek, Winooski, VT, USA) and a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. ( ) g Two different Next Generation Sequencing (NGS) Illumina protocols were used to ob- tain the complete genome sequences of the two ASF strains used in this study. The NU1984 viral DNA libraries were prepared using the Illumina Nextera XT DNA sample prepara- tion protocol (Illumina, San Diego, CA, USA). The libraries of NU1984 were sequenced using the HiSeq 2500 platform (Illumina) generating paired-end reads 2 × 150 at the Center for Advanced Studies, Research and Development in Sardinia, Pula (CRS4) on 17 September 2018. p The libraries of the LO2018 virus were prepared using a Nextera DNA Flex Library Prep Kit (Illumina Inc., San Diego, CA, USA) and sequencing was carried out on the Novaseq 6000 (Illumina) generating paired-end reads 2 × 150 at AMES Group, Instrumental Polydiagnostic Center Srl Naples, Italy on 11 May 2019. The sequences of the B602L (bases 96,322–97,938) and the EP402R genes (bases 68,928–70,112) were conﬁrmed by repeated Sanger sequencing using the primers and the methods described previously [22]. 2.3. Bioinformatic Analysis Genome data processing was performed using an in-house bioinformatic pipeline. The bcl2fastq program (https://support.illumina.com/sequencing/sequencing_software/ bcl2fastq-conversion, access date: 10 March 2021) was used to convert BCL ﬁles generated by the sequencing systems to standard FASTQ ﬁle formats. Trim Galore (https://github. com/FelixKrueger/TrimGalore, access date: 10 March 2021) was used to quality trim the data and remove sequencing adaptors. The reads were then aligned to the pig reference genome (Sus scrofa 10.2) [43] using the bwa-mem algorithm [44]. Only reads mapping uniquely to the ASFV genome were retained and realigned using GEM [45]. Aligned bam ﬁles were sorted and indexed with SAMtools [46] and deduplicated with Picard tools (https: //broadinstitute.github.io/picard, access date: 10 March 2021). To obtain high-quality variants, FreeBayes [47] was used to call variants for each sample using the KX354450 [24] sequence as reference genome (parameters: “–ploidy 1 -X -u -m 20 -q 20 -F 0.2”). WGSs were aligned using MAFFT v. 7.427 [48] and polymorphism positions were visually inspected using Jalview v. 2.10.3 B.1 software [49]. Bam ﬁles of both NU1984 and LO2018 were aligned with the KX354450 sequence and visually inspected with IGV v. 2.4.14 [50] to detect dual infections. The program GC Content Calculator (https://jamiemcgowan.ie/bioinf/gc.html, access date: 10 March 2021) was used to calculate the % G~C content. Genome annotation was performed using GATU software [51] using KX354450 as the reference genome. 2.1. Ethic Statement, Sampling, and Virus Isolation 2.1. Ethic Statement, Sampling, and Virus Isolation Fresh monocytes/macrophages from healthy crossbred pigs (Sus scrofa domesticus), 6 to 24 months old, were used for virus isolation from on-ﬁeld samples. Healthy cross- bred pigs were housed at the Experiment Station of Istituto Zooproﬁlattico Sperimen- tale (IZS) of Sardinia (‘Surigheddu’, Sassari, Italy). Animal husbandry and handling procedures were performed according to Legislative Decree n.26 of 4 March 2014 and in agreement with the Guide of Use of Laboratory Animals issued by the Italian Min- istry of Health (available at: https://www.salute.gov.it/imgs/C_17_EventiStampa_355_ intervisteRelatori_itemInterviste_1_ﬁleAllegatoIntervista.pdf, access date: 20 January 2021), under the authorization No. 1232/2020-PR of 31/12/2020 by the Italian Ministry of Health. No animals were killed for the present work. Virus isolation has been carried out since 1978 on samples collected during ASF case notiﬁcation in Sardinia (i.e., domestic pig outbreaks on farms, hunted wild boar, or carcasses). The presence of infectious ASFV was assessed using the Malmquist test (haemoadsorption test) as described in the OIE Terrestrial Manual [42]. ASFV isolation was performed on homogenized spleen tissues from naturally infected animals. Tested samples were added to porcine two-day-old monocytes/macrophage monolayers, and cells were monitored daily for ﬁve days for hemadsorption effect. In the presence of haemadsorp- tion, culture supernatant was collected and stored at −80 ◦C (ASF Virus Archive, IZS of Sardinia, Italy). For negative samples, the Malmquist test was repeated by adding culture supernatants into fresh monocytes/macrophages; after three negative results, the absence of live ASFV virus was declared [42]. In this study, we obtained the WGS of the historical strain NU1984 isolated from a wild boar sampled in Nuoro province (unknown municipality) in 1984 and of the strain LO2018 isolated in 2018 from a domestic pig carcass (lat 39.97506, long 9.66024, Lotzorai municipality, Nuoro province) [26]. Sequencing of these strains was performed within the remit of a research project aimed at obtaining the WGS of several Sardinian ASFV strains Viruses 2021, 13, 2145 4 of 13 collected between 1978 and 2018 [26]. The analyses are still ongoing, but all strains were checked for dual infections. collected between 1978 and 2018 [26]. The analyses are still ongoing, but all strains were checked for dual infections. 2.2. DNA Extraction, Quantiﬁcation, and Sequencing 2.4. Inference of Strain Frequency and Composition from Variant Frequencies There are well-known approaches to detect dual infections from a collection of intra- host sequences [52]. However, when it is not possible to reconstruct such sequences due to short reads and low diversity, dual infections from two different strains can be detected by a different approach based on the frequency distribution of variants, as inferred from deep sequencing of short reads [53]. This approach is based on the observation of a large number of variants of similar, intermediate frequency. For NU1984 and LO2018, we selected all nucleotide variants with minor allele frequencies of >10% and covered by at least two reads, located in bases with minimum coverage 10. Variants were pre-called using SiNPle [54] with posterior probability >90%. To infer the frequencies of the two strains and their most likely sequences, we considered all possible frequencies of the two strains and all possible Viruses 2021, 13, 2145 5 of 13 assignments of variants. We inferred the most likely using an ML approach, based on baseline frequencies and composition of all the Sardinian full genomes sequenced and published so far [17,23–25] (Table S1). For the selected variants, the likelihood was based on random sampling of both sets of variants from the initial set of Sardinian sequences and the binomial sampling of reads from the two sets of variants. More precisely, if a sequence composition s appears ns times among Sardinian sequences, with an additional pseudo- count of ns = 1 for combinations not present among Sardinian sequences, the likelihood is the product of ns/Σs,ns and of the product among all variants of the binomial likelihood c1 + c2 c1 f1c1 f2c2 (1) (1) with f1 and f2 denoting the frequencies of the two strains in the viral population (which must satisfy f1 + f2 = 1) and c1, c2 the read counts. Confidence intervals were inferred from likelihood profiling and the Likelihood-Ratio test using Wilks’ theorem (1938) [55]. The whole analysis was implemented in R-software (Version 3.6.2, R-Foundation for Statistical Computing, Vienna, Austria). Once the two coinfecting virus variants within the NU1984 and LO2018 isolates were assembled by ML, the two haplotypes resulted in four different sequences named NU1984_major, NU1984_minor, LO2018_major, and LO2018_minor. The four reconstructed sequences and all the Sardinian ASFV whole genomes were then aligned to analyze differences between strains, their location, and their function. 2.5. Phylogenetic Analysis The ML tree was built from all the whole genome Sardinian strains sequenced so far [17,23–25] plus two outgroups (LI/1960 from Portugal and ES/1975 from Spain), and details are provided in Table S1. Sequences were aligned with MAFFT v. 7.427 [48]. The more likely assignment was deﬁned based on AIC, and the inferred ML frequency for minor and major haplotypes are presented as frequency (%) and a 95% conﬁdence interval (95% CI). The ﬁrst and last 2000 bases of the genome were removed to avoid alignment artifacts. The ML tree was built using RAxML v. 8.2.12 [56] with 1000 bootstraps and GTR-CAT as the evolutionary model. The NJ tree was inferred using bioNJ* [57] from distances computed according to the TN93 model implemented in APE [58]. 2.4. Inference of Strain Frequency and Composition from Variant Frequencies Their genome sequences were deposited in GenBank under the following accession number: MW647171/SRR15179098 (LO2018_major) and MW647172/ SRR15179098 (LO2018_minor); SRR13785534 (NU1984). 3.2. Evidence for Dual Infection 3.2. Evidence for Dual Infection We analyzed reads from d 3.2. Evidence for Dual Infection 3.2. Evidence for Dual Infection We analyzed reads from d We analyzed reads from deep sequencing of the samples NU1984 and LO2018, com- paring them with 13 Sardinian genome sequences previously published [17,25] and reads from 58 further Sardinian ASFV strains collected between 1978 and2018 [26], scaffolded against the KX354450 reference genome. NU1984 and LO2018 are the only two samples showing a large number of intra-host nucleotide polymorphisms, illustrated in Figure 1 and Table 1. We analyzed reads from deep sequencing of the samples NU1984 and LO2018, com- paring them with 13 Sardinian genome sequences previously published [17,25] and reads from 58 further Sardinian ASFV strains collected between 1978 and2018 [26], scaffolded against the KX354450 reference genome. NU1984 and LO2018 are the only two samples showing a large number of intra-host nucleotide polymorphisms, illustrated in Figure 1 and Table 1. Figure 1. Sequence logo for intra-host polymorphisms. Each column represents the nucleotide con- tent of a polymorphic site, with nucleotide size proportional to the relative frequencies among mapped reads. Genomic positions relative to the KX354450 sequence are reported below for each it Figure 1. Sequence logo for intra-host polymorphisms. Each column represents the nucleotide content of a polymorphic site, with nucleotide size proportional to the relative frequencies among mapped reads. Genomic positions relative to the KX354450 sequence are reported below for each site. si e In the NU1984 sample, we find a set of seven intra-host variants at intermediate fre- quencies between 13% and 47%, with an average frequency of ~28% (SD = 12%). In the LO2018 sample, we find 22 variants with a range of minor allele frequencies between 23% and 34%. Given the low mutation rate and very low intra-host variability of ASFV [5], such variants are unlikely to be the result of intra-host mutations. The natural alternative is that they originated from multiple variants infecting the same animal at some point; therefore, these ASFV infections are excellent candidates for dual infections. For NU1984, the frequencies look more dispersed than expected (overall SD is about 2.3 times the one expected under binomial sampling of reads), but the modest coverage of the positions In the NU1984 sample, we ﬁnd a set of seven intra-host variants at intermediate frequencies between 13% and 47%, with an average frequency of ~28% (SD = 12%). 3.1. Bioinformatic Analysis 3.1. Bioinformatic Analysis We obtained the consensus genome sequence for samples LO2018 [26] and NU1984. Both sequences were deposited in the NCBI Short Read Archive (SRA) (accession numbers are reported in Table S1). The median coverage for NU1984 and LO2018 was estimated and reported in Table S2. The inverted terminal repeats (ITRs) that included KP86R, KP96L, DP93R, and DP86L genes were missed at both ends (this is probably linked to the difﬁculties in assembling reads due to low coverage of these regions). The comparison of LO2018 and NU1984 with the genotype I genome sequences (Table S1) available from the NCBI GenBank repository allowed us to observe point mutations, including insertions or deletions (indels), described in Table S3 and in Torresi et al. (2020) [25]. The genomic content of all known Sardinian sequences is very similar [25], and the same is true for NU1984 and LO2018. The sequence of the LO2018 strain was 181,758 bp long with GC content of 38.57%. Following annotation by GATU software, a total of 231 ORF in LO2018 were identiﬁed, with 165 protein-coding genes. The replacement of A > C base (27%) in position 107474 (relative to KX354450) causes a start codon loss in the URF 33 gene, resulting in the absence of this hypothetical protein; this ﬁnding was also observed in other Sardinian isolates (22,653, 103,917 and DE/2018 [17,25]). Viruses 2021, 13, 2145 6 of 13 e URF so ob- 3.2. Evidence for Dual Infection 3.2. Evidence for Dual Infection We analyzed reads from d In the LO2018 sample, we ﬁnd 22 variants with a range of minor allele frequencies between 23% and 34%. Given the low mutation rate and very low intra-host variability of ASFV [5], such variants are unlikely to be the result of intra-host mutations. The natural alternative is that they originated from multiple variants infecting the same animal at some point; therefore, these ASFV infections are excellent candidates for dual infections. For NU1984, the frequencies look more dispersed than expected (overall SD is about 2.3 times the one expected under binomial sampling of reads), but the modest coverage of the positions where the variants are located (11 reads only for some variants) and possibly the additional noise due to viral DNA extraction and ampliﬁcation are likely explanations for this over- dispersion in frequencies. The variants and their allele counts are reported in Table 1. No further evidence suggestive of coinfections was found among the other Sardinian strains analyzed. Known variable regions in B602L and EP402R of NU1984 and LO2018 samples were also sequenced several times. Repeated Sanger sequencing of NU1984 for the B602L region provided a consistent result, ﬁnding a deletion in all seven replicates analyzed. However, sequences of the EP402R region obtained by Sanger sequencing showed clear genetic differences: the same deletion that is present among consensus sequences is found in approximately 70% (21 out of 30) of them, 3 out of 30 replicates clearly showed no deletion, and 6 out of 30 replicated gave inconclusive results due to the presence of several double peaks within the deletion region. Since it is very unlikely that intra-host processes would generate precisely the same deletion found in sequences Viruses 2021, 13, 2145 7 of 13 of other viruses isolated from the same epidemic, this is clear evidence of a dual infection at intermediate frequencies. On the other hand, repeated LO2018 Sanger sequencing for B602L and EP402R regions consistently showed deletions in all sequences for both regions. Table 1. Position with respect to the reference sequence KX354450, major and minor allele and their read counts for all intermediate variants, in NU1984 and LO2018 samples. 3.3. Inference of Strain Frequency and Composition from Variant Frequencies . Inference of Strain Frequency and Composition from Variant Frequencies The Maximum Likelihood (ML) analysis for NU1984 conﬁrms that the most likely sequence contains all major alleles TAACAAC and all minor alleles CGGGGGG. This is the most likely assignment, since the next most likely one (a switch of the major and minor alleles in position 12,452) is strongly penalized by the difference in the Akaike Informa- tion Criterion (AIC) score of 27.4. The inferred ML frequency for the minor haplotype is 31% (95% CI: 28–35%) and therefore 69% for the major haplotype (95% CI: 65–72%). The same analysis for LO2018 indicates that the most likely sequence containing all major alleles was TCCTGTGTGTAACGCTCCCCCT and the corresponding minor alleles was GTTCACACACGGTATCAGTTTC. This is by far the most likely assignment given that the difference in the AIC score of 324.4 strongly disfavors the next most likely one (a switch of the major and minor alleles in the last position 180,061). The inferred ML frequency for the minor haplotype is 28% (95% CI: 27–29%) and therefore 72% for the major haplotype (95% CI: 71–73%). 3.2. Evidence for Dual Infection 3.2. Evidence for Dual Infection We analyzed reads from d , p Sample Position in Reference Major Variant Minor Variant Major Read Count Minor Read Count Major Frequency Minor Frequency NU1984 12452 T C 11 4 0.73 0.27 24,115 A G 13 2 0.87 0.13 53,275 A G 52 42 0.55 0.45 63,153 C G 144 76 0.65 0.35 82,709 A G 57 9 0.86 0.14 133,600 A G 28 17 0.62 0.38 161,784 C G 70 22 0.76 0.24 LO2018 6930 T G 200 88 0.69 0.31 9187 C T 257 84 0.75 0.25 23,900 C T 275 101 0.73 0.27 25,014 T C 228 104 0.69 0.31 27,680 G A 259 98 0.73 0.27 33,008 T C 227 89 0.72 0.28 34,564 G A 228 90 0.72 0.28 66,032 T C 233 102 0.70 0.30 71,406 G A 220 96 0.70 0.30 77,775 T C 263 97 0.73 0.27 77,903 A G 244 102 0.71 0.29 80,076 A G 243 87 0.74 0.26 81,605 C T 217 87 0.71 0.29 82,112 G A 239 85 0.74 0.26 100,721 C T 270 83 0.76 0.24 106,379 T C 240 124 0.66 0.34 107,474 C A 230 85 0.73 0.27 116,165 C G 203 89 0.70 0.30 154,851 C T 294 118 0.71 0.29 166,904 C T 262 103 0.72 0.28 180,038 C T 102 16 0.86 0.14 180,061 T C 110 26 0.81 0.19 3.3. Inference of Strain Frequency and Composition from Variant Frequencies 3.4. Phylogenetic Analysis From the comparison of the polymorphisms in NU1984 and the consensus bases in other Sardinian strains (Table S1), it is evident that the major haplotype is very sim- ilar to those sequences sampled after the mid-1980s but with two exclusive variants, while the minor one is more closely related to those viruses circulating during the early years of the Sardinian epidemic. We reconstructed the phylogenetic tree including the se- quences inferred for major and minor strains in these dual infections found in both samples. Viruses 2021, 13, 2145 8 of 13 the the The Neighbor Joining (NJ) tree and ML tree in Figure 2 show how both sequences in NU1984 are consistently grouped among other sequences sampled between 1985 and1995. j p Neighbor Joining (NJ) tree and ML tree in Figure 2 show how both sequences in NU1984 re consistently grouped among other sequences sampled between 1985 and1995. igure 2. Maximum Likelihood and Neighbor Joining phylogenies reconstructed from Sardinian ASFV genotype I sequences. Tips characterizing the dual infections are represented in blue LO\|2018) and orange (NU\|1984). Nodes with supported bootstrap values of > 90 are colored in ed Figure 2. Maximum Likelihood and Neighbor Joining phylogenies reconstructed from Sardinian ASFV genotype I sequences. Tips characterizing the dual infections are represented in blue (LO\|2018) and orange (NU\|1984). Nodes with supported bootstrap values of >90 are colored in red. gure 2. Maximum Likelihood and Neighbor Joining phylogenies reconstructed from Sardinian FV genotype I sequences. Tips characterizing the dual infections are represented in blue O\|2018) and orange (NU\|1984). Nodes with supported bootstrap values of > 90 are colored in d Figure 2. Maximum Likelihood and Neighbor Joining phylogenies reconstructed from Sardinian ASFV genotype I sequences. Tips characterizing the dual infections are represented in blue (LO\|2018) and orange (NU\|1984). Nodes with supported bootstrap values of >90 are colored in red. The major and minor alleles of LO2018 are spaced out in both the NJ and ML trees: e minor groups with other sequences from 2014 to2018, while the major is assigned sis- r to the clade that includes isolates from 1997 to 2008, with six exclusive variants. While The major and minor alleles of LO2018 are spaced out in both the NJ and ML trees: the minor groups with other sequences from 2014 to2018, while the major is assigned sister to the clade that includes isolates from 1997 to 2008, with six exclusive variants. 3.4. Phylogenetic Analysis While the most frequent haplotype of NU1984 in the dual infection is assigned within a clade of sequences from 1985 to1995 (which correspond to those samples collected after the 1984 epidemic peak), the less frequent haplotype corresponds to a virus that can be considered an evolutionary intermediate between the viruses circulating pre-1984 and those occurring from 1985 to 1995. Focusing on LO2018, the least frequent haplotype is a close relative of known sequences from the same year and geographical area, while the most frequent one is a basal lineage for the later phase of the Sardinian epidemic. The consistent phylogenetic placement of the two coinfecting viruses is further conﬁrmation of the correct inference of their sequences and that these intra-host polymorphisms originate from dual infections rather than evolution within hosts. 3.5. Genomic Differences between Coinfecting Strains In this study we conﬁrm the genome differences between Sardinian strains previ- ously detected [17,25,26], and we further report newly identiﬁed point mutations. Genetic differences between Sardinian viruses with the strains analyzed in this study are sum- marized in Table S3 (NU1984 major and minor), S4, and S5 (LO2018 major and minor). Viruses 2021, 13, 2145 9 of 13 9 of 13 A total of 14 mutations in 13 genes and 1 hypothetical protein or URF (unassigned reading frame) were found (8 synonymous mutations and 6 non-synonymous mutations). As dis- cussed, only seven nucleotide variants were observed between the two coinfecting viruses (NU1984_major and NU1984_minor), corresponding to a very low divergence of about 4 × 10−5 mutations per base. Two variants exclusive to NU1984_major were observed, located in the region coding for the genes EP1242L (involved in nucleic acid synthesis) and in the gene D250R (coding for the mRNA-decapping protein g5R). The replacement of the G > C base (65%) in position 63153 (relative to KX354450), within the EP1242L gene, is a silent point mutation. The replacement of the A > G base (62%) in position 133,600 produced an amino acid mutation at position 157 in the D250R gene, corresponding to a E > K substitution. This mutation is located within the Nudix hydrolase signature domain, which has been shown to regulate the virulence of ASFV by altering or inhibiting the expression of host proteins through the degradation of mRNA by means of decap- ping the methylated cap attached to mRNA [59,60]. A BLAST analysis revealed that this non-synonymous nucleotide change is present only in NU1984_major respective to all the D250R gene sequences present in GenBank. The assignment of the deletion in the EP402R region is further evidenced in the tree topologies presented above. In fact, the deleted allele is consistently present in the cluster of sequences phylogenetically associated to the major strain. Hence, the most parsimonious explanation is that the major strain presents only the deletion in B602L, while the minor strain presents deletions in both EP402R and B602L regions. Regarding the sample LO2018, a total of 22 variants relative to KX354450 were observed, but only 8 were exclusive (3 non-synonymous and 5 synonymous mutations, Table S3). 3.5. Genomic Differences between Coinfecting Strains These are located within the genes MGF110-1L and MGF505-5R (MGF families), EP84R (coding for a transmembrane protein), C717R (belonging to a PK-like protein super- family), M1249L (coding for a capsid component) [28], and C122R (coding for a structural component of the virus particle) [29]. Two mutations (one non-synonymous and one synonymous) were found within the gene DP96R, involved in virus virulence [61]. Further, two non-synonymous and two synonymous mutations, already described [25,26], are lo- cated within the genes MGF110-13L_1 (MGF 110 family), Lis117, URF11, and QP509L and are restricted to both LO2018_minor and DE/2018 (GenBank MT932579) [17]. The deletions found in the CD2 and B602L regions are present in both major and minor strains. 4. Discussion In this study, we analyzed two Sardinian ASF viruses, which were collected from the most endemic area of Sardinia and were isolated from a wild boar and a domestic pig during the 1984 and 2018 endemic peaks, respectively. The analysis of NU1984 and LO2018 [26] WGSs revealed the presence of two different viral strains in each sample that we named NU1984_major and NU1984_minor and LO2018_major and LO2018_minor, respectively. Dual infections require a superinfection event, which happens when two or more viral strains are transmitted to the same animal at different times [12]. Our results suggest that even if ASFV superinfections are very difﬁcult to detect, these could be more common than expected, at least in the ASF endemic context of Sardinia characterized by several epidemic peaks. In fact, most superinfections will involve viruses with almost identical sequences, and the two viruses are unlikely to be found in similar proportions unless the second infection occurs shortly after the ﬁrst one. This observation has two important bases. First, there is a high incidence and prevalence of the disease in the cen- tral areas of Sardinia during the 1984 and 2018 peaks; otherwise, occurrence of multiple transmissions to the same animal in a short period of time would not have been possible. Second, during the same years, there was abundant co-circulation of multiple viral strains within the local host populations; otherwise, such dual infections could not originate. There is no reason to believe that these ﬁndings would be unique to ASFV genotype I or to the Sardinian epidemic. We expect that coinfections could be found in other ASF endemic areas having a similar epidemiological context. The presence of dual infections is a pre- condition for recombination; hence, the evidence in this paper implies that recombination Viruses 2021, 13, 2145 10 of 13 10 of 13 between ASFV strains is likely to occur and supports the recent phylogenetic evidence for recombination as a relevant evolutionary force acting on ASFV genomes. Note that the presence of such dual infections suggests that either superinfection exclusion does not occur in ASFV infections, despite the similarity of the coinfecting strains, or that the occurrence of these dual infections may have also been facilitated by functional differ- ences among the coinfecting haplotypes, e.g., because their genetic variation could have triggered differences in virulence or intra-host growth. 4. Discussion Some of these variants are found exclusively in these samples, for example, in NU1984, the silent mutation for EP1242L gene encoding RNA polymerase and the amino acid substitution in D250R gene encoding the mRNA-decapping protein g5R. The latter mutation occurs within the Nudix hydrolases signature domain, which has been shown to play a role in the virulence of ASFV by altering or inhibiting the expression of host proteins through the degradation of mRNA by means of decapping the methylated cap attached to mRNA [59]. This non-synonymous change was unique to the NU1984_major haplotype. The sample LO2018 contained several variants that could cause a functional difference between haplotypes, located in MGF505-5R (MGF 505 family), C717R, and DP96R, as well as synonymous silent mutation for MGF110-1L (MGF 110 family), Ep84R, M1249, C122R, QP509L, and DP96R genes. LO2018_minor contained the variant between haplotypes, located in MGF110-13L_1 and Lis117, as well as synonymous silent mutation for URF11 and QP509L present only in DE/2018 (GenBank accession number MT932579) isolated in 2018 [17]. A recent study [62] suggests that a mutant of QP509L, the DEAH box ATP-dependent RNA helicase gene, which is essential for viral replication, can be a good candidate for a live attenuated vaccine by not producing progeny and inducing a protective immune response. As described in previous studies, the ASF prevalence detected within illegal free-ranging pigs in Sardinia was much higher than the prevalence detected in the other two ASF host populations (wild boar and domestic pigs) [35,41]. The increasing probability of detecting coinfection in an epidemiological context of high disease prevalence could partially explain this ﬁnding in LO2018. Given the distant location of the minor and major strains in the ML tree, further investigations are ongoing to evaluate the variability of the ASFV population within the closed ecosystem of Sardinia. In conclusion, this is the ﬁrst report of the presence of ASFV dual infections based on clear genomic evidence. Given the technical difﬁculties in detecting such infections, our ﬁnding of two different dual infections out of less than a hundred samples strongly suggests that dual ASFV infections are relatively common events in Sardinia and linked to areas of high disease prevalence and frequent co-circulation of multiple strains. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/ v13112145/s1, Table S1: African swine fever virus (ASFV) whole-genome sequences available from public databases; Table S2: Sample coverage; Table S3: Point mutation. 4. Discussion Acknowledgments: The authors would like to thank Paola Nicolussi, Antonio Pilo, and all the Animal Service Unit (‘Stabulario’, IZS della Sardegna, Italy) for the care of animals and provision of blood samples and Susanna Zinellu and Pier Paolo Angioi (Virological Diagnostic Laboratory-IZS della Sardegna, Italy) for invaluable technical support. Acknowledgments: The authors would like to thank Paola Nicolussi, Antonio Pilo, and all the Animal Service Unit (‘Stabulario’, IZS della Sardegna, Italy) for the care of animals and provision of blood samples and Susanna Zinellu and Pier Paolo Angioi (Virological Diagnostic Laboratory-IZS della Sardegna, Italy) for invaluable technical support. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Dixon, L.K.; Chapman, D.A.; Netherton, C.L.; Upton, C. African swine fever virus replication and genomics. Virus Res. 2013, 173, 3–14. [CrossRef] 2. 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Discussion Author Contributions: Data curation, M.S.F., L.F., M.F., A.M.S., A.L., G.P., M.L.S., M.P.M., R.S., E.E. and S.D.G.; formal analysis, M.S.F., L.F., M.F. and S.D.G.; funding acquisition, F.L. and A.O.; investigation, M.S.F., L.F., M.F., F.L., A.O. and S.D.G.; methodology, M.S.F., L.F., M.F., F.L., A.D.N., C.T., A.O. and S.D.G.; project administration, F.L., A.O. and S.D.G.; resources, A.O. and S.D.G.; software, L.F., M.F. and S.D.G.; supervision, M.S.F., L.F., M.F., F.L., A.D.N., A.O. and S.D.G.; validation, M.S.F., L.F., M.F., A.D.N., C.T., E.E., A.O. and S.D.G.; visualization, M.S.F., L.F., M.F., A.D.N., A.M.S., A.L., G.P., D.S., F.S., M.L.S., M.P.M., R.S., A.O. and S.D.G.; writing—original draft, M.S.F., L.F., M.F., F.L., C.T., A.O. and S.D.G.; writing—review and editing, M.S.F., L.F., M.F., F.L., A.D.N., A.M.S., A.L., G.P., D.S., F.S., M.L.S., M.P.M., C.T., R.S., E.E., A.O. and S.D.G. All authors have read and agreed to the published version of the manuscript. Author Contributions: Data curation, M.S.F., L.F., M.F., A.M.S., A.L., G.P., M.L.S., M.P.M., R.S., E.E. and S.D.G.; formal analysis, M.S.F., L.F., M.F. and S.D.G.; funding acquisition, F.L. and A.O.; investigation, M.S.F., L.F., M.F., F.L., A.O. and S.D.G.; methodology, M.S.F., L.F., M.F., F.L., A.D.N., C.T., A.O. and S.D.G.; project administration, F.L., A.O. and S.D.G.; resources, A.O. and S.D.G.; software, L.F., M.F. and S.D.G.; supervision, M.S.F., L.F., M.F., F.L., A.D.N., A.O. and S.D.G.; validation, M.S.F., L.F., M.F., A.D.N., C.T., E.E., A.O. and S.D.G.; visualization, M.S.F., L.F., M.F., A.D.N., A.M.S., A.L., G.P., D.S., F.S., M.L.S., M.P.M., R.S., A.O. and S.D.G.; writing—original draft, M.S.F., L.F., M.F., F.L., C.T., A.O. and S.D.G.; writing—review and editing, M.S.F., L.F., M.F., F.L., A.D.N., A.M.S., A.L., G.P., D.S., F.S., M.L.S., M.P.M., C.T., R.S., E.E., A.O. and S.D.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Italian Ministry of Health, grant number IZSUM PSRC 1/2018: “Miglioramento delle strategie e degli strumenti di prevenzione e controllo della Peste suina africana in Italia”. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. 11 of 13 11 of 13 Viruses 2021, 13, 2145 Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. References 2008, 153, 2303–2306. [CrossRef] 20. Giammarioli, M.; Gallardo, C.; Oggiano, A.; Iscaro, C.; Nieto, R.; Pellegrini, C.; Giudici, S.D.; Arias, M.; De Mia, G.M. Genetic characterisation of African swine fever viruses from recent and historical outbreaks in Sardinia (1978–2009). Virus Genes 2011, 42, 377–387. [CrossRef] [PubMed] 20. Giammarioli, M.; Gallardo, C.; Oggiano, A.; Iscaro, C.; Nieto, R.; Pellegrini, C.; Giudici, S.D.; Arias, M.; De Mia, G.M. 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https://openalex.org/W4361817793	https://figshare.com/articles/journal_contribution/Figure_S1_from_Dopamine_Receptor_D5_is_a_Modulator_of_Tumor_Response_to_Dopamine_Receptor_D2_Antagonism/22472337/1/files/39923829.pdf	English	null	Figure S2 from Dopamine Receptor D5 is a Modulator of Tumor Response to Dopamine Receptor D2 Antagonism	null	2,023	cc-by	464	Supplemental Figure 1 A B DRD2 mRNA log2 (RNA seq v2) AML Liver DLBCL Thyroid Cholangiocarcinoma Bladder ccRCC pRCC Cervical Lung (adeno) Melanoma Head & Neck Thymoma Mesothelioma ACC Breast Prostate Lung (squamous) Uterine Testicular Pancreas Sarcoma Uveal melanoma Uterine CS Colorectal chRCC Ovarian Glioma GBM PCPG Melanoma Lung (adeno]) Testicular Ovarian Uterine Esophageal Stomach Sarcoma Stomach/Esophogeal Lung (squamous) Prostate Head & Neck Colorectal pRCC AML DLBCL Breast Liver chRCC GBM Uveal melanoma Glioma Pancreatic ccRCC Bladder ACC Cholangiocarcinoma Mesothelioma PCPG Thyroid Thymoma Alteration Frequency D DRD2 mRNA log2 (RNA seq v2) E Disease-Free Survival (months) DRD2 mRNA log2 (RNA seq v2) Shallow Diploid Gain Amplification Deletion C 0 50 100 150 200 250 300 DRD1 DRD2 DRD3 DRD4 DRD5 RNA Seq (SEM) Supplemental Figure 1 A B DRD2 mRNA log2 (RNA seq v2) AML Liver DLBCL Thyroid Cholangiocarcinoma Bladder ccRCC pRCC Cervical Lung (adeno) Melanoma Head & Neck Thymoma Mesothelioma ACC Breast Prostate Lung (squamous) Uterine Testicular Pancreas Sarcoma Uveal melanoma Uterine CS Colorectal chRCC Ovarian Glioma GBM PCPG Melanoma Lung (adeno]) Testicular Ovarian Uterine Esophageal Stomach Sarcoma Stomach/Esophogeal Lung (squamous) Prostate Head & Neck Colorectal pRCC AML DLBCL Breast Liver chRCC GBM Uveal melanoma Glioma Pancreatic ccRCC Bladder ACC Cholangiocarcinoma Mesothelioma PCPG Thyroid Thymoma Alteration Frequency D DRD2 mRNA log2 (RNA seq v2) E Disease-Free Survival (months) DRD2 mRNA log2 (RNA seq v2) Shallow Diploid Gain Amplification Deletion C 0 50 100 150 200 250 300 DRD1 DRD2 DRD3 DRD4 DRD5 RNA Seq (SEM) Supplemental Figure 1 Supplemental Figure 1 B Melanoma Lung (adeno]) Testicular Ovarian Uterine Esophageal Stomach Sarcoma Stomach/Esophogeal Lung (squamous) Prostate Head & Neck Colorectal pRCC AML DLBCL Breast Liver chRCC GBM Uveal melanoma Glioma Pancreatic ccRCC Bladder ACC Cholangiocarcinoma Mesothelioma PCPG Thyroid Thymoma Alteration Frequency Supplemental Figure 1 A B DRD2 mRNA log2 (RNA seq v2) AML Liver DLBCL Thyroid Cholangiocarcinoma Bladder ccRCC pRCC Cervical Lung (adeno) Melanoma Head & Neck Thymoma Mesothelioma ACC Breast Prostate Lung (squamous) Uterine Testicular Pancreas Sarcoma Uveal melanoma Uterine CS Colorectal chRCC Ovarian Glioma GBM PCPG Melanoma Lung (adeno]) Testicular Ovarian Uterine Esophageal Stomach Sarcoma Stomach/Esophogeal Lung (squamous) Prostate Head & Neck Colorectal pRCC AML DLBCL Breast Liver chRCC GBM Uveal melanoma Glioma Pancreatic ccRCC Bladder ACC Cholangiocarcinoma Mesothelioma PCPG Thyroid Thymoma Alteration Frequency B A DRD2 mRNA log2 (RNA seq v2) AML Liver DLBCL Thyroid Cholangiocarcinoma Bladder ccRCC pRCC Cervical Lung (adeno) Melanoma Head & Neck Thymoma Mesothelioma ACC Breast Prostate Lung (squamous) Uterine Testicular Pancreas Sarcoma Uveal melanoma Uterine CS Colorectal chRCC Ovarian Glioma GBM PCPG A Alteration Frequency D DRD2 mRNA log2 (RNA seq v2) Shallow Diploid Gain Amplification Deletion E Disease-Free Survival (months) DRD2 mRNA log2 (RNA seq v2) C 0 50 100 150 200 250 300 DRD1 DRD2 DRD3 DRD4 DRD5 RNA Seq (SEM) E D C
https://openalex.org/W2786981732	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0191859&type=printable	English	null	Anthropogenic marine litter composition in coastal areas may be a predictor of potentially invasive rafting fauna	PloS one	2,018	cc-by	12,270	Anthropogenic marine litter composition in coastal areas may be a predictor of potentially invasive rafting fauna Sabine Rech, Yaisel J. Borrell Pichs, Eva Garcı´a-Vazquez Sabine Rech, Yaisel J. Borrell Pichs, Eva Garcı´a-Vazquez Department of Functional Biology, University of Oviedo, Oviedo, Asturias, Spain * rechsabine@uniovi.es * rechsabine@uniovi.es a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract Anthropogenic plastic pollution is a global problem. In the marine environment, one of its less studied effects is the transport of attached biota, which might lead to introductions of non-native species in new areas or aid in habitat expansions of invasive species. The goal of the present work was to assess if the material composition of beached anthropogenic litter is indicative of the rafting fauna in a coastal area and could thus be used as a simple and cost-efficient tool for risk assessment in the future. Beached anthropogenic litter and attached biota along the 200 km coastline of Asturias, central Bay of Biscay, Spain, were analysed. The macrobiotic community attached to fouled litter items was identified using genetic barcoding combined with visual taxonomic analysis, and compared between hard plastics, foams, other plastics and non-plastic items. On the other hand, the material compo- sition of beached litter was analysed in a standardized area on each beach. From these two datasets, the expected frequency of several rafting taxa was calculated for the coastal area and compared to the actually observed frequencies. The results showed that plastics were the most abundant type of beached litter. Litter accumulation was likely driven by coastal sources (industry, ports) and river/sewage inputs and transported by near-shore currents. Rafting vectors were almost exclusively made up of plastics and could mainly be attributed to fishing activity and leisure/ household. We identified a variety of rafting biota, including species of goose barnacles, acorn barnacles, bivalves, gastropods, polychaetes and bryo- zoan, and hydrozoan colonies attached to stranded litter. Several of these species were non-native and invasive, such as the giant Pacific oyster (Crassostrea gigas) and the Aus- tralian barnacle (Austrominius modestus). The composition of attached fauna varied strongly between litter items of different materials. Plastics, except for foam, had a much more diverse attached community than non-plastic materials. The predicted frequency of several taxa attached to beached litter significantly correlated with the actually observed fre- quencies. Therefore we suggest that the composition of stranded litter on a beach or an area could allow for predictions about the corresponding attached biotic community, includ- ing invasive species. RESEARCH ARTICLE H2020 AQUAINVAD-ED; grant agreement no. 642197]. H2020 AQUAINVAD-ED; grant agreement no. 642197]. Since plastics have been made available to a broad spectrum of consumers after the Second World War, their global production has risen to 322×109 kg in 2015 [1]. Although plastic pro- duction is concentrated in China, Europe, the USA, Canada and Mexico, plastics and recycla- ble plastic waste, which are not classified as hazardous [2], are exported internationally [1,3,4], posing a global threat to human health, interests, and ecosystems [2,5]. The pollution by plastic litter has advanced to such a level that today it is present in virtually every environment and every location of the Earth [6,7]. The marine environment is especially affected, as it receives not only direct pollution from sea-based activities, but also land-based plastics [7–9]. Plastic pollution causes the death of a high number of marine animals, as well as severe damages to ecosystems and human health and interests, like tourism, fishing, or leisure activities at beaches [10–13]. Plastics do not degrade naturally but fragment to smaller pieces, which multi- plies their abundance [6]. In recent decades, campaigns are being conducted to combat the excessive production and consumption of single-use plastics, for example plastic bags from supermarkets, microbeads in cosmetic products, or PET (Polyethylene terephthalate) beverage bottles (e.g. http://storyofstuff.org/, http://www.beatthemicrobead.org/). Policy changes have been requested after increasing scientific evidence and public awareness about the pollution problem [14,15]. Competing interests: The authors have declared that no competing interests exist. While research and actions on several aspects of the plastic litter problem are steadily advancing, there are still many important aspects that have gained little scientific attention so far. One problem that has received less attention is the role of anthropogenic litter items serv- ing as artificial rafts for non-native and possibly invasive species. Notably, rafting has been mentioned in several publications [16] and public media, but at present there is no clear understanding of the scale and the underlying processes of this phenomenon. Research priori- ties include an estimation of its global impact, the localization of natural sink areas, and the identification of high-risk anthropogenic litter items/materials and sources [17]. Rafting of biota on floating objects, like driftwood, macro algae or volcanic pumice has importantly shaped the species composition of islands [16,18,19]. Floatable litter items of anthropogenic origin greatly enhance the number of stable rafts, particularly in areas where natural vectors are scarce. Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna OPEN ACCESS Citation: Rech S, Borrell Pichs YJ, Garcı´a-Vazquez E (2018) Anthropogenic marine litter composition in coastal areas may be a predictor of potentially invasive rafting fauna. PLoS ONE 13(1): e0191859. https://doi.org/10.1371/journal.pone.0191859 Editor: Christopher A. Lepczyk, Auburn University, UNITED STATES Editor: Christopher A. Lepczyk, Auburn University, UNITED STATES Received: May 25, 2017 Accepted: January 13, 2018 Published: January 31, 2018 Copyright: © 2018 Rech 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: The FASTA sequences of the analyzed individuals are published in the GenBank database with the accession numbers KY607884-KY607909, KY614195-KY614223, KY628986, KY661434- KY661534, KY683467-KY683511, KY944812- KY944984, KY963587-KY963595, KY986731- KY986745, MF037237-MF037246, MF043915. All other relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the European Commission [Marie Curie 2014 ITN 1 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna glass surfaces were found in another study and object softness, rather than the type of material, was suggested to be an important factor for biota attachment [31]. On the other hand, labora- tory experiments and controlled field studies with fixed floaters do not incorporate the buoy- ancy or floating behaviour of the different materials, which may also influence the biotic colonization by some taxonomic groups [16,27,29,32]. The ability of items to float over long distances depends not only on their buoyancy, but also on their stability and shape, with thin- ner and more flexible plastic items (like plastic bags and packaging material) sinking faster than thicker and more robust plastic items [33]. The origin of litter could have an influence in the attached biota. Marine anthropogenic litter stems from various sources, like households, beach-based leisure activities, sea- going activities, industries, and sewage [34]. The contribution of each source to anthropo- genic litter has been investigated at many locations [9,35–37], but the main sources of lit- ter rafts with biota are less known. For particular items, macroscopic attached biota has been reported. Examples are lines, ropes, nets and bait pots [38–40], aquaculture and other buoys [39,41], plastic packaging bands used in Antarctic bases and fishing boats [42], virgin plastic pellets [43], glass bottles [39], a gas cylinder reported above [21], a plas- tic spool [40], and tennis shoes and slippers [44], amongst others. Those reports might point to a higher contribution of litter items originated from sea-based activities such as aquaculture and fisheries. However, this first impression needs to be investigated in depth and on a larger geographic scale. Floating objects displace along with currents and tides, thus their role in the dispersal of attached species may be important. Rafting on marine litter has been suggested to be involved in regional dispersal of several invertebrates [23,45,46]. For example, juveniles of the bivalve Pinctada imbricata and adults of Isognomon bicolor, which are considered inva- sive in Brazil, were found attached to anthropogenic litter for the first time at the Uru- guayan coast, where they are regarded as potentially invasive as well [38,44]. In the Spanish part of the Bay of Biscay, several alien invasive species are registered [47], some of which are already known to attach to floating anthropogenic litter in other regions [16]. The invasive pygmy mussel Xenostrobus securis was first reported in the Bay of Biscay in 2012, attached to natural as well as plastic and metal objects, among others [48]. The invasive Crassostrea gigas and the exotic Ostrea stentina were also found attached to artificial materials on regional ports [49]. According to EU Regulation (EU) No 1143/2014 there are about 12,000 alien species in European countries, of which 10–15% are regarded as invasive and pose a serious threat to the environment and human interests [50]. Such species can be regarded as ecosystem infestations or epidemics, with the anthropogenic litter carrying it, being infested vectors. Given the concern of anthropogenic beach litter our goal was to determine whether the composition of anthropogenic beach litter can predict macrobiotic communities attached to stranded litter items in a region. In answering this goal, we had three main objectives. First, determine which native, non-native, and potentially invasive macroscopic animal species are present on stranded anthropogenic litter items. Second, determine the principal material and sources of the infested vectors. Third, test if the occurrence of a certain species/ taxon can be predicted based on the general litter composition at a beach or a coastal area. H2020 AQUAINVAD-ED; grant agreement no. 642197]. Anthropogenic litter pollution is estimated to double marine rafting opportunities [16,20] and on some beaches more than 60% of all anthropogenic litter items carried attached organisms [6]. Although the vast majority of anthropogenic litter used as rafts are plastic items, there are also cases of macrobiotic rafting on glass, metal, and paper objects [16]. Notably, a metal gas cylinder encrusted by the stony coral Favia fragum had probably crossed the Atlantic Ocean from the USA to the Netherlands [21]. Another invading coral, Oculina patagonica, is commonly found on submerged metal objects [22], while some pelagic barnacles are frequently recorded on glass and metal objects [23]. Biofouling was also reported for air-filled glass floats, used in (mainly Japanese) fisheries before plastics became widely available and still afloat in the world´s oceans nowadays [21,24–26]. Differences between materials in the abundance and composition of the micro fauna in early stages of biofouling have been found [27,28]. Particularly, polystyrene seems to carry a higher number of both species and individuals than other types of plastics, which may be due to its higher surface roughness [27,29]. Settlement of individuals of the invasive species Bugula neritina was significantly higher on several plastic surfaces [Polyvinylchloride (PVC), Polypro- pylene (PP), Polycarbonate (PC), Polyethylene terephthalate (PET) and Polystyrene (PS)] than on glass surfaces, under both field and laboratory conditions, whereas the invasive barnacle Austrominius modestus settled more on glass than on plastic surfaces (tested under field condi- tions) [30]. In contrast, no significant differences between biofilm composition on PET and 2 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 Material and methods No specific permissions were required for sampling because all the organisms analysed in this study were obtained from litter items. Those items must be removed from the beaches as they are not natural substrate. The field studies did not involve endangered or protected species. 3 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna Fig 1. Map of the sampled area including waterways, national ports, fishing ports and marinas, sewage treatment plants, and principal industrial sites. Sampling sites are numbered and are specified in Table 1. https://doi.org/10.1371/journal.pone.0191859.g001 Fig 1. Map of the sampled area including waterways, national ports, fishing ports and marinas, sewage treatment plants, and principal industrial sites. Sampling sites are numbered and are specified in Table 1. https://doi.org/10.1371/journal.pone.0191859.g001 Beach litter samplings and analysis A total of fifteen sandy beaches, covering a linear distance of 190 km along the Cantabrian coastline in Asturias, Spain, were sampled in a 26-day period between February and March 2016 (Fig 1). Each beach was sampled one day during low tide and daylight. We conducted two independent surveys: 1) A sampling of fouled beached items along the whole area of each beach to test if there are material-related differences in the taxonomic composition of the macro fauna attached to beached litter, and 2) a count and material-based classification of bea- ched anthropogenic litter in general (both fouled and non-fouled) in a smaller standardized area. Please see the supporting figure for a graphic sampling scheme (S1 Fig). Survey 1: The whole area of each beach was searched for anthropogenic litter items with attached macrofauna (visible fauna). Each of the items found was photographed with a Motor- ola Moto G3 camera (resolution 13 MP) next to a size reference (a finger or any other object of known dimensions) and given an identification code. The type of object (e.g. buoy, fragment, rope; Table 2), type of material and colour was noted down for each item. We did not only classify the fouled items by material as plastic and non-plastic (e.g. metal, paper, glass; abbrevi- ated NPl), but moreover separated plastic items in three categories, based on their stability and surface roughness: Hard plastics (abbreviated HPl), synthetic foams (e.g. Polystyrene; abbrevi- ated foams), and other plastics (abbreviated OPl). Litter items found on the beaches were asso- ciated to three sources: Sewage, Fishing/Aquaculture and Household/Leisure. All objects or fragments that were not identifiable or not attributable to one of the categories above were classified as N/A (not attributable; Table 2). Attached biota was visually assigned to the most specific distinguishable taxonomic group based on morphology and the number of individuals (colonies for bryozoans and hydrozoans) was counted and noted down for each group. A representative number of individuals ( 50) of each morphotype was detached from each litter item using forceps and a scraper. They were stored in commercially available hard plastic sampling pots in 50–500 ml (depending on the size and number of stored individuals) of ethanol 80% for further analysis and labelled with the identification code of the corresponding litter item. Sampling area To address our main research goal and objectives, we evaluated the coast of Asturias region in the south-central Bay of Biscay (north of Spain). The coast is under the influence of currents going eastwards [51], with a boundary in Cape Peñas (central cape marked in Fig 1) that divides the coast into the colder west and the warmer east zone [52]. The sampling sites cover a wide spectrum of factors that may influence marine litter distribution, like land-use, distance to human settlements, industry, and geomorphology [53–55]. There are two international cargo ports in the sampled area (Gijo´n and Avile´s), as well as shellfish aquaculture areas in two estuaries (Rı´a del Eo and Villaviciosa). There are several villages and two bigger cities, Gijo´n and Avile´s, along the coastline in Spain (Fig 1). The central area of the region is strongly pol- luted by industrial activities [56,57], which are mainly based in the area of Avile´s. Among the several rivers discharging into the Cantabrian Sea in the sampling area, the rivers Nalo´n, Navia, Sella, and Esva have the largest stream basins (Fig 1). Table 1. Sampled beaches as shown in Fig 1, with geographic position. Number Beach name Longitude [˚W] Latitude [˚N] 1 Figueras -7.02 43.54 2 Penarronda -6.99 43.55 3 Navia -6.72 43.55 4 Barayo -6.62 43.56 5 Silencio -6.29 43.57 6 Bayas -6.04 43.57 7 Salinas -5.95 43.58 8 Nieva -5.94 43.59 9 Xago´ -5.92 43.60 10 Bañugues -5.81 43.63 11 Xivares -5.72 43.57 12 Rodiles -5.38 43.53 13 Sta. Marina -5.07 43.47 14 Poo -4.78 43.43 15 Andrı´n -4.71 43.41 Table 1. Sampled beaches as shown in Fig 1, with geographic position. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 4 / 22 Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna Survey 2: A standardized quantification and characterization of anthropogenic beach lit- ter (not restricted to fouled objects) was done at all beaches, except for Figueras, Silencio, and S. Juan de Nieva (for location of the beaches see Fig 1). On the other 12 beaches, of sim- ilar sandy granulation, standardized litter counts were conducted in 2 horizontal transects at every beach, each consisting of four adjoined quadrats of 3×3m2 each. The two transects were placed parallel to the water line, the upper transect along the most recognizable higher tideline, and the lower transect along the most recognizable lower tideline, to account for possible differences in litter composition with shore height [64] and to include both recently stranded litter (lower tide line) and litter stranded less recently (most recognizable high tide line). The area for the counts was defined at every beach after visual inspection, where accu- mulation of flotsam (both natural and anthropogenic) was representative of the whole beach (i.e. neither exceptionally high, nor exceptionally low, relating to the rest of the beach). This method was chosen over a random approach to avoid bias due to the small transect area (36 m2 per transect) and the limited number of replicates (two transects per beach), as anthropogenic litter and other flotsam is often distributed heterogeneously along the beach [64,65]. The sampling quadrats were defined with a tape measure and their outlines were marked in the sand using a stick. In each quadrat all macro litter (items and fragments bigger than 1.5 cm) was inspected and sorted by object type (e.g. lid, drinking straw, fragment) and material. Then the number of items of each combination of object type and material (e.g. hard plastic lids, metal lids, paper fragments; Table 2) was counted and noted down for each quadrat in situ. All items and fragments were then assigned to a source category. The mate- rial categories and source categories used for classification were the same as described above for Survey 1. Beach litter samplings and analysis Some smaller litter items and items of complex shapes were stored in plastic bags and taken to the laboratory for measurement, while the dimensions of bigger items and of items with a simple shape were estimated based on the photos, and the surface area was calculated for each item. The native distribution area and the potential invasive capacity of each attached species were examined from relevant current liter- ature [49,58–62] and databases, namely the global invasive species database (GISD, http:// www.issg.org/database) and World Register of Marine Species [63]. Table 2. Categories of beach litter sources and associated litter objects. Sewage Fishing/Aquaculture HH/Leisure N/A Cotton buds Buoys Sandals Fragments Menstrual hygiene Netfloats Cosmetics container Unknown objects products + packaging Cage nets Shoes Boxes Wet wipes Jerrycans Shoe soles Bottles Nets Cigarette stubs Buckets Ropes Lighters Lids Paper and carton Beverage crates Textiles Drinking straws HH = household, N/A = not attributable. https://doi.org/10.1371/journal.pone.0191859.t002 Table 2. Categories of beach litter sources and associated litter objects. ategories of beach litter sources and associated litter objects. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 5 / 22 Statistical analysis Analysis of rafting fauna was done at regional level after confirming large dispersal capacity of the species found. Comparison among materials for the attached biotic community was done using the number of individuals per object as a standardized unit. To compare among commu- nities we classified biota as goose barnacles, acorn barnacles, bryozoan and hydrozoan colo- nies, decapods, molluscs and polychaetes. Composition and sources of beach litter found along the main accumulation lines (from standardized samplings) were compared to composition and sources of the litter items used as rafts, employing the PERMANOVA function of PRIMER 6 software [69,70]. PERMANOVA results were regarded as statistically significant at a p-value of 0.05. The contribution of each litter source to the differences was tested by SIMPER (= similiarity percentage) analysis. Both analyses were based on Bray- Curtis similarities. The abundance of anthropogenic litter was compared between and within beaches using boxplots, showing the mean value, quartiles and variability for each beach. Hetero- geneity in composition and abundance of anthropogenic beach litter in general, and of items used as artificial rafts by biota, were tested using PERMANOVA, based on Euclidean distances. Multidimensional scaling (MDS) based on Bray-Curtis similarities was used to graphically represent the grouping of the sampled beaches, based on dominant litter mate- rial: beaches dominated by hard plastics (termed HPl–dominant), beaches dominated by other plastics (termed OPl-dominant), and beaches with mixed litter composition and less than 25 litter items in the standardized sampling area (> 0.35 items×m2; termed Mix). These analyses were done for the subsample of beaches where standardized litter analysis was carried out. Since litter composition and litter with rafting biota in a beach were independent datasets, a correlation approach was followed to determine if rafting biota in a beach area can be inferred from litter composition. Biota expectation from litter composition was estimated for 12 beaches based on the characteristic community profile of the beaches’ litter materials. The goodness of adjustment between estimated and observed taxa was tested using a correlation approach, based on Spearman´s rank correlation coefficient and the linear correlation was graphically illustrated in a scatter plot. Genetic barcoding DNA was extracted from a small piece of tissue (about 2×2 mm) using Chelex (Bio Rad BT Chelex1 100 Resin). For DNA extraction from very small individuals with non-tissue parts, like shells (e.g., molluscs), the complete individual was treated with E.Z.N.A1 Mol- lusc DNA Kit. PCRs were performed with the universal primers detailed in Table 3. When necessary, the PCR product was purified using EURx1 Gene Matrix Agarose Out DNA Purification Kit. DNA sequencing was performed by Macrogen Europe, Amsterdam, Netherlands. Sequence editing and alignment was done using the freeware BIOEDIT Version 7.2.5 [66]. From the DNA Barcode the species was assigned using the BLAST database [67] and the best match with the maximum hit score (minimum 97% nucleotide identity). Phylogenetic trees for confirming species assignation were built with MEGA 7 [68] from the sequences obtained in this study and reference sequences of voucher specimens taken from GenBank (https:// www.ncbi.nlm.nih.gov/nucleotide/), based on the maximum likelihood reconstruction method, with 500 bootstraps. Table 3. Primers used for DNA amplification in different taxa. Taxon Primers Sequence Molluscs, Arthropods jgLCO1490 jgHCO2198 5’TITCIACIAAYCAYAARGAYATTGG3’ 5’TAIACYTCIGGRTGICCRAARAAYCA3' Polychaetes 18s EukF 18s EukR 5’WAYCTGGTTGATCCTGCCAGT3’ 5’TGATCCTTCYGCAGGTTCACCTAC3’ Bryozoans Hydrozoans 16s HF 16s HR 5’ATAACACGAGAAGACCCT3’ 5’CCCRCGGTCGCCCCAAC3’ https://doi.org/10.1371/journal.pone.0191859.t003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 6 / 22 Table 3. Primers used for DNA amplification in different taxa. 6 / 22 Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna Statistical analysis We calculated the expected number of individuals by taxa at each of the twelve beaches as: TBðxÞ ¼ Pn i¼1fMðiÞ fTBMði; rÞ NtðxÞ ð1Þ ð1Þ TBðxÞ ¼ Pn i¼1fMðiÞ fTBMði; rÞ NtðxÞ Where TB (x) is the expected number of individuals for taxon B on beach x, fM(i) is the fre- quency of litter material i (HPI, OPI, Foams or NPI) found on beach x, fTBM (i, r) is the fre- quency of taxon B on material i in the region r and Nt (x) is the total number of rafting biota found on beach x. Where TB (x) is the expected number of individuals for taxon B on beach x, fM(i) is the fre- quency of litter material i (HPI, OPI, Foams or NPI) found on beach x, fTBM (i, r) is the fre- quency of taxon B on material i in the region r and Nt (x) is the total number of rafting biota found on beach x. Standardized quantification and categorization of anthropogenic beach litter All the sampled beaches were polluted with anthropogenic litter. The mean abundance of anthropogenic litter ranged from 0.17 ± 0.21 items×m-2 (Barayo) to 5 ± 3.95 items×m-2 (Xivares). The abundance of anthropogenic litter varied strongly, not only between beaches, but also between quadrats within beaches, indicating a patchy distribution (Fig 2). The compo- sition of beached litter in the region was not significantly different of the composition of litter rafts with biota (Table 4: PERMANOVA 1). 7 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna Fig 2. Abundance of anthropogenic litter, counted in a standardized area at the sampled beaches. Data are presented in a box-and whisker plot, with the middle box representing 50% of the values and the upper and lower whiskers representing the values outside of the 50% range. The median and outliers are indicated by a middle line and a circle (◦), respectively. Litter items were counted in a standardized area at each beach. h //d i /10 1371/j l 0191859 002 Fig 2. Abundance of anthropogenic litter, counted in a standardized area at the sampled beaches. Data are presented in a box-and whisker plot, with the middle box representing 50% of the values and the upper and lower whiskers representing the values outside of the 50% range. The median and outliers are indicated by a middle line and a circle (◦), respectively. Litter items were counted in a standardized area at each beach. Fig 2. Abundance of anthropogenic litter, counted in a standardized area at the sampled beaches. Data are presented in a box-and whisker plot, with the middle box representing 50% of the values and the upper and lower whiskers representing the values outside of the 50% range. The median and outliers are indicated by a middle line and a circle (◦), respectively. Litter items were counted in a standardized area at each beach. Fig 2. Abundance of anthropogenic litter, counted in a standardized area at the sampled beaches. Data are presented in a box-and whisker plot, with the middle box representing 50% of the values and the upper and lower whiskers representing the values outside of the 50% range. The median and outliers are indicated by a middle line and a circle (◦), respectively. Litter items were counted in a standardized area at each beach. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna Table 4. Detailed results of PERMANOVA analyses. PERMANOVA Variable Factor df SS MS Pseudo-F P(perm) Unique perms 1 Material composition General litter vs Rafts 1 809.71 809.71 2.6639 0.073 998 Residuals 22 6687 303.96 Total 23 7496.7 2 Litter abundance, Beaches 11 382.66 34.788 8.7906 0.001 998 composition Residuals 84 332.42 3.9574 Total 95 715.08 3 Litter abundance, Beach groups 2 224.28 112.14 21.2500 0.001 999 composition Residuals 93 490.8 5.2774 Total 95 715.08 4 Litter source General litter vs Rafts 1 5573.5 5573.5 6.1282 0.003 995 Residuals 22 20009 909.49 Total 23 25582 5 Attached biota Raft material 3 30743 10248 2.7185 0.001 998 Residuals 87 3.2795E5 3769.5 Total 90 3.5869E5 Df = degrees of freedom, SS = sum of squares, MS = mean sum of squares, Pseudo-F = F value by permutation, perm = permutation. htt //d i /10 1371/j l 0191859 t004 Table 4. Detailed results of PERMANOVA analyses. Df = degrees of freedom, SS = sum of squares, MS = mean sum of squares, Pseudo-F = F value by permutation, perm = permutation. Plastics (including foams) made up the highest share of anthropogenic litter on all beaches (75% to 100%), except at Andrı´n beach, where non-plastic litter was more abundant (55%; Table 5). The sampled beaches differed significantly from each other regarding both abun- dance and composition of anthropogenic litter (Table 4: PERMANOVA 2). Beaches were clas- sified based on the prevalent litter material, forming three groups in the sampling area that significantly differed from each other (Table 4: PERMANOVA 3) and could be graphically dis- tinguished by multidimensional scaling (MDS; Fig 3). The treatment of beaches in categories facilitated further analyses. Most anthropogenic litter items found on the sampled beaches could not be attributed to a source, as many of them were small fragments. For the objects that could be likely assigned to a source, most were sewage-related. At Xago´ and Penarronda fishing and aquaculture activities were also important sources of beached litter (Table 5). Standardized quantification and categorization of anthropogenic beach litter The highest pollution levels were found in direct proximity to the coastal region´s main industrial and populational centers, Gijo´n (Xivares beach: 5 ± 3.95 items×m-2) and Avile´s (Salinas and Xago´ beaches: 2 ± 1 items×m-2 and 2.7 ± 1.9 items×m-2, respectively) both of which have a national port and a sewage treatment plant, as well as at the river mouth of the Navia river, in proximity to a fishing port and a marina (Navia beach: 4.3 ± 4 items×m-2, see map in Fig 1). The abundance of beach litter at the other sampled beaches along the Canta- brian coastline seems to reflect the geomorphology of the coastline and its exposure to the pre- vailing eastward surface current, with a maximum peak in the northernmost Cape Peñas: Pollution rose from Barayo eastwards up to Xago´, situated on the western side of Cape Peñas, which is more exposed to the eastward surface current, and subsequently declined on the east- ern side of the cape, which is more protected from the prevailing currents (Fig 1, Fig 2). 8 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna Table 5. Composition and likely source of anthropogenic beach litter from standardized beach litter counts (in white, at left), and fouled litter items along the whole beach area (in grey, at right). Anthropogenic beach litter (from standardized sampling in quadrats) Fouled items (from whole beach area) MATERIAL [%] SOURCE [%] MATERIAL [%] SOURCE [%] Beach group Beach Litter [items m-2] HPl OPl Foam NPl ∑Pl S F HH N/A Fouled vectors [total] HPl OPl Foam NPl ∑Pl S F HH N/A Mix Andrı´n 0.31 23 5 18 55 45 0 0 0 100 5 40 40 20 0 100 0 20 60 20 Mix Sta. Marina 0.22 44 6 31 19 81 6 0 13 81 2 50 50 0 0 100 0 0 50 50 Mix Barayo 0.17 58 17 0 25 75 0 8 8 83 5 40 20 40 0 100 0 40 0 60 OPl-dom Xago´ 2.68 18 74 8 0 100 34 24 0 41 4 75 0 25 0 100 0 75 0 25 OPl-dom Penarronda 1.25 43 54 2 0 100 3 42 0 54 20 75 10 10 5 95 0 25 10 65 OPl-dom Bayas 1.15 41 45 10 5 95 8 4 2 86 7 71 14 14 0 100 0 33 0 67 OPl-dom Navia 4.25 26 68 1 6 94 17 5 6 72 1 100 0 0 0 100 0 100 0 0 HPl-dom Salinas 1.50 80 11 7 2 98 13 3 3 81 25 48 20 20 12 88 0 4 12 84 HPl-dom Xivares 5.00 89 3 8 1 99 18 2 0 80 4 75 25 0 0 100 0 25 25 50 HPl-dom Bañugues 1.03 72 14 12 3 97 9 7 5 78 2 100 0 0 0 100 0 0 0 100 HPl-dom Rodiles 0.47 71 21 6 3 97 9 6 3 82 8 88 0 13 0 100 0 75 0 25 HPl-dom Poo 0.50 78 11 8 3 97 19 3 6 72 1 100 0 0 0 100 0 0 0 100 X Silencio x x x x x x x x x x 4 100 0 0 0 100 0 25 0 75 X Nieva x x x x x x x x x x 1 100 0 0 0 100 0 0 0 100 X Figueras x x x x x x x x x x 5 0 80 0 20 80 0 0 20 80 MEAN 54 27 9 10 90 11 9 4 76 71 17 9 2 98 0 28 12 60 ST. DEV. 24 26 8 16 16 10 12 4 15 30 24 12 6 6 0 32 19 32 x = no data available. dom = dominant, HPl = Hard plastics, OPl = Other plastics, NPl = Nonplastic, S = Sewage, F = Fishing and aquaculture, HH = Household and leisure, N/A = Not attributable, ST. DEV = Standard deviation. https://doi.org/10.1371/journal.pone.0191859.t005 particularly important role of fishing/aquaculture related litter for the rafting of biota in the sampling area was especially noticeable at the beaches of Xago´, Navia and Rodiles, where all the identifiable items with attached biota were from this source (Table 5). Leisure and house- hold-related items also had a high share in rafting vectors. Items from this source were found on six beaches and consisted of 20 shoes/sandals and one cosmetic container. Leisure and household was the main litter source for Andrı´n beach (Table 5). On the other hand, sewage- related litter made up to 11% (mean) of all anthropogenic beach litter, although none of the biota rafts was related to this source (Tables 5 and 6). Anthropogenic litter items used as rafts A total of 94 litter objects with attached fauna were found on the surveyed beaches (Fig 4). High prevalence of hard plastics and plastics in general (71 ± 30% and 98 ± 6%, respectively), was found among rafting vectors, while the share of non-plastic objects was very low (2 ± 6%, Table 5). In fact, only five non-plastic objects with attached fauna were found on three beaches: three glass bottles (one with a metal cap), one piece of processed wood, and one sandal, which was counted as nonplastic as the attached organism was found on its textile part. Within the plastics the share of other plastics tended to be less abundant in rafting vectors than in general beach litter (17 ± 24% versus 27 ± 26%), while the share of foams was rather similar in rafting vectors and general litter (9 ± 12% and 9 ± 8%, respectively). The standard deviation between beaches however was high (Table 5). The main sources of fouled litter items were significantly different from the main sources of other non-fouled beach litter (Table 5, Table 4: PERMANOVA 4). SIMPER showed that the source category with the highest contribution to the differences (after unidentified litter NA, contribution: 37%) was Fishing and Aquaculture (contribution: 34%; Table 6). This PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 9 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 https://doi.org/10.1371/journal.pone.0191859.t005 Fauna attached to anthropogenic rafts More than 3300 individuals (or colonies for bryozoans and hydrozoans) were found attached to the litter objects found in the beaches surveyed (Table 7). With genetic analyses, more than 400 DNA barcodes were obtained, identifying 23 species of attached animals from four phyla (Fig 5, Table 7). The Barcodes were submitted to GenBank database, where they are available with the Accession Numbers KY607884-KY607909, KY614195-KY614223, KY628986, KY661434-KY661534, KY683467-KY683511, KY944812-KY944984, KY963587-KY963595, KY986731-KY986745, MF037237-MF037246, MF043915. Crustaceans (Phylum Arthropoda) such as Lepadidae (Goose barnacles), Balanidae and Verrucidae (Barnacles), and the amphi- pod Caprella andreae were the most abundant animals in this study (> 1000 individuals; Table 7), followed by annelids, which all belonged to the family Serpulidae (~700 individuals). PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 10 / 22 Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna Fig 3. Multi-dimensional plot of the sampled beaches, based on abundance and composition of anthropogenic litter counted in a standardized area at each beach. HPl = hard plastics, OPl = Other plastics, Mix = beaches with mixed litter composition and less than 25 litter items in the standardized sampling area (> 0.35 items×m2). https //doi o g/10 1371/jo nal pone 0191859 g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna Table 6. Contributions of several litter sources to the differences between the general beach litter counted in a standardized area (group General) and litter used as biota raft (group Rafts), calculated by SIMPER analysis. Groups General & Rafts Average dissimilarity = 44.61 General Rafts Litter source Abundance Abundance Dissimilarity Diss. / SD Contribution [%] Cumulative [%] Not identified 75.83 53.83 16.67 1.45 37.37 37.37 Fishing/Aquaculture 8.67 33.08 15.36 1.06 34.44 71.81 HH / Leisure 3.83 13.08 6.90 0.77 15.47 87.28 Sewage 11.33 0.00 5.68 1.22 12.72 100.00 HH = household, Diss. = Dissimilarity, SD = standard deviation https://doi.org/10.1371/journal.pone.0191859.t006 Table 7. Overview of species attached to stranded litter, identified in the present study. Visual identification N Phylum/ Subphylum Class Order Family Genetic identification Barcodes Geographic origin Goose barnacles 676 Arthropoda/ Crustacea Maxillopoda Pedunculata Lepadidae Lepas anatifera 170 COS Lepas anserifera 2 Lepas pectinata 44 Dosima fascicularis 3 Acorn barnacles 308 Arthropoda/ Crustacea Maxillopoda Sessilia Balanidae Austrominius modestus 57 Australia, NZ Chthamalus stellatus 30 NAT Chthamalus montagui 26 Balanidae sp., (Perforatus perforatus) 2 Verrucidae Verruca stroemia 4 Caprellids 75 Arthropoda/ Crustacea Malacostraca Amphipoda Caprellidae Caprella andreae 10 COS ∑ARTHROPODS 1059 Mytilidae 70 Mollusca Bivalvia Mytiloida Mytilidae Mytilus edulis 5 NAT Mytilus galloprovincialis 1 Mytilus sp. 10 x Ostreidae 21 Mollusca Bivalvia Ostreoida Ostreidae Crassostrea gigas 16 NE-Pacific Ostrea stentina 1 S-Atlantic, Med Gastropods 2 Mollusca Gastropoda x Trochidae Gibbula umbilicalis 2 NAT ∑MARINE MOLLUSCS 93 Polychaetes 699 Annelida Polychaeta Canalipalpata Serpulidae Spirobranchus triqueter 3 NAT Spirobranchus taeniatus 17 Serpula columbiana 1 N-Pacific Neodexiospira sp. 1 S-Atlantic Spirobranchus sp. 3 x ∑ANNELIDS 699 Hydrozoan and Bryozoan colonies 396 Cnidaria Hydrozoa Anthoathecata Bougainvilliidae Bougainvillia muscus 1 NAT Leptomedusae Campanulariidae Obelia dichotoma 1 COS Table 6. Contributions of several litter sources to the differences between the general beach litter counted in a standardized area (group General) and litter used as biota raft (group Rafts), calculated by SIMPER analysis. Table 6. Contributions of several litter sources to the differences between the general beach litter counted in a standardized area (group General) and litter used as biota raft (group Rafts), calculated by SIMPER analysis. https://doi.org/10.1371/journal.pone.0191859.t006 https://doi.org/10.1371/journal.pone.0191859.t006 Table 7. Overview of species attached to stranded litter, identified in the present study. Fig 3. Multi-dimensional plot of the sampled beaches, based on abundance and composition of anthropogenic litter counted in a standardized area at each beach. HPl = hard plastics, OPl = Other plastics, Mix = beaches with mixed litter composition and less than 25 litter items in the standardized sampling area (> 0.35 items×m2). https://doi org/10 1371/journal pone 0191859 g003 Fig 3. Multi-dimensional plot of the sampled beaches, based on abundance and composition of anthropogenic litter co HPl = hard plastics, OPl = Other plastics, Mix = beaches with mixed litter composition and less than 25 litter items in the st Fig 3. Multi-dimensional plot of the sampled beaches, based on abundance and composition of anthropogenic litter counted in a standardized area at each beach. HPl = hard plastics, OPl = Other plastics, Mix = beaches with mixed litter composition and less than 25 litter items in the standardized sampling area (> 0.35 items×m2). https://doi.org/10.1371/journal.pone.0191859.g003 https://doi.org/10.1371/journal.pone.0191859.g003 Hydrozoan and bryozoan colonies were also very numerous (~400) and might be underesti- mated in this study, due to the difficulty of counting them individually. As most of the colonies were dried out and in a state of advanced degradation, DNA was degraded in most cases and Fig 4. Examples of fouled litter items. a) Hard plastic object with oyster, polychaetes and acorn barnacles b) PET bottle with goose barnacles c) float of fishing net with bryozoan colonies and polychaetes, d) shoe sole with oyster, snail and acorn barnacles, e) duct tape with goose barnacles. Fig 4. Examples of fouled litter items. a) Hard plastic object with oyster, polychaetes and acorn barnacles b) PET bottle with goose barnacles c) float of fishing net with bryozoan colonies and polychaetes, d) shoe sole with oyster, snail and acorn barnacles, e) duct tape with goose barnacles. https://doi.org/10.1371/journal.pone.0191859.g004 https://doi.org/10.1371/journal.pone.0191859.g004 https://doi.org/10.1371/journal.pone.0191859.g004 11 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 Visual identification N Phylum/ Subphylum Class Order Family Genetic identification Barcodes Geographic origin Goose barnacles 676 Arthropoda/ Crustacea Maxillopoda Pedunculata Lepadidae Lepas anatifera 170 COS Lepas anserifera 2 Lepas pectinata 44 Dosima fascicularis 3 Acorn barnacles 308 Arthropoda/ Crustacea Maxillopoda Sessilia Balanidae Austrominius modestus 57 Australia, NZ Chthamalus stellatus 30 NAT Chthamalus montagui 26 Balanidae sp., (Perforatus perforatus) 2 Verrucidae Verruca stroemia 4 Caprellids 75 Arthropoda/ Crustacea Malacostraca Amphipoda Caprellidae Caprella andreae 10 COS ∑ARTHROPODS 1059 Mytilidae 70 Mollusca Bivalvia Mytiloida Mytilidae Mytilus edulis 5 NAT Mytilus galloprovincialis 1 Mytilus sp. 10 x Ostreidae 21 Mollusca Bivalvia Ostreoida Ostreidae Crassostrea gigas 16 NE-Pacific Ostrea stentina 1 S-Atlantic, Med Gastropods 2 Mollusca Gastropoda x Trochidae Gibbula umbilicalis 2 NAT ∑MARINE MOLLUSCS 93 Polychaetes 699 Annelida Polychaeta Canalipalpata Serpulidae Spirobranchus triqueter 3 NAT Spirobranchus taeniatus 17 Serpula columbiana 1 N-Pacific Neodexiospira sp. 1 S-Atlantic Spirobranchus sp. 3 x ∑ANNELIDS 699 Hydrozoan and Bryozoan colonies 396 Cnidaria Hydrozoa Anthoathecata Bougainvilliidae Bougainvillia muscus 1 NAT Leptomedusae Campanulariidae Obelia dichotoma 1 COS ∑HYDROZOANS + BRYOZOANS 396 Gastropod, terrestrial 4 Mollusca Gastropoda x Helicidae Helix aspersa aspersa 4 NAT N = total number of individuals found, NAT = native, COS = cosmopolitan distribution, N = North, S = South, Med = Mediterranean sea. Non-native species are marked by bold writing. = Species (both native and non-native to study area) listed in the global invasive species database (GISD, http://www.issg.org/database). https://doi.org/10.1371/journal.pone.0191859.t007 Table 7. Overview of species attached to stranded litter, identified in the present study. 12 / 22 Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna Fig 5. Phylogenetic trees reconstructed from sequences obtained in this study and reference sequences from GenBank database (bold style). a) molluscs, b) crustaceans, c) polychaetes, d) hydrozoans. Frame = Species not native to the study area; = Species listed in the invasive species database; + = Terrestrial species; # = reference without species voucher. https://doi org/10 1371/journal pone 0191859 g005 Fig 5. Phylogenetic trees reconstructed from sequences obtained in this study and reference sequences from GenBank database (bold style). a) molluscs, b) crustaceans, c) polychaetes, d) hydrozoans. Frame = Species not native to the study area; = Species listed in the invasive species database; + = Terrestrial species; # = reference without species voucher. https://doi.org/10.1371/journal.pone.0191859.g005 https://doi.org/10.1371/journal.pone.0191859.g005 only two species of Cnidarians were identified from genetic techniques: Bougainvillia muscus and Obelia dichotoma. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 On other plastics, the main share of attached biota was made up of polychaetes (66%) and goose barnacles (23%), while barnacles, bryozoan colonies, and molluscs were less common (5%, 5%, 13 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna Fig 6. Particular profile of attached biota for each litter material. Fig 6. Particular profile of attached biota for each litter material. https://doi.org/10.1371/journal.pone.0191859.g006 and 2%, respectively). Differences between materials regarding the biota profile were indeed highly statistically significant (Table 4: PERMANOVA 5). and 2%, respectively). Differences between materials regarding the biota profile were indeed highly statistically significant (Table 4: PERMANOVA 5). Inference of litter-related biotic community from beach litter composition We tested if the composition of an area´s macrobiotic communities attached to stranded litter items can be predicted based on its composition of anthropogenic beach litter, using the data of the 12 beaches where standardized litter counts have been conducted. The predicted fre- quency of attached biota of several taxa, estimated from litter composition significantly corre- lated with the actually observed frequencies on both sides of cape Peñas (Western side: Spearman‘s rank correlation coefficient (R) = 0.498; p = 0.002; Eastern side: R = 0.629; p = 0.027), as well as for the whole sampling area (R = 0.565; p < 0.001; Fig 7). For the exact fig- ures of estimated and observed biota, please see the Supporting table (S1 Table). The animals found in the present study were morphologically diverse and it is possible that the hydrozoan and bryozoan colony group actually included more spe- cies and taxa. Around 100 molluscs were found attached to anthropogenic litter items, with the majority of them belonging to the genus Mytilus, followed by the oysters Crassostrea gigas and Ostrea stentina. Moreover, we found two species of gastropods: the marine species Gibbula umbilicalis, and the land snail Helix aspersa. For the latter, which is terrestrial, taking into account its common occurrence in the sampled area, it seems likely it did not arrive on the beach by rafting but from the land. Most of the rafting animals were native to the study region or recognized as cosmopolitans (Lepadidae). Five species were not native: Crassostrea gigas, Ostrea stentina, Austrominius mod- estus, Serpula columbiana, and Neodexiospira sp. C. gigas and A. modestus are listed in the global invasive species database (GISD, http://www.issg.org/database). The native M. gallopro- vincialis and the terrestrial species H. aspersa are included in GISD as well. The species identifi- cation provided by BLAST was confirmed from phylogenetic analysis after clustering analyses including voucher species references from GenBank (Fig 5). Regarding the type of material carrying each species, differences occurred in this region between taxonomic groups. While molluscs like Mytilus and Crassostrea were found on all types of anthropogenic litter, Polychaetes were exclusively found on hard plastic and other plastic items. Barnacles, like Austrominius, were found on all materials except foams, but were most important on hard plastic items. Therefore, each type of litter seemed to exhibit a particu- lar profile of attached biota (Fig 6). Foams carried almost exclusively goose barnacles (99%) and, to a much lesser extent, molluscs (1%). Non-plastic items contained a similar biota pro- file, with an additional small share of barnacles (2%). Hard plastic and other plastic objects on the other hand carried a broad spectrum of attached taxa. On hard plastic items the main share of attached biota were barnacles (37%), polychaetes (31%) and bryozoan colonies (18%). They also carried goose barnacles, molluscs, and decapods (7%, 4%, and 2%, respectively). Discussion In this study six rafting species were recorded for the first time on anthropogenic beach litter: Verruca stroemia, Ostrea stentina, Gibbula umbilicalis, Spirobranchus taeniata, Serpula columbiana, and Neodexiospira sp. Although many rafting species have been documented on anthropogenic marine litter during the last years [16] and the recent discovery of 289 living 14 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna Fig 7. Correlation between expected and observed numbers of individuals of several taxa, calculated from data of 12 Asturian beaches. https://doi.org/10.1371/journal.pone.0191859.g007 Fig 7. Correlation between expected and observed numbers of individuals of several taxa, calculated from data of 12 Asturian beaches. https://doi.org/10.1371/journal.pone.0191859.g007 marine species, which had crossed the Pacific Ocean on objects detached by a tsunami, showed the importance of floating marine litter as a rafting vector [71], many rafting species are not known or reported yet and knowledge of the actual dimension and impact of marine litter raft- ing is still far from complete. The finding of Perforatus perforatus on anthropogenic litter is particularly interesting, as large numbers of this species, probably originating from NW Spain, have been found on beach litter in Wales [72]. A similar range expansion might also occur for invasive barnacles, such as Austrominius modestus. marine species, which had crossed the Pacific Ocean on objects detached by a tsunami, showed the importance of floating marine litter as a rafting vector [71], many rafting species are not known or reported yet and knowledge of the actual dimension and impact of marine litter raft- ing is still far from complete. The finding of Perforatus perforatus on anthropogenic litter is particularly interesting, as large numbers of this species, probably originating from NW Spain, have been found on beach litter in Wales [72]. A similar range expansion might also occur for invasive barnacles, such as Austrominius modestus. Besides the species listed above, most of the taxa found in our study are known rafters and have already been found on anthropogenic litter (floating or stranded) in other regions [16]. The predominance of cosmopolitan stalked barnacles among marine rafters is a common phe- nomenon, with the small and light-weight species L. pectinata and D. fascicularis being espe- cially suited for the colonization of smaller rafts [16,23]. Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna port of Avile´s [49]. These two findings with a linear distance of less than 100 km may indicate that this species is already established in the region, and may use anthropogenic litter for dis- persal beyond the range of its propagules. The results show a link between the composition of anthropogenic beach litter in an area and the frequency of several taxa of fauna attached to stranded litter objects. This finding should be valid for a broad range of coastal regions, as it is based on taxa composition and general litter materials, rather than on particular species and/ or litter items, which may vary more strongly between regions. , y y g y g The strong prevalence of (hard) plastic rafts confirms the results of previous studies [73]. The very low share of non-plastic rafts may be due to the fact that the majority of these items are not buoyant and/or of very little persistence. Plastic foams, despite being highly buoyant and having rather rough surfaces, which facilitate initial colonization [16], are less stable and persistent than hard plastics [29]. This may explain their low share amongst rafting vectors. For the potential sources of litter with rafting biota, there was a high share of unidentified items but still some important conclusions may be drawn from our results. Firstly, rafting vec- tors could be identified and attributed to a source much more frequently than other items of anthropogenic beach litter. The reason is probably that small plastic fragments whose source cannot be identified, which are quite common in beach litter in general, are too small for serv- ing as rafts. Fazey and Ryan (2016) proposed size and buoyancy as predictors of dispersal dis- tance for floating debris [74]. Given that biofouling reduces an item´s buoyancy, smaller items will sink faster than bigger items and travel much smaller distances [75]. This phenomenon may also explain why sewage litter, although quite abundant on beaches, was never found as a rafting vector. Rafting vectors from fishing and aquaculture, as well as other sea-based activi- ties, have been reported in other studies [41,76]. An explanation for the high occurrence of items from these sources among rafts may be their buoyancy, stability, size and persistence. Discussion Lepas barnacles may influence the rafting community on plastic debris: the ratio Lepas cover /surface area was found positively correlated with the diversity of mobile rafters, while negatively with sessile rafters’ diversity, in a study by Gil and Pfaller (2016). Our results were concordant with this study, since the debris dominated by goose barnacles contained a very low diversity of other sessile rafting species (only molluscs and acorn barnacles), while materials with a lower share of goose barnacles exhibited a relatively diverse attached community (Fig 6). Another common rafter found in this study was the amphipod Caprella andreae. The genus Caprella is generally adapted to raft- ing because of their reduced abdominal appendages, and C. andreae is the only known obligate rafter in its genus [60]. Two non-native oysters were found on Figueras beach, close to the region´s only active site of mollusc aquaculture. While C. gigas is a recognized invasive species and quite common along the Asturian coast, O. stentina has only been reported in the region once before, in the PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 15 / 22 Anthropogenic marine litter composition in coastal areas as a predictor of potentially invasive rafting fauna was a difficult task because the majority of items could not be clearly related to a litter cate- gory, either because the item could stem from several sources, or because the item was not identifiable (i.e. fragments). Notwithstanding it, our results indicate that sewage-related lit- ter is a problem in the sampled area. In fact, waste-water discharging pipelines and accumu- lations of preproduction pellets in the sand below such pipelines were noted on several of the sampled beaches (personal observation SR), but did not enter in the present study due to their small size. Fishing and aquaculture have also been identified as important litter sources in the sampling area. This finding is consistent with the fact that pollution by lost or discarded fishing gear is a common problem in the world’s seas (including the benthos) and on beaches [37,82–84]. There is a high activity of small-scale fishery, with 19 fishing ports along Asturias coastline and a large area of fishing grounds near- and off-shore, plus one active site of mollusc aquaculture (mainly oysters) near Figueras, and several crustacean ponds (http://www.sigmarinoasturias.es/). The exposure to the prevalent currents may make the sampling area a sink for anthropo- genic floating litter and attached biota from other areas. In fall and winter, the sampling area is dominated by a warm poleward surface current, referred to as ‘Navidad’, which enters near Cape Finisterre and moves eastward along the Cantabrian shelf and slope [51]. As the sam- plings presented in this study were conducted from mid-February to mid-March, it could be assumed that the overall accumulation pattern, particularly the increase of litter abundances from more western beaches towards the tip of Cape Peñas, was driven by this current. On the eastern side of cape Peñas, sediments are transported from the coastal currents to the beaches [85]. This transport may explain the observed abundances of litter on these beaches, which are not directly exposed to the prevalent current. Apart from this main driver, there seems to be an effect of rivers in the area, contributing to the high litter abundance on the beaches Navia and Xivares. Both are situated at the mouth of rivers (Rio Navia and Rio Aboño, respectively). Riverine influence was also reflected in the relatively high share of sewage-linked litter on both beaches. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 12 of the 23 fishing/aquaculture-related rafting vectors were buoys or netfloats, which are obvi- ously highly buoyant and seven were grids or cages made from stable plastic wire, which are big items with a rather small surface/volume ratio. The other four rafts were rather big items (min. 10x2x2 cm3) made from hard plastics. Leisure and household-related litter is quite diffi- cult to define, because many of the items which might stem from this source might as well stem from sea-based sources (e.g. PET bottles). These items have not been assigned to a source category, so perhaps the actual contribution of this source was higher. Shoes and sandals, clearly sourced household or leisure, are known to be able to float over large distances and have already been reported as rafting vectors [44,77–79]. The patchy abundance of beach litter, with high variances both within and between beaches was congruent with the situation reported in many other studies [7,9,80]. Although compari- sons of abundance between different locations, observers, and studies with different approaches (regarding for example transect size, choice of strand lines and/or ground between strand lines sampled, minimum size of items counted, biological material present in the sampled area etc.) are rather difficult [7,65,81], the abundance of beach litter found in this study falls within the same range as reported for many other sampling sites around the globe. As this study focuses on stranded litter which had already been at sea, the litter counts were conducted in transects targeting tidelines, where natural and anthropogenic litter is deposited by the sea. Targeting areas of litter accumulations, the results are likely overestimating the total litter abundances of the sampled beaches, and are not representative for the whole area of the beaches. They do how- ever allow for comparisons of stranded litter abundances between the beaches sampled during this study, where the same method was used for all beaches. Plastics (including plastic foams) are reported as the main constituents of beach litter in most studies [7]. According with that, the share of plastics found on beaches along the Can- tabrian coast (present study) was rather high. Source attribution of the stranded litter items PLOS ONE \| https://doi.org/10.1371/journal.pone.0191859 January 31, 2018 16 / 22 Author Contributions Conceptualization: Sabine Rech, Yaisel J. Borrell Pichs, Eva Garcı´a-Vazquez. Data curation: Sabine Rech. Formal analysis: Sabine Rech, Yaisel J. Borrell Pichs, Eva Garcı´a-Vazquez. Project administration: Yaisel J. Borrell Pichs, Eva Garcı´a-Vazquez. Resources: Yaisel J. Borrell Pichs, Eva Garcı´a-Vazquez. Supervision: Yaisel J. Borrell Pichs, Eva Garcı´a-Vazquez. Visualization: Sabine Rech. Writing – original draft: Sabine Rech. Writing – review & editing: Sabine Rech, Yaisel J. Borrell Pichs, Eva Garcı´a-Vazquez. S1 Table. Expected vs. observed numbers of each taxon of the community attached to stranded litter. S1 Table. Expected vs. observed numbers of each taxon of the community attached to stranded litter. (DOCX) S1 Table. Expected vs. observed numbers of each taxon of the community attached to stranded litter. Although the present study clearly showed the relation between anthropogenic beach litter composition and attached fouling biota in a coastal area, it had some limitations. The sam- plings were restricted to one geographic area (the south-central Bay of Biscay) and season (feb- ruary to march), and each beach was sampled only once. Moreover, our study concentrated on stranded anthropogenic litter and did not include litter which was still floating in the water. Thereby we ensured to sample only taxa/species which are still present after a beaching event and might therefore pose a risk of invasion. On the other hand, it should be considered that the biota found on beach litter in this study probably do not represent the complete macrobi- otic rafting community of the respective items before the beaching event, as beached litter is often biased towards sessile biota [16]. In summary, the results presented here give several important insights in the mecha- nisms on biota rafting on anthropogenic marine litter. Plastic items, except for foams, house a much more diverse biota community than non-plastic items and foams, which may be due to their stability and buoyancy. Several non-native and invasive species were present on litter items along the sampled beaches. 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https://openalex.org/W4388323108	https://masujournal.org/store_file/archive/49-9-9-293-298.pdf	Maltese	null	Influence of long term manuring on cation exchange properties of soils	Madras Agricultural Journal	1,962	cc-by	2	https://doi.org/10.29321/MAJ.10.A03991 https://doi.org/10.29321/MAJ.10.A03991
https://openalex.org/W4387882199	https://journal.binus.ac.id/index.php/EMACS/article/download/10602/4925	English	null	Predictive Modeling of Jakarta's Social Cohesion: GBDT Leads Comparative Analysis	Engineering, Mathematics and Computer Science Journal (EMACS)	2,023	cc-by-sa	3,839	I. INTRODUCTION Abstract – In this study, we address the challenge of predicting the Social Cohesion Index in Jakarta through a comprehensive analysis of machine learning models. Finding the most accurate and effective predictive model for this crucial urban evaluation task is the primary goal of our research. We use a variety of machine learning algorithms, comparing their performance using metrics like Mean Absolute Error (MAE), Root Mean Square Error (RMSE), Mean Absolute Percentage Error (MAPE), and computational cost. These algorithms include Gradient Boosted Decision Trees (GBDT), Polynomial Regression, Random Forest, Support Vector Machine (SVM), and Multi-Layer Perceptron (MLP). It should be noted that GBDT stands out as a top performer, regularly displaying outstanding accuracy with a competitive MAE of 0.692, RMSE of 0.887, and MAPE of 25.59%. The computational efficiency of GBDT is also impressive, with predictions taking only 0.05 seconds. These results underscore the potential of GBDT as a practical and precise tool for real-time assessments of social cohesion in large urban environments like Jakarta. The findings offer a data-driven way to guide policy decisions and community development activities, with important implications for urban planning and governance. Overall, this research emphasizes the promise of GBDT in boosting social cohesion evaluation approaches and increases our understanding of the application of machine learning in addressing complex urban difficulties. Social cohesion is crucial in determining any urban community’s harmony, resilience, and development (Jewett et al., 2021) (Steiner et al., 2018). Its evaluation provides crucial insights into individuals’ collective health and interdependence within a city’s diverse fabric. In megacities like Jakarta, where population density is surging, it is challenging to collect comprehensive data on social cohesion using conventional survey techniques (Rybak, 2023). The overwhelming number of individuals, geographical complexities, and logistical constraints have necessitated assessing social cohesion. This study employs a data-driven methodology to forecast and examine the Social Cohesion Index of Jakarta’s neighborhoods. Using machine learning capabilities, we intend to revolutionize the method by which we gain insight into the complex social dynamics of this enormous metropolis. Despite their value, traditional survey techniques frequently need help to accommodate the vast size of Jakarta’s population (Andariesta & Wasesa, 2022) (Viljanen et al., 2022) (Sarker, 2021). Consequently, our research arises as a timely response, offering an alternative method for data acquisition that is both scalable and efficient.. Predictive Modeling of Jakarta’s Social Cohesion: GBDT Leads Comparative Analysis Muhammad Rizki Nur Majiid1, Karli Eka Setiawan2* 1,2 Computer Science of Department, School of Computer Science, Bina Nusantara University, Jakarta, Indonesia 11480 muhammad.majiid@binus.ac.id; karli.setiawan@binus.ac.id Correspondence: karli.setiawan@binus.ac.id Correspondence: karli.setiawan@binus.ac.id e-ISSN: 2686-2573 DOI: 10.21512/emacsjournal.v5i3.10602 JURNAL EMACS (Engineering, MAthematics and Computer Science) Vol.5 No.3 September 2023: 169-174 e-ISSN: 2686-2573 DOI: 10.21512/emacsjournal.v5i3.10602 JURNAL EMACS (Engineering, MAthematics and Computer Science) Vol.5 No.3 September 2023: 169-174 e-ISSN: 2686-2573 DOI: 10.21512/emacsjournal.v5i3.10602 JURNAL EMACS (Engineering, MAthematics and Computer Science) Vol.5 No.3 September 2023: 169-174 DOI: 10.21512/emacsjournal.v5i3.10602 (Engineering, MAthematics and Computer Science) Vol.5 No.3 September 2023: 169-174 DOI: 10.21512/emacsjournal.v5i3.10602 ematics and Computer Science) Vol.5 No.3 September 2023: 169-174 Keywords: Social Cohesion; Machine Learning; Urban Assessment; GBDT; Predictive Accuracy 2.1 Jakarta Social Cohesion Dataset scope of our investigation, the Gradient Boosted Decision Tree (GBDT) model surfaces prominently, demonstrating remarkable predictive abilities (Zhang & Jung, 2021). This accomplishment has the potential to advance the field of predictive modeling considerably and highlights the expanding role of machine learning techniques in addressing significant urban challenges comprehensively. The dataset by (Amir et al., 2023) is an extensive compilation of factors associated with social cohesion in Jakarta’s urban life. The dataset comprises information regarding 2,052 respondents from 44 Jakarta districts. Various questions were posed to the respondents regarding their social interactions, participation in community activities, and perceptions of trust and reciprocity. The dataset also contains information on the demographic characteristics of the respondents, such as their age, gender, level of education, and income. This research serves as a beacon of innovation for urban planners, policymakers, and public data management entities grappling with the complexity of accurately documenting social cohesion in Jakarta. Our findings could facilitate more enlightened decision-making processes and targeted interventions that promote social harmony. By investigating the relationship between data science and social dynamics, this study represents a proactive step toward developing comprehensive and effective urban assessment methods. The dataset was collected using a technique of stratified random sampling. The respondents were selected from different districts in Jakarta in proportion to the population of each district. The data compilation occurred between January and February of 2022. The dataset includes eight cohesion factor variables, including gender, age, level of education, income, two district variables, religion, and residential location. There are three variables for each: Trust, Recognition, Participation, Reciprocity, and Insertion among the cohesion variables. All data types are categorical or ordinal, and no complex data cleansing is required. The model will be trained to predict all 15 variables of cohesion. II. METHODS This study aimed to develop a machine learning model for predicting Jakarta’s Social Cohesion Index (SCI), which assesses the degree of social integration and solidarity among city residents. Figure 1 depicts the six primary stages involved in developing and evaluating our model. First, this study conducted a literature review to comprehend the concept and dimensions of SCI and identify the variables and indicators that can be used to measure it. Secondly, we gathered data from a publicly available dataset paper to ensure validity. Based on the data analysis and the literature review, we then planned the design and architecture of our model. Our primary machine learning technique is a polynomial regression model with optimal degree selection because it can capture the nonlinear and complex relationship between the SCI and its predictors. Fourth, we constructed and trained our model with the collected data using Python and Sci-Kit-Learn libraries. Fifth, we utilized K-Fold Cross-Validation to assess the performance and precision of our model. Several metrics, including Mean Squared Error (MSE), Root Mean Squared Error (RMSE), and Mean Average Percentage Error (MAPE), were utilized to evaluate the model’s fit. In conclusion, this research compared our model to existing models that employ various machine learning techniques, such as Decision Trees, Random Forests, Support Vector Machines (SVM), and Multi-Layer Perceptron (MLP). I. INTRODUCTION We predicted the Social Cohesion Index using a variety of well-known machine learning algorithms and a large dataset containing numerous socioeconomic and demographic characteristics. This study compared the effectiveness of Polynomial Regression (Narayan & Daniel, 2022), Decision Tree (Pekel, 2020), Random Forest (Tzenios, 2020), Support Vector Machine (SVM) (Parbat & Chakraborty, 2020), and Multi-Layer Perceptron (MLP) models (Ouma et al., 2020). Notably, within the Keywords: Social Cohesion; Machine Learning; Urban Assessment; GBDT; Predictive Accuracy Copyright © 2023 169 URNAL EMACS (Engineering, MAthematics and Computer Science) Vol.5 No.3 September 2023: 169-17 2.3 Gradient Boosted Decision Tree This comprehensive experiment uses several different machine-learning models, which is a diversity that inevitably creates issues when attempting to develop a uniform parameter situation relevant to all models. To address this level of complexity, a more subtle technique has been employed, wherein each model has been painstakingly fine-tuned over a specific number of trials. By making this tactical change, we are allowed to maximize the parameters of each model and, as a result, make full use of the inherent benefits and distinguishing qualities of these models. We ensure a solid foundation for our comparative research by adapting the tuning process to the specific requirements of each model. This decision enables us to identify the intricacies of the performance of each model. Gradient boosting develops sequentially weaker (simpler) prediction models, each attempting to predict the error left over by the previous model. Weak learners who perform slightly better than random chance is used in boosting. Gradient Boosting focuses on adding these weak learners one at a time and eliminating the observations a learner gets right at every step. The focus is on teaching new, weaker learners how to handle the remaining difficult observations at each step. Figure 3. Gradient Boosted Decision Tree (GBDT) The replies to eight survey questions regarding respondents’ places of residence make up the primary dataset used for all machine learning models. These characteristics, which function as independent variables, are essential components of the predictive models we use. The answers to multiple-choice questions go through an essential step called one-hot encoding so machines may process them more easily. This transformation guarantees that the data are compatible with the algorithms used in machine learning and maintains the critical nuances embedded in the responses. In the meantime, our research depends on several aspects of social cohesion, represented by the dependent variables. Each of these dimensions consists of two parameters, and the machine learning models’ overall goal is to forecast both characteristics to contribute to a comprehensive knowledge of the dynamics of social cohesiveness. In the framework of our experiment, the evaluation set comprises around 410 rows (or 20% of the overall dataset), whereas the training set comprises the remaining 1642 rows. This segmentation ensures a thorough examination of the predicted performance of each model on data that has yet to be observed. 2.2 K-Fold Cross-Validation We have used the K-Fold Cross-Validation method in all of our experimental settings to reduce the potential for variance bias caused by randomly splitting the test set. This bias may result in inaccurate results. Specifically, we have divided our dataset into five folds, resulting in a test data ratio equaling twenty percent of the overall dataset for each split. The purpose of this method is to systematically evaluate the performance of our predictive models across several different data folds. The test data error values were recorded throughout each iteration of the K-Fold Cross- Validation procedure. After all folds have been completed, these values are then averaged in order to produce an overall evaluation of the model’s performance for the scenario that was provided. p Figure 2. K-Fold Cross-Validation Principle Figure 2. K-Fold Cross-Validation Principle Figure 1. Machine Learning Model Research and Development Plan Figure 2. K-Fold Cross-Validation Principle Figure 1. Machine Learning Model Research and Development Plan 170 In this study, the application of K-Fold Cross- Validation, depicted in Figure 2, is one of the most critical factors contributing to improving our experimental findings’ reliability and validity. This method ensures that our models are carefully tested across various subsets of the data, delivering a more comprehensive evaluation of their ability to predict future outcomes as a result. We can evaluate the model’s generalization performance more accurately by averaging the test error values from numerous folds. This method also lessens the impact of the data’s variability on our findings and increases the overall reliability of our conclusions. statistical learning. The weak learners are adjusted so that each new learner fits into the leftovers from the step before. As the model gets better over iteration, the weak learners fit better. Each step’s results are combined in the final model to produce a strong learner. The residuals are found using a loss function. For example, logarithmic loss (log loss) can be used for classification tasks and mean squared error (MSE) can be used for regression tasks. It is important to note that adding a new tree does not impact any existing trees in the model. The additional decision tree fits the current model’s residuals. III. RESULTS AND DISCUSSIO Figure 5. GBDT, Polynomial Regression, and Random Forest (RF) Error Comparison Examining the performance between the Gradient Boosted Decision Trees (GBDT) model and the Decision Tree (DT) model, which is more generic, was the first stage in designing our experiment. The necessity of laying a basic standard for our predictive modeling system led to the conscious decision to use this benchmark. When we first evaluate how well a traditional DT performs, we can get essential insights into the intrinsic complexity of the social cohesiveness prediction problem. Using this comparison to establish a baseline, we can evaluate the additional value that the GBDT, in its capacity as an ensemble learning approach, brings to the table. In addition, it offers a helpful point of reference for assessing the efficiency of more sophisticated machine learning models that were subsequently included in the scope of our investigation. This method of strategically comparing models enables us to determine that GBDT is superior and can improve prediction accuracy considerably. As a result, verify its function as the proposed model for forecasting the Social Cohesion Index in Jakarta. Figure 5. GBDT, Polynomial Regression, and Random Forest (RF) Error Comparison Polynomial Regression and Random Forest (RF) are three additional machine learning models that we include in Figure 5 to further our comparative research. The graph shows the typical Mean Absolute Error (MAE) and Root Mean Square Error (RMSE) for these models across cross- validation folds. With an average MAE of 0.692 and RMSE of 0.887, GBDT remains the best-performing model. With an average MAE of 0.6974 and RMSE of 0.8963, Polynomial Regression comes in second place, demonstrating its proficiency in capturing complex relationships. Random Forest performs admirably while somewhat trailing with an average MAE of 0.7329 and RMSE of 0.9559. These outcomes demonstrate how GBDT consistently provides higher accuracy and precision, reiterating its effectiveness in predicting social cohesion scores. Furthermore, the competitive performance of Polynomial Regression and Random Forest highlights the significance of investigating several modeling strategies in tackling complex urban challenges, where nuanced insights and reliable forecasts are of utmost relevance. Figure 4. GBDT and Decision Tree (DT) Error Comparison Figure 4. 2.3 Gradient Boosted Decision Tree Performance metrics, including the Mean Average Error (MAE), Root Mean Squared Error (RMSE), Mean Average Percentage Error (MAPE), and model processing duration in seconds, are systematically recorded to assess each model’s efficacy comprehensively. In addition, it is of the utmost importance to point out that all tests are carried out on the Google Colab platform without employing GPUs. This strategic decision ensures the validity and comparability of our metric measurements while emphasizing the applicability and accessibility of our research findings for a broad audience of users and scholars. Figure 3. Gradient Boosted Decision Tree (GBDT) As shown by Figure 3, GBDT combines several weak learners into a single strong learner. The weak learners, in this case, are the individual decision trees. Each tree attempts to reduce the error of the one that comes before it, and all the trees are connected in series. Boosting algorithms are typically slow to learn but also highly accurate because of this sequential connection. Slower learning models outperform faster learning ones in 171 Predictive Modeling of Jakarta’s Social Cohesion… (Muhammad Rizki Nur Majiid & Karli Eka Setiawan) III. RESULTS AND DISCUSSIO GBDT and Decision Tree (DT) Error Comparison In Figure 4, we present a comparative analysis of the performance of Gradient Boosted Decision Trees (GBDT) and traditional Decision Trees (DT) based on their average Mean Absolute Error (MAE) and Root Mean Square Error (RMSE) across cross-validation folds. The findings show that the GBDT model has a significant advantage over its DT counterpart. Remarkably, GBDT outperforms DT in terms of accuracy in predicting social cohesion scores, with an average MAE of 0.692 compared to 0.7848 for DT. This result shows that GBDT captures more intricate correlations and patterns in the data, leading to more accurate predictions. Similarly, GBDT surpasses DT in terms of RMSE, with an average RMSE of 0.887 as opposed to DT’s 1.0726, indicating that GBDT’s predictions are more accurate and have less volatility. These results highlight the effectiveness of ensemble approaches, such as GBDT, in improving predictive performance, which is crucial in applications where precision is crucial, such as evaluating social cohesion in urban situations. Figure 6. GBDT, Support Vector Machine (SVM), and Multi-Layer Perceptron (MLP) Error Comparison Figure 6. GBDT, Support Vector Machine (SVM), and Multi-Layer Perceptron (MLP) Error Comparison Support Vector Machine (SVM) and Multi-Layer Support Vector Machine (SVM) and Multi-Layer Perceptron (MLP) were two additional machine learning models that were included in our comparison study, as shown in Figure 6. The average Mean Absolute Error (MAE) and Root Mean Square Error (RMSE) for these models throughout cross-validation folds are shown in the graph. The average MAE and RMSE for GBDT are noteworthy at 0.692 and 0.887, respectively. However, SVM shines out because of its exceptional ability to identify underlying patterns in the data, as evidenced by its incredibly low average MAE of 0.6536 and RMSE of 0.9188. With an average MAE of 0.701 and RMSE of 0.9179, MLP, despite slightly lagging, nevertheless exhibits good predictive performance. These 172 AL EMACS (Engineering, MAthematics and Computer Science) Vol.5 No.3 September 2023: 169-174 examined models. The Gradient Boosted Decision Trees (GBDT) model is an outstanding example of efficiency, which can provide predictions in about 0.05 seconds. This efficiency is awe-inspiring, given its strong prediction abilities and the competitive MAE, RMSE, and MAPE values. In comparison, the Decision Trees (DT) model requires only 0.02 seconds, showing quick processing. IV. CONCLUSION Figure 7. Models Error Percentage Comparison In conclusion, this study has investigated in-depth machine learning models for estimating social cohesiveness scores in Jakarta’s dynamic metropolitan environment. The Gradient Boosted Decision Trees (GBDT) model, put out as a strong candidate, has repeatedly shown exceptional prediction ability among the models tested. In terms of Mean Absolute Error (MAE), Root Mean Square Error (RMSE), and Mean Absolute Percentage Error (MAPE), GBDT attained competitive results, demonstrating its competence in capturing the complex patterns underlying social cohesiveness dynamics. Additionally, GBDT demonstrated admirable computing efficiency, providing a helpful edge for large-scale and real-time applications. We analyze the average Mean Absolute Percentage Error (MAPE) throughout cross-validation folds for all the models considered in this study, and the results are shown in Figure 7. With a MAPE of 25.59%, the Gradient Boosted Decision Trees (GBDT) model, which is the suggested model, stands out. This result shows that, on average, the social cohesion scores predicted by GBDT differ by about 25.59% from the actual values. Although GBDT has good predictive performance, placing this finding within the larger pool of models tested is essential. The MAPE for Decision Trees (DT) is somewhat higher at 27.11%, while the MAPE for Polynomial Regression and Support Vector Machine (SVM) are both competitive at 24.79%. With a MAPE of 25.59%, Random Forest (RF) exhibits a comparable level of accuracy, while Multi-Layer Perceptron (MLP) trails slightly with a MAPE of 25.82%. Different MAPE values provide helpful information about the relative accuracy of different models and can help determine which model is best for forecasting Jakarta’s Social Cohesion Index. Despite performance differences, GBDT’s steady performance upholds its reputation as a reliable and accurate option for this crucial forecasting activity. Although GBDT stood out as a performer, it is crucial to recognize the larger context of our research. Alternative models with different strengths and weaknesses in terms of accuracy and efficiency included Polynomial Regression, Random Forest, Support Vector Machine (SVM), and Multi-Layer Perceptron (MLP). Specific application needs and constraints should guide when selecting the best model. Overall, the study emphasizes the critical role that machine learning plays in tackling complex urban issues like social cohesiveness evaluation and draws attention to the potential of data-driven insights to guide community development projects. III. RESULTS AND DISCUSSIO Polynomial Regression is the most time-efficient among the models, taking only 0.01 seconds, while Random Forest (RF) and Support Vector Machine (SVM) incur slightly longer processing times at 0.29 and 0.51 seconds, respectively. Incredibly, Multi-Layer Perceptron (MLP) performs predictions in just 0.0081 seconds. These results demonstrate a trade-off between computational expense and prediction precision. In real-time or large-scale applications where accuracy and speed are critical factors, GBDT appears attractive because it strikes a compromise between predictive strength and computational efficiency. findings highlight the adaptability of machine learning methods; of notice are the excellent accuracy of SVM and the robustness of GBDT. In the case of forecasting social cohesiveness scores, the choice of the most relevant model may depend on certain application needs, such as the need for precision or computational efficiency, underlining the significance of carefully selecting the right method for the task at hand. Figure 7. Models Error Percentage Comparison REFERENCES tegration on Academic Performance Using Ran dom Forest Regression. ResearchBerg Review of Science and Technology, 3(1), 94–106. https:// researchberg.com/index.php/rrst/article/view/84 Amir, S., Hidayana, I., Rahvenia, Z., & Haydar, S. (2023). Dataset on factors associated with social cohesion of urban life in Jakarta. Data in Brief, 49, 109339. https://doi.org/10.1016/j.dib.2023.109339 Viljanen, Viljanen, M., Meijerink, L., Zwakhals, L., & van de Kassteele, J. (2022). A machine learning ap proach to small area estimation: predicting the health, housing and well-being of the popula tion of Netherlands. 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Energy Efficient Pro tocol for Lifetime Prediction of Wireless Sensor Network using Multivariate Polynomial Regres sion Model. Journal of Scientific and Industri al Research, 81(12), 1297–1309. https://doi. org/10.56042/jsir.v81i12.54908 Ouma, Y. O., Okuku, C. O., & Njau, E. N. (2020). Use of Artificial Neural Networks and Multiple Linear Regression Model for the Prediction of Dissolved Oxygen in Rivers: Case Study of Hydrograph ic Basin of River Nyando, Kenya. Complexity, 2020. https://doi.org/10.1155/2020/9570789 Parbat, D., & Chakraborty, M. (2020). A python based sup port vector regression model for prediction of COVID19 cases in India. Chaos, Solitons and Fractals, 138, 109942. https://doi.org/10.1016/j. chaos.2020.109942 Pekel, E. (2020). Estimation of soil moisture using deci sion tree regression. Theoretical and Applied Climatology, 139(3–4). https://doi.org/10.1007/ s00704-019-03048-8 Rybak, A. (2023). Survey mode and nonresponse bias: A meta-Analysis based on the data from the inter national social survey programme waves 1996 2018 and the European social survey rounds 1 to 9. PLoS ONE, 18(3 March). https://doi. org/10.1371/journal.pone.0283092 Sarker, I. H. (2021). Machine Learning: Algorithms, Re al-World Applications and Research Directions. SN Computer Science, 2(3), 1–21. https://doi. org/10.1007/s42979-021-00592-x Steiner, A., Woolvin, M., & Skerratt, S. (2018). IV. CONCLUSION As we look to the future, the continuous study of cutting-edge machine learning methods and their integration with urban planning and governance processes holds the possibility of producing more resilient and peaceful urban settings. Figure 8. Models Computational Cost Comparison Figure 8. Models Computational Cost Comparison By calculating the time each model took to forecast the whole evaluation set, Figure 8 provides essential insights into the computational effectiveness of the 173 Predictive Modeling of Jakarta’s Social Cohesion… (Muhammad Rizki Nur Majiid & Karli Eka Setiawan) URNAL EMACS (Engineering, MAthematics and Computer Science) Vol.5 No.3 September 2023: 169-17 REFERENCES Measuring community resilience: Developing and apply ing a “hybrid evaluation” approach. Commu nity Development Journal, 53(1). https://doi. org/10.1093/cdj/bsw017 Tzenios, N. (2020). Examining the Impact of EdTech In 174
https://openalex.org/W2530969823	https://www.frontiersin.org/articles/10.3389/fmicb.2016.01636/pdf	English	null	The Influence of Sporulation Conditions on the Spore Coat Protein Composition of Bacillus subtilis Spores	Frontiers in microbiology	2,016	cc-by	8,352	The Inﬂuence of Sporulation Conditions on the Spore Coat Protein Composition of Bacillus subtilis Spores Wishwas R. Abhyankar1,2, Kiki Kamphorst1, Bhagyashree N. Swarge1,2, Henk van Veen3, Nicole N. van der Wel3, Stanley Brul2, Chris G. de Koster1 and Leo J. de Koning1 1 Department of Mass Spectrometry of Bio-macromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, Netherlands, 2 Department of Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, Netherlands, 3 Department of Cell Biology and Histology, Electron Microscopy Centre Amsterdam, Academic Medical Center, Amsterdam, Netherlands Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions, especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared under a liquid medium condition. It has also been shown that the more mature a spore is, the higher is its heat resistance most likely mediated, at least in part, by the progressive cross-linking of coat proteins. The current study for the ﬁrst time assesses, at the proteomic level, the effect of two commonly used sporulation conditions on spore protein presence. 14N spores prepared on solid Schaeffer’s-glucose (SG) agar plates and 15N metabolically labeled spores prepared in shake ﬂasks containing 3-(N- morpholino) propane sulfonic acid (MOPS) buffered deﬁned liquid medium differ in their coat protein composition as revealed by LC-FT-MS/MS analyses. The former condition mimics the industrial settings while the latter conditions mimic the routine laboratory environment wherein spores are developed. As seen previously in many studies, the spores prepared on the solid agar plates show a higher thermal resistance than the spores prepared under liquid culture conditions. The 14N:15N isotopic ratio of the 1:1 mixture of the spore suspensions exposes that most of the identiﬁed inner coat and crust proteins are signiﬁcantly more abundant while most of the outer coat proteins are signiﬁcantly less abundant for the spores prepared on solid SG agar plates relative to the spores prepared in the liquid MOPS buffered deﬁned medium. Sporulation condition- speciﬁc differences and variation in isotopic ratios between the tryptic peptides of expected cross-linked proteins suggest that the coat protein cross-linking may also be condition speciﬁc. Since the core dipicolinic acid content is found to be similar in both the spore populations, it appears that the difference in wet heat resistance is connected to the differences in the coat protein composition and assembly. The Inﬂuence of Sporulation Conditions on the Spore Coat Protein Composition of Bacillus subtilis Spores Corroborating the proteomic analyses, electron microscopy analyses show a signiﬁcantly thinner outer coat layer of the spores cultured on the solid agar medium. Keywords: Bacillus, sporulation conditions, spores, proteomics, quantitative proteomics Edited by: Edited by: Simon Michael Cutting, Royal Holloway, University of London, UK Reviewed by: Daniel Paredes-Sabja, Universidad Andres Bello, Chile Susan Schlimpert, Biotechnology and Biological Sciences Research Council, UK Correspondence: Wishwas R. Abhyankar w.r.abhyankar@uva.nl Specialty section: This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology Specialty section: This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology Received: 30 June 2016 Accepted: 30 September 2016 Published: 13 October 2016 ORIGINAL RESEARCH published: 13 October 2016 doi: 10.3389/fmicb.2016.01636 Citation: Abhyankar WR, Kamphorst K, Swarge BN, van Veen H, van der Wel NN, Brul S, de Koster CG and de Koning LJ (2016) The Inﬂuence of Sporulation Conditions on the Spore Coat Protein Composition of Bacillus subtilis Spores. Front. Microbiol. 7:1636. doi: 10.3389/fmicb.2016.01636 INTRODUCTION prepared in 2x Schaeﬀer’s-glucose (2x SG) liquid and solid medium. The spores made on plates were more resistant to a temperature of 90◦C in water than the spores made in liquid medium (Rose et al., 2007). The same group also concluded that there was no diﬀerence in the core water, DPA levels between two spore populations but these populations diﬀered in their germination behavior. Yet, the eﬀect of sporulation conditions on the spore’s (coat) proteome remains elusive. Bacterial spores are of high interest because of their extreme resistance properties, especially toward nutritional and environmental stresses (Rose et al., 2007), and the heterogeneity amongst them. Due to their resistant nature, the methods to prevent bacterial contamination do not eliminate spores easily. This is not only a concern to the food processing and production industries but also to the cleaning rooms of industries as well as the medical sectors (Checinska et al., 2015). To develop spore prevention methods, the mechanisms of development and resistance of spores have to be understood completely. For a long time, the heat resistance of spores is of high interest in microbiology and therefore this property has been highly examined (Mundra et al., 2014). Stress resistant spores can germinate to form vegetative cells when the conditions become favorable resulting in food spoilage or sometimes food intoxication (Checinska et al., 2015). The spore germination behavior is heterogeneous and therefore an additional challenge to the food industries. The multiple layers of spores all contribute in some way to their resistance properties (Driks, 2002). It is believed that next to the dehydrated core, the cortex and coat contribute to the spores’ heat resistance. The core consists of the cell’s DNA, ribosomes, dipicolinic acid (DPA), and small acid-soluble proteins or SASPs (Setlow, 1988). The SASPs protect the DNA, while DPA forms a chelate complex with cations like calcium (Checinska et al., 2015). These chelate complexes are suggested to play a role in dehydration of the core, which in turn protects the spore from heat stress and UV light (Setlow et al., 2006). The major function of the external coat layers is to protect the spore from any enzyme or chemical assault and preserve the genome (Mckenney et al., 2013). A peculiarity of these proteinaceous layers is that ∼30% of it is said to be composed of insoluble cross-linked proteins. INTRODUCTION The current research has focused on three main questions – (1) What is the inﬂuence of diﬀerent sporulation conditions used in industries and research laboratories on the spore coat protein composition? (2) What is the inﬂuence of diﬀerent sporulation conditions on spore coat inter-protein cross-linking and coat ultrastructure? (3) Does the (sporulation) condition- induced speciﬁc changes in spore thermal resistance, correlate with the changes in the spore coat proteome and changes in the extent of cross-linking of proteins such as CotG, CotU, GerQ, etc.? To answer these questions a quantitative proteomic analysis of B. subtilis spores made in liquid and on agar plates has been set-up under two commonly used sporulation conditions using two of the most common sporulation media. To that end 14N and metabolically labeled 15N vegetative cells are sporulated on 14N 2x SG agar solid plates or in a 15N-labeled 3-(N- morpholino) propane sulfonic acid (MOPS) buﬀered deﬁned liquid medium, respectively. The former condition is generally used in industry as a condition known to yield most stress resistant spores and one that bears similarity to the conditions in the food chain. The latter condition is commonly used in fundamental microbiology laboratories as it allows for the dissection of the eﬀects of individual medium components as well as provides homogenous conditions to maximize spore homogeneity useful for numerous biochemical studies. The spore suspensions are mixed 1:1 based on the optical density values and the protein ratios for the two diﬀerent sporulation conditions are determined as the 14N: 15N protein abundance ratios with LC-FTMS analyses. DPA contents of two spore populations are determined to check the eﬀect of each sporulation condition on the DPA content. Finally, to support our proteomics data, electron microscopy (EM) has been used and the thickness of diﬀerent spore layers is measured. It appears that the spore coat protein composition of the two spore populations diﬀers signiﬁcantly. Especially, the outer coat proteome is aﬀected by the sporulation conditions. The EM analyses show that the spores developed on the solid medium possess thinner outer coats compared to the spores formed in the liquid sporulation medium. Though the DPA levels of both spore populations are similar, the spores formed on plates are signiﬁcantly more heat resistant. Citation: Citation: Abhyankar WR, Kamphorst K, Swarge BN, van Veen H, van der Wel NN, Brul S, de Koster CG and de Koning LJ (2016) The Inﬂuence of Sporulation Conditions on the Spore Coat Protein Composition of Bacillus subtilis Spores. Front. Microbiol. 7:1636. doi: 10.3389/fmicb.2016.01636 October 2016 \| Volume 7 \| Article 1636 1 Frontiers in Microbiology \| www.frontiersin.org Spore Proteomics in Different Sporulation Conditions Abhyankar et al. Frontiers in Microbiology \| www.frontiersin.org Spore Harvesting g After 5 days of incubation, the 14N spores were collected by scraping them oﬀfrom the 2x SG agar plates and centrifugation whereas the 15N spores were harvested by centrifugation and removal of the supernatant. Both sets of spores were further puriﬁed as described in the literature (Abhyankar et al., 2011). In short, the spores were washed once with 0.01% Tween to kill the vegetative cells and further washed seven times with Milli-Q water. Any remaining vegetative cells were removed using a Histodenz gradient centrifugation, if needed as described elsewhere (Abhyankar et al., 2015). The yield of 14N spores was lower (109spores/mL) compared to the 15N spores (1011spores/mL). The puriﬁed spore crops contained >99% phase bright spores. FIGURE 1 \| Workﬂow of quantitative proteomics of spores prepared under the most common sporulation conditions used in industries (solid agar plates) and in routine microbiology laboratories (liquid medium). The magenta color indicates presence of 15N in to the medium, cells, and therefore also in spores. linked proteins and intracellular contaminants, isolated layers of insoluble spore material were washed extensively with 1 M NaCl and thermally treated for 10 min at 80◦C in a water bath, with 50 mM Tris-HCl, pH 7.8 containing 2% SDS, 100 mM Na-EDTA, 150 mM NaCl, and 100 mM β-mercaptoethanol (Abhyankar et al., 2011). SDS-treated coats were washed four times with Milli-Q water and freeze dried overnight for immediate use for mass spectrometric (MS) analysis. MATERIALS AND METHODS FIGURE 1 \| Workﬂow of quantitative proteomics of spores prepared under the most common sporulation conditions used in industries (solid agar plates) and in routine microbiology laboratories (liquid medium). The magenta color indicates presence of 15N in to the medium, cells, and therefore also in spores. Bacterial Strain and Sporulation Conditions The B. subtilis wild-type strain PY79 used in this work was obtained from Dr. Eichenberger’s lab (New York University, USA). The aim was to study the eﬀect of two of the most common yet drastically diﬀerent sporulation conditions used in practice in industries and fundamental studies, respectively. Therefore we chose 2x SG agar as a solid medium and liquid deﬁned medium buﬀered with MOPS in our study. Initially, the cells were cultured on a Tryptic Soy agar plate and incubated overnight at 37◦C. A single colony was then inoculated in the liquid Tryptic Soy broth medium and cultivated until early exponential phase. The exponentially growing cells were then subjected to growth in serial dilutions (as shown in Figure 1) of 2x SG liquid medium (14N medium; further referred to as 14N cells) and a deﬁned medium, buﬀered with MOPS supplemented with 15NH4Cl (further referred to as 15N cells). Further, the culture enrichment was done in 20 ml 2x SG and MOPS liquid media, respectively. For sporulation, the conditions diﬀered signiﬁcantly. The enriched 14N cells (0.1 ml) were spread on 10 plates of 2x SG agar (incubated at 37◦C) whereas the enriched 15N cells were inoculated (1% ﬁnal volume) to 500 ml of MOPS medium (incubated at 37◦C at 200 rpm). The cells were allowed to sporulate for 5 days when the sporulation was induced by glucose exhaustion as described elsewhere (Abhyankar et al., 2011). Four independent biological replicates were prepared and analyzed. INTRODUCTION Interestingly, the crust proteins and certain coat proteins known to be involved in cross-linking in the spores are identiﬁed in higher abundance in the spores developed on solid medium relative to those developed in liquid medium. For these proteins, variations in peptide ratios are seen. This suggests that the enhanced heat resistance is possibly linked to the speciﬁc cross-linked coat proteins and the degree of cross-linking therein. The resistance of spores is aﬀected by more than one factor during their development. The group of Sanchez-Salas et al. (2011) compared the wet heat resistance of young spores with the total produced (matured) spores from the same culture and found young spores to have a signiﬁcantly lower wet heat resistance. Sanchez-Salas et al. (2011) concluded that changes in the spore coat during maturation are responsible for the higher wet heat resistance. Furthermore, a recent study from our group suggested that the inter-protein cross- linking might contribute to the heat resistance of the spores (Abhyankar et al., 2015). However, the inﬂuence of initial sporulation conditions on spore resistance properties was here not explicitly assessed. In a previous study, it had been shown that spores made at higher temperatures had a higher wet heat resistant than spores generated at lower sporulation temperatures (Melly et al., 2002). In addition, eﬀects of the sporulation medium on spore’s stress resistance properties have been assessed in a number of studies. Dadd et al. (1983) showed that the level of nutrients and the amount of available divalent cations aﬀect spore thermal stress resistance properties (Dadd et al., 1983). Rose et al. (2007) performed a study comparing the wet heat resistance of Bacillus subtilis spores October 2016 \| Volume 7 \| Article 1636 2 Spore Proteomics in Different Sporulation Conditions Abhyankar et al. Mixing of 14N and 15N- Labeled Spores and Spore Coat Isolation The harvested 14N and 15N spores were mixed in 1:1 ratio based on OD600 and re-suspended in cold 10mM Tris-HCl (pH 7.5). Subsequently the spores were disintegrated with 0.1-mm Zirconia Silica beads in a Bio-Savant Fast Prep 120 machine (Qbiogene, Carlsbad, CA, USA). A program of nine Fast-prepping rounds was set. One round lasted for 40 s at a maximum speed of 6. After every three rounds, the samples were placed on ice for 5 min to prevent protein degradation by over-heating. To remove non-covalently Sample Preparation for MS Analysis The program facilitates organization and data mining of large sets of proteomics data. The identiﬁed proteins for each of the four replica of 14N/15N spore mixture coat isolate samples are listed in Supplementary Table S1A, together with their protein MASCOT score and number of peptide spectrum matches. These proteins were identiﬁed in at least two out of four replicates with at least two unique peptides. Using the quantiﬁcation toolbox, the isotopic ratios for a subset of identiﬁed proteins were determined as an average of the isotopic ratios of the corresponding light over heavy tryptic peptides. This subset represents a selection of known and putative spore coat proteins, based on literature, sigma regulations, and localization studies. Critical settings were: require bold red: on, signiﬁcance threshold: 0.05: Protocol type: precursor; Correction: Element 15N; Value 99.6; Report ratio L/H; Integration method: Simpson’s integration method; Integration source: survey; Allow elution time shift: on; Elution time delta: 20 s; Std. Err. Threshold: 0.15, Correlation Threshold (Isotopic distribution ﬁt): 0.98; XIC threshold: 0.1; All charge states: on; Max XIC width: 200 s; Threshold type: at least homology; Peptide threshold value: 0.05; unique pepseq: on. All reported isotopic peptide ratios were manually validated by inspection of the spectral MS data. The MASCOT DISTILLER protein quantiﬁcation reports were exported in excel format and then imported in the Protein Browser program. To correct for possible errors in 14N/15N 1:1 culture mixing, the 14N/15N ratios were normalized on the median for each replicate dataset. For quantiﬁcation a sub-selection of putative and known coat proteins was made based on previous literature (Abhyankar et al., 2015), localization studies and their regulating sigma factors. The quantiﬁed putative coat proteins for each of the four replica 14N/15N spore mixture coat isolate samples are included in Supplementary Table S1B, together with their protein MASCOT score and light over heavy (L/H) isotopic ratio. The proteins that were quantiﬁed at least in two of the four replicates have been included in the results. The proteomics data has been deposited to the ProteomeXchange Consortium (Vizcaíno et al., 2014) via the PRIDE partner repository with the dataset identiﬁer PXD004473. Measurement of Thermal Resistance of Liquid Chromatography-Fourier Transform Ion Cyclotron Tandem Mass Spectrometry (LC-FT-ICR MS/MS) Analysis y LC-MS/MS data were acquired with an Bruker ApexUltra Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a 7T magnet and a nano-electrospray Apollo II DualSourceTM coupled to an Ultimate 3000 (Dionex, Sunnyvale, CA, USA) High- Performance Liquid Chromatography system. For each digest, samples containing up to 200 ng of tryptic peptides were injected as a 20 µl 0.1%TFA, 3% ACN aqueous solution, and loaded onto a PepMap100 C18 (5 µm particle size, 100 Å pore size, 300 µm inner diameter × 5 mm length) precolumn. Following injection, the peptides were eluted via an Acclaim PepMap 100 C18 (3 µm particle size, 100 Å pore size, 75 µm inner diameter × 500 mm length.) analytical column (Thermo Scientiﬁc, Etten-Leur, The Netherlands) to the nano-electrospray source. LC gradient proﬁles of up to 120 min were used with 0.1% formic acid/99.9% H2O (A) and 0.1% formic acid/80% ACN/19.9% H2O (B) at a ﬂow rate of 300 nL/min at a column temperature of 60◦C. The gradient proﬁles were as follows 0 min 97%A/3%B, 2 min 94%A/6%B, 110 min 70%A/30%B, 120 min 60%A/40%B, 125 min 100%B. Data dependent Q-selected peptide ions were fragmented in the hexapole collision cell at an Argon pressure of 6 × 10−6 mbar (measured at the ion gage) and the fragment ions were detected in the ICR cell at a resolution of up to 60,000. In the MS/MS duty cycle, three diﬀerent precursor peptide ions were selected from each survey MS. The MS/MS duty cycle time for one survey MS and three MS/MS acquisitions was about 2 s. Instrument mass calibration was better than 2 ppm over an m/z range of 250–1500. Raw FT-MS/MS data were processed with the MASCOT DISTILLER program, version 2.4.3.1 (64 bits), MDRO 2.4.3.0 (MATRIX science, London, UK), including the Search toolbox and the Quantiﬁcation toolbox. Peak-picking for both MS and MS/MS spectra were optimized for the mass resolution of up to 60,000. Peaks were ﬁtted to a simulated isotope distribution with a correlation threshold of 0.7, with minimum signal to noise of 2. The processed data, from the four independent biological replicates, were searched with the MASCOT server program 2.3.02 (MATRIX science, London, UK) against a complete B. subtilis 168 ORF translation database Sample Preparation for MS Analysis All reported isotopic peptide ratios were manually validated by inspection of the spectral MS data. The MASCOT DISTILLER protein quantiﬁcation reports were exported in excel format and then imported in the Protein Browser program. To correct for possible errors in 14N/15N 1:1 culture mixing, the 14N/15N ratios were normalized on the median for each replicate dataset. For quantiﬁcation a sub-selection of putative and known coat proteins was made based on previous literature (Abhyankar et al., 2015), localization studies and their regulating sigma factors. The quantiﬁed putative coat proteins for each of the four replica 14N/15N spore mixture coat isolate samples are included in Supplementary Table S1B, together with their protein MASCOT score and light over heavy (L/H) isotopic ratio. The proteins that were quantiﬁed at least in two of the four replicates have been included in the results. The proteomics data has been deposited to the ProteomeXchange Consortium (Vizcaíno et al., 2014) via the PRIDE partner repository with the dataset identiﬁer PXD004473. were digested for 18 h at 37◦C with trypsin (Trypsin Gold Promega, Madison, WI, USA) using a 1:60 (w/w) protease: protein ratio. After 18 h, the tryptic digests were desalted using Omix µC18 pipette tips (80 µg capacity, Varian, Palo Alto, CA, USA) according to the manufacturer’s instructions. The peptides were collected in 25 µL 50% acetonitrile (ACN), 0.1% triﬂuoroacetic acid (TFA), and stored at −80◦C (Abhyankar et al., 2011). Before analysis a fraction of eluted peptide material was freeze-dried and concentrated in 10 µL of 0.1% TFA and peptide concentration was measured at 205 nm (Scopes, 1974) with a Nanodrop ND1000 spectrophotometer (Isogen Life Sciences, De Meern, The Netherlands). (Uniprot 2011 update, downloaded from http://www.uniprot. org/uniprot) combined with a protein contamination data base (compiled by and downloaded from Max Planck Institute of Biochemistry, Martinsried). Quantiﬁcation using 15N-Metabolic [MD] labeling was included. Trypsin was used as enzyme and one missed cleavage was allowed. Carbamidomethylation of cysteine was used as a ﬁxed modiﬁcation and oxidation of methionine as a variable modiﬁcation. The peptide mass tolerance was set to 25 ppm and the peptide fragment mass tolerance was set to 0.03 Dalton. MASCOT MudPIT peptide identiﬁcation score was set to a cut-oﬀof 18. The MASCOT protein identiﬁcation reports were exported as XML and then imported in custom made Visual Basic (VBA) software program Protein Browser running in Microsoft Excel. Frontiers in Microbiology \| www.frontiersin.org Sample Preparation for MS Analysis The freeze-dried coat material was subjected to reduction with 10 mM dithiothreitol in 100 mM NH4HCO3 (1 h at 55◦C). To avoid the re-formation of the di-sulﬁde bridges, alkylation of the reduced coat material was performed with 55 mM iodoacetamide in 100 mM NH4HCO3 for 45 min at room temperature in the dark. Subsequently the proteins October 2016 \| Volume 7 \| Article 1636 Frontiers in Microbiology \| www.frontiersin.org 3 Spore Proteomics in Different Sporulation Conditions Abhyankar et al. Spore Proteomics in Different Sporulation Conditions (Uniprot 2011 update, downloaded from http://www.uniprot. org/uniprot) combined with a protein contamination data base (compiled by and downloaded from Max Planck Institute of Biochemistry, Martinsried). Quantiﬁcation using 15N-Metabolic [MD] labeling was included. Trypsin was used as enzyme and one missed cleavage was allowed. Carbamidomethylation of cysteine was used as a ﬁxed modiﬁcation and oxidation of methionine as a variable modiﬁcation. The peptide mass tolerance was set to 25 ppm and the peptide fragment mass tolerance was set to 0.03 Dalton. MASCOT MudPIT peptide identiﬁcation score was set to a cut-oﬀof 18. The MASCOT protein identiﬁcation reports were exported as XML and then imported in custom made Visual Basic (VBA) software program Protein Browser running in Microsoft Excel. The program facilitates organization and data mining of large sets of proteomics data. The identiﬁed proteins for each of the four replica of 14N/15N spore mixture coat isolate samples are listed in Supplementary Table S1A, together with their protein MASCOT score and number of peptide spectrum matches. These proteins were identiﬁed in at least two out of four replicates with at least two unique peptides. Using the quantiﬁcation toolbox, the isotopic ratios for a subset of identiﬁed proteins were determined as an average of the isotopic ratios of the corresponding light over heavy tryptic peptides. This subset represents a selection of known and putative spore coat proteins, based on literature, sigma regulations, and localization studies. Critical settings were: require bold red: on, signiﬁcance threshold: 0.05: Protocol type: precursor; Correction: Element 15N; Value 99.6; Report ratio L/H; Integration method: Simpson’s integration method; Integration source: survey; Allow elution time shift: on; Elution time delta: 20 s; Std. Err. Threshold: 0.15, Correlation Threshold (Isotopic distribution ﬁt): 0.98; XIC threshold: 0.1; All charge states: on; Max XIC width: 200 s; Threshold type: at least homology; Peptide threshold value: 0.05; unique pepseq: on. Measurement of Thermal Resistance of 14N and 15N Spores The error bars indicate the standard deviation within two technical replicates. By deﬁnition, the values are within 95.40% conﬁdence interval in the range of the error bars. FIGURE 3 \| Thermal resistance of spores developed under different sporulation conditions. Log10 values of colony forming units (CFU) obtained after 48 h incubation per OD600 of spores. The values represent the mean averages for three biological replicates where the error bars represent the standard deviation observed for two technical replicates. By deﬁnition, the values are within 95.40% conﬁdence interval in the range of the error bars. vegetative cells, if any, were inactivated by the heat activation. After heat activation the 1 ml of spores were injected with a syringe into metal screw-cap tubes with 9 ml of sterile Milli-Q water which were pre-heated for 20 min in a glycerol bath (85◦C). The spore suspensions from three biological replicates were heated for 10 min (85◦C) in the glycerol bath inducing thermal stress. After 10 min, the tubes were cooled on ice. One milliliter of the spore suspension was serial diluted in sterile Milli- Q water and 100 µl of sample from last three dilutions was spread on Tryptic Soy agar plates. The heat resistance was determined by counting the number of colonies after 48 h incubation at 37◦C (Kooiman et al., 1973). Two technical replicates were performed for each biological replicate. Measurement of DPA Concentration in 14N and 15N Spores FIGURE 3 \| Thermal resistance of spores developed under different FIGURE 3 \| Thermal resistance of spores developed under different sporulation conditions. Log10 values of colony forming units (CFU) obtained after 48 h incubation per OD600 of spores. The values represent the mean averages for three biological replicates where the error bars represent the standard deviation observed for two technical replicates. By deﬁnition, the values are within 95.40% conﬁdence interval in the range of the error bars. FIGURE 3 \| Thermal resistance of spores developed under different sporulation conditions. Log10 values of colony forming units (CFU) obtained after 48 h incubation per OD600 of spores. The values represent the mean averages for three biological replicates where the error bars represent the standard deviation observed for two technical replicates. By deﬁnition, the values are within 95.40% conﬁdence interval in the range of the error bars. Dipicolinic acid analysis was performed according to an established protocol (Janssen et al., 1958). Spore suspension with a ﬁxed OD600 was freeze dried and weighed. Consequently, the spores were suspended in 5 ml of sterile Milli-Q water and autoclaved for 15 min at 121◦C. The spores were then cooled on ice, acidiﬁed with 100 µl of 1 N acetic acid and incubated for 1 h at room temperature. After 1 h, the spores were centrifuged (10 min at 1500 × g) and 4 ml of the supernatant was transferred to new test tube. One milliliter of the color reagent [1 % Fe(NH4)2(SO4)2.6H2O along with 1 % ascorbic acid in 0.5 M acetate buﬀer of pH 5.5] was added to the supernatant. By measuring the A440, a standard curve was prepared for the concentration range of 20–140 µg/ml DPA. The A440 of the mixture was measured and the amounts were calculated from the standard curve. FIGURE 4 \| Core dipicolinic acid (DPA) content as % per spore dry weight of spores developed under different sporulation conditions. The error bars indicate the standard deviation within two technical replicates. By deﬁnition, the values are within 95.40% conﬁdence interval in the range of the error bars. Electron Microscopy For each sporulation condition, the spores were ﬁxed in 1% glutaraldehyde and 4% paraformaldehyde in 0.1 M Phosphate buﬀer (pH 7.4) for 60 min After ﬁxation the spores were washed in distilled water, block stained overnight in 1.5% uranyl acetate to enhance contrast in the electron microscope, washed in distilled water, and osmicated for 60 min in 1% OsO4 in water (Electron Microscopy Sciences, Hatﬁeld, PA, USA). Subsequently, the spores were dehydrated in an alcohol series (of ethanol) and embedded into propylene oxide/Epon 1:1 before embedding into LX-112 resin (Ladd Research, Williston, VT, USA). Between each step, the spores were centrifuged down. After polymerization at 60◦C the Epon blocks with the spores were prepared for ultrathin sectioning. Ultrathin sections of 90 nm were cut on a Reichert EM UC6 with a diamond knife, collected on Formvar coated grids and stained with uranyl acetate and lead citrate. Sections were examined with a FEI Technai- 12 G2 Spirit Biotwin transmission electron microscope (TEM). Images were taken with a Veleta camera (Olympus, SIS). For measurement of the thickness of the coat layers, using the iTEM software (Emsis) the images of cross-sectioned spores were taken at a magniﬁcation of 120,000×. For these measurements, 80 spores per replicate were used. FIGURE 4 \| Core dipicolinic acid (DPA) content as % per spore dry weight of spores developed under different sporulation conditions. The error bars indicate the standard deviation within two technical replicates. By deﬁnition, the values are within 95.40% conﬁdence interval in the range of the error bars. The error bars indicate the standard deviation within two technical replicates. By deﬁnition, the values are within 95.40% conﬁdence interval in the range of the error bars. Measurement of Thermal Resistance of 14N and 15N Spores One milliliter of spores (14N as well as 15N spores) with an OD600 of 2 were heat activated (70◦C, 30 min) in a water bath. The October 2016 \| Volume 7 \| Article 1636 Frontiers in Microbiology \| www.frontiersin.org 4 Spore Proteomics in Different Sporulation Conditions Abhyankar et al. FIGURE 2 \| Fold changes in spore coat protein expression in spores cultured on solid agar plates and in shake ﬂask with liquid medium. The bars indicate the mean ratios of proteins across four replicates while error bars indicate the standard deviations in these ratios. By deﬁnition, the values are within 95.40% conﬁdence interval in the range of the error bars. The protein localization is based upon their sigma factor regulations and the available literature (van Ooij et al., 2004; Imamura et al., 2010; McKenney et al., 2010; Driks and Eichenberger, 2016). Cs, Crust; OC, Outer coat; IC, Inner coat; Ct. asso., Spore coat associated FIGURE 2 \| Fold changes in spore coat protein expression in spores cultured on solid agar plates and in shake ﬂask with liquid medium. The bars indicate the mean ratios of proteins across four replicates while error bars indicate the standard deviations in these ratios. By deﬁnition, the values are within 95.40% conﬁdence interval in the range of the error bars. The protein localization is based upon their sigma factor regulations and the available literature (van Ooij et al., 2004; Imamura et al., 2010; McKenney et al., 2010; Driks and Eichenberger, 2016). Cs, Crust; OC, Outer coat; IC, Inner coat; Ct. asso., Spore coat associated proteins; OM, Outer membrane; Cx, Cortex; IM, Inner membrane; Cr, Core; Sp. asso., Spore associated proteins. October 2016 \| Volume 7 \| Article 1636 5 Frontiers in Microbiology \| www.frontiersin.org Spore Proteomics in Different Sporulation Conditions Abhyankar et al. Spore Proteomics in Different Sporulation Conditions FIGURE 3 \| Thermal resistance of spores developed under different sporulation conditions. Log10 values of colony forming units (CFU) obtained after 48 h incubation per OD600 of spores. The values represent the mean averages for three biological replicates where the error bars represent the standard deviation observed for two technical replicates. By deﬁnition, the values are within 95.40% conﬁdence interval in the range of the error bars. FIGURE 4 \| Core dipicolinic acid (DPA) content as % per spore dry weight of spores developed under different sporulation conditions. Frontiers in Microbiology \| www.frontiersin.org Thermal Resistance of 14N and 15N Spores Obtained from SG Plates and MOPS in Shake Flask Conditions The results for the thermal resistance test for spores at 85◦C for 10 min are shown in Figure 3. The data shows the Log10 number of colonies, grown on the TSA plates 48 h after the thermally induced stress had been given. Shown are mean values, averaged over two technical replicates performed for three biological replicates. It appears that the spores prepared on the 2x SG solid medium plates yield signiﬁcantly more colonies thereby indicating a higher thermal resistance than observed for the spores generated under liquid medium condition. concentrations were measured. For both spore populations, the DPA content analyses show no signiﬁcant diﬀerences. As shown in Figure 4 the DPA amount, mean averaged over two technical replicate analyses in both spore populations, is between the previously reported range of 5–15% (by weight) per spore (Gerhardt and Marquis, 1989). Differences in Coat Protein Levels of Spores Developed under Solid and Liquid Medium Conditions A total of 351 proteins have been identiﬁed across four replicates (Supplementary Table S1A). Because part of the identiﬁed proteins are co-isolated cytosolic proteins, only a selected group of spore proteins have been quantiﬁed by calculating their 14N/15N isotopic ratio (Supplementary Table S1B). This sub- selection includes outer coat, inner coat, and crust proteins as identiﬁed based on their sigma factor regulation and/or October 2016 \| Volume 7 \| Article 1636 Frontiers in Microbiology \| www.frontiersin.org 6 Spore Proteomics in Different Sporulation Conditions Abhyankar et al. FIGURE 5 \| Phase contrast microscopic images of spores cultured on solid agar plates (A) and in shake ﬂasks containing liquid medium (B). The spores are observed under 100× magniﬁcation with oil under simple phase contrast microscope. Scale bars indicate 10 µm. FIGURE 5 \| Phase contrast microscopic images of spores cultured on solid agar plates (A) and in shake ﬂasks containing liquid medium (B). The spores are observed under 100× magniﬁcation with oil under simple phase contrast microscope. Scale bars indicate 10 µm. localization (Abhyankar et al., 2011). The mean average of light/heavy ratios of the proteins over the four replicates are plotted on a Log2 scale (Figure 2) as coat protein fold changes between the spores cultured under solid and in liquid medium conditions. The error bars indicate the standard deviation per protein. The results in Figure 2 show that most of the outer coat proteins such as CotA, CotG, CotC, CotU, etc., are lower in abundance in spores developed on solid sporulation medium (represented in the upper part of the ﬁgure with isotopic ratios lower than 1). Also all known crust proteins CotX, CotY, and CotZ as well as many of the inner coat proteins such as CotJA, CotJB, etc., are seen to be highly abundant in the spores obtained from solid sporulation media (represented in the lower part of the ﬁgure with isotopic ratios greater than 1). FIGURE 6 \| Appearance of liquid suspensions of spores cultured on 2x SG agar plates (A) and in 3-(N-morpholino) propane sulfonic acid (MOPS) buffered deﬁned liquid medium in shake ﬂask (B). Two spore crops differ in their color as liquid suspensions in sterile Milli-Q water with equal OD600. FIGURE 6 \| Appearance of liquid suspensions of spores cultured on 2x SG agar plates (A) and in 3-(N-morpholino) propane sulfonic acid (MOPS) buffered deﬁned liquid medium in shake ﬂask (B). Differences in Coat Protein Levels of Spores Developed under Solid and Liquid Medium Conditions Two spore crops differ in their color as liquid suspensions in sterile Milli-Q water with equal OD600. The Core DPA Content of 14N and 15N Spores Obtained from SG Plates and MOPS in Shake Flask Conditions The Core DPA Content of 14N and 15N Spores Obtained from SG Plates and MOPS in Shake Flask Conditions The spores prepared on solid and in liquid media under the phase contrast microscope show no signiﬁcant morphological diﬀerence in terms of size and shape of both spore populations In order to check the role of DPA on thermal resistance of spore cultured under diﬀerent sporulation conditions, the DPA October 2016 \| Volume 7 \| Article 1636 Frontiers in Microbiology \| www.frontiersin.org 7 Spore Proteomics in Different Sporulation Conditions Abhyankar et al. FIGURE 7 \| Electron microscopic images of spores developed under solid (A) and liquid (B) medium conditions with the corresponding thickness of the spore coat layers (C). The coat layer thicknesses are averaged over 80 individual spores. The error bars indicate the standard deviations between two biological replicates. By deﬁnition, the values are within 95.40% conﬁdence interval in the range of the error bars. Cr, core; Cx, cortex; Ic, inner coat; Oc, outer coat. FIGURE 7 \| Electron microscopic images of spores developed under solid (A) and liquid (B) medium conditions with the corresponding thickness of the spore coat layers (C). The coat layer thicknesses are averaged over 80 individual spores. The error bars indicate the standard deviations between two biological replicates. By deﬁnition, the values are within 95.40% conﬁdence interval in the range of the error bars. Cr, core; Cx, cortex; Ic, inner coat; Oc, outer coat. Frontiers in Microbiology \| www.frontiersin.org DISCUSSION (Figure 5). However, the spores prepared on the 2x SG agar plates and those prepared in MOPS liquid medium in shake ﬂasks show a clear color diﬀerence when seen as liquid suspensions of similar OD (Figure 6). The spores prepared on the 2x SG agar plates have a light brown/beige color, while spores prepared in the liquid MOPS medium are colored dark brown. The electron microscopic images of the spores (Figure 7) clearly show characteristic diﬀerences between the spores cultured under solid and liquid medium conditions. While the outer coat layer of the spores cultured under solid medium condition is signiﬁcantly thinner, the thicknesses of the inner coat and the cortex layers are comparable to the corresponding layers in the spores prepared under liquid medium condition (Figure 7). (Figure 5). However, the spores prepared on the 2x SG agar plates and those prepared in MOPS liquid medium in shake ﬂasks show a clear color diﬀerence when seen as liquid suspensions of similar OD (Figure 6). The spores prepared on the 2x SG agar plates have a light brown/beige color, while spores prepared in the liquid MOPS medium are colored dark brown. The electron microscopic images of the spores (Figure 7) clearly show characteristic diﬀerences between the spores cultured under solid and liquid medium conditions. While the outer coat layer of the spores cultured under solid medium condition is signiﬁcantly thinner, the thicknesses of the inner coat and the cortex layers are comparable to the corresponding layers in the spores prepared under liquid medium condition (Figure 7). The protein fold changes depicted in Figure 2 show that most of the inner coat and all the known crust morphogenetic proteins (CotX, CotY, and CotZ; Imamura et al., 2011; Driks and Eichenberger, 2016) are more abundant (14N:15N ratio > 1) and most of the outer coat proteins such as CotA, CotB, CotC, CotG, CotQ, and CotU (Driks and Eichenberger, 2016) are less abundant (14N:15N ratio < 1) in spores obtained from cultures grown on solid agar plates relative to the cells that were sporulated in liquid medium. Signiﬁcantly, CotE levels are not aﬀected. CotE is a pivotal protein coordinating the formation of the outer coat (Driks, 2002) and directing the assembly of a large October 2016 \| Volume 7 \| Article 1636 Frontiers in Microbiology \| www.frontiersin.org 8 Spore Proteomics in Different Sporulation Conditions Abhyankar et al. DISCUSSION set of inner and outer coat proteins (Kim et al., 2006). Diﬀerent regions of CotE are responsible for the assembly and deposition of spore coat proteins. Proteins CotA, YaaH, CotR, CotG, CotB, CotS, and CotSA are dependent on the C-terminal of CotE for their deposition (Little and Driks, 2001). It is remarkable that most of these proteins are found to be less abundant in spores cultured on solid 2x SG agar plates. No tryptic peptide from C-terminal region of CotE has been identiﬁed in this study. This can be either due to the lack of lysine and arginine cleavage sites in this region or due to the involvement of the C-terminal in linking and depositing other proteins. Both CotJC and CotJB (∼17 kDa) are found to be signiﬁcantly abundant in spores prepared on solid agar plates which is consistent with the report that CotJC and CotJA, which are part of the same operon as CotJB, directly inﬂuence each other (Kim et al., 2006). Setlow, 2004; Checinska et al., 2015), and variations in peptide ratios suggest that cross-linking during maturation hampers the eﬃcient digestion of these proteins (Abhyankar et al., 2015) and that the progress of cross-linking is critical to reproduce. Similarly, the cysteine rich crust proteins CotX, CotY, and CotZ are also assumed to be cross-linked via di-sulﬁde bonds. Yet, no large variations of protein isotopic ratios are observed for these proteins. This is because the di-sulﬁde links in the crust are cut during processing of the spores before the tryptic digestion. These proteins like the ones mentioned above are present at higher abundance in the spores cultured on the solid agar plates. This suggests that the enhanced heat resistance is likely to be linked to the sum of speciﬁc cross-linked coat proteins and the degree of cross-linking. In the current study, we compare the two well-known sporulation conditions, one used often in industries to generate high heat resistant spores on solid plates, and one in deﬁned liquid medium that provides a basis for laboratory experiments that seek for the molecular mechanisms of spore resistance. ACKNOWLEDGMENT Authors thank the Netherlands Organization for Scientiﬁc Research (NWO) for ﬁnancial support. CONCLUSION It appears that the spores developed on solid 2x SG agar plates and those in liquid deﬁned medium in a shake ﬂask, have a distinct coat protein composition, which is correlated, and likely functionally connected, to the diﬀerence in thermal resistance. Despite the higher abundance of most of the outer coat proteins and consequently a thicker outer coat layer, the spores obtained from cells sporulated under liquid medium conditions have a lower thermal resistance. Coat and crust proteins typically involved in cross-linking are more abundant in the thermally resistant spores (prepared on agar plates) that possess thinner outer coats. With the DPA contents of both spore crops being comparable, the diﬀerence in thermal resistance could well be linked to the diﬀerence in the degree of cross-linking in the coat. DISCUSSION Although, the phase contrast microscopic images in Figure 5 show no signiﬁcant diﬀerences in the morphology and spore size of the two spore crops, the spore suspensions in Figure 6 show a more intense browning for the spores cultured in the shake ﬂask, which agrees with the observed increased abundance of the outer coat protein CotA in those spores. This protein is known to produce the brown melanin-like pigment in spores (Hullo et al., 2001). These results are in contrast with those obtained by Rose et al.(2007) as they observed no major diﬀerence in the CotA levels but a large diﬀerence in pigment production, i.e., CotA activity when they compared two diﬀerent sporulation conditions using the same medium. This indicates that the CotA activity and CotA levels might not always be comparable. This observation also calls for a detailed EM studies of these spore populations. Upon zooming in with an electron microscope, the images clearly show that the crust and outer coat layer, averaged over 80 spore images is about 10 nm thicker for the spores cultured in the liquid deﬁned medium (Figure 7). This agrees with the quantitative proteomic results, which show high abundance for most of the crust and outer coat proteins in these spores. The EM images further show no signiﬁcant diﬀerences between the thickness of the inner coat and cortex layers, while no signiﬁcant diﬀerence has been found in the DPA content in the core of both spore crops (Figure 4). Also Rose et al. (2007) have found no signiﬁcant diﬀerence in the core water levels of spores prepared in the same medium but with diﬀerent sporulation conditions. The DPA content, linked to the thermal resistance of spores (Orsburn et al., 2008), is comparable in both 14N and 15N spores in our study and yet the thermal resistance of the spores cultured on the solid agar is signiﬁcantly higher. For the coat proteins Tgl, GerQ, KatX (catalase), YjqC (catalase), SodM unusual large variations in the isotopic ratios have been found between the four replicates as shown by their large standard deviation (Figure 2). These proteins are suggested to be involved in inter-protein cross- linking during spore maturation (Driks, 1999; Ragkousi and AUTHOR CONTRIBUTIONS WA and LdK formulated the research concept, contributed equally to drafting of the manuscript, and performed data analysis. KK and BS performed the laboratory work. HvV and NvW carried out the electron microscopy measurements. SB and CdK contributed by guiding the laboratory work and by critically reading the manuscript. Frontiers in Microbiology \| www.frontiersin.org REFERENCES previously uncharacterized layer of the Bacillus subtilis spore coat. Curr. Biol. 20, 934–938. doi: 10.1016/j.cub.2010.03.060 previously uncharacterized layer of the Bacillus subtilis spore coat. Curr. Biol. 20, 934–938. doi: 10.1016/j.cub.2010.03.060 Abhyankar, W., Pandey, R., Ter Beek, A., Brul, S., De Koning, L. J., and De Koster, C. G. (2015). Reinforcement of Bacillus subtilis spores by cross-linking of outer coat proteins during maturation. Food Microbiol. 45, 54–62. doi: 10.1016/j.fm.2014.03.007 Melly, E., Genest, P. C., Gilmore, M. E., Little, S., Popham, D. L., Driks, A., et al. (2002). Analysis of the properties of spores of Bacillus subtilis prepared at diﬀerent temperatures. J. Appl. Microbiol. 92, 1105–1115. doi: 10.1046/j.1365- 2672.2002.01644.x Abhyankar, W., Ter Beek, A., Dekker, H., Kort, R., Brul, S., and De Koster, C. G. (2011). Gel-free proteomic identiﬁcation of the Bacillus subtilis insoluble spore coat protein fraction. Proteomics 11, 4541–4550. doi: 10.1002/pmic.201100003 Mundra, R. V., Mehta, K. K., Wu, X., Paskaleva, E. E., Kane, R. S., and Dordick, J. S. (2014). Enzyme-driven Bacillus spore coat degradation leading to spore killing. Biotechnol. Bioeng. 111, 654–663. doi: 10.1002/bit.25132 Biotechnol. Bioeng. 111, 654–663. doi: 10.1002/bit.25132 Checinska, A., Paszczynski, A., and Burbank, M. (2015). Bacillus and other spore- forming genera: variations in responses and mechanisms for survival. Annu. Rev. Food Sci. Technol. 6, 351–369. doi: 10.1146/annurev-food-030713-092332 Orsburn, B., Melville, S. B., and Popham, D. L. (2008). Factors contributing to heat resistance of Clostridium perfringens endospores. Appl. Environ. Microbiol. 74, 3328–3335. doi: 10.1128/AEM.02629-07 Ragkousi, K., and Setlow, P. (2004). Transglutaminase-mediated cross-linking of GerQ in the coats of Bacillus subtilis spores. J. Bacteriol. 186, 5567–5575. doi: 10.1128/JB.186.17.5567-5575.2004 Dadd, A. H., Mccormick, K. E., and Daley, G. M. (1983). Factors inﬂuencing the resistance of biological monitors to ethylene oxide. J. Appl. Bacteriol. 55, 39–48. doi: 10.1111/j.1365-2672.1983.tb02645.x j 9). Bacillus subtilis spore coat. Microbiol. Mol. Biol. Rev. 63, 1–20. Rose, R., Setlow, B., Monroe, A., Mallozzi, M., Driks, A., and Setlow, P. (2007). Comparison of the properties of Bacillus subtilis spores made in liquid or on agar plates. J. Appl. Microbiol. 103, 691–699. doi: 10.1111/j.1365- 2672.2007.03297.x Driks, A. (2002). Maximum shields: the assembly and function of the bacterial spore coat. Trends Microbiol. 10, 251–254. doi: 10.1016/S0966-842X(02) 02373-9 Driks, A., and Eichenberger, P. (2016). The spore coat. Microbiol. Spectr. 4, 1–22. doi: 10.1128/microbiolspec.TBS-0023-2016 Sanchez-Salas, J. L., Setlow, B., Zhang, P., Li, Y. Q., and Setlow, P. (2011). SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.01636 October 2016 \| Volume 7 \| Article 1636 Frontiers in Microbiology \| www.frontiersin.org 9 Spore Proteomics in Different Sporulation Conditions Abhyankar et al. REFERENCES Maturation of released spores is necessary for acquisition of full spore heat resistance during Bacillus subtilis sporulation. Appl. Environ. Microbiol. 77, 6746–6754. doi: 10.1128/AEM.05031-11 Gerhardt, P., and Marquis, R. E. (1989). “Spore thermoresistance mechanisms,” in Regulation of Prokaryotic Development, eds I. Smith, R. A. Slepecky, and P. Setlow (Washington, DC: American Society for Microbiology), 43–63. Scopes, R. K. (1974). Measurement of protein by spectrophotometry at 205 nm. Anal. Biochem. 59, 277–282. doi: 10.1016/0003-2697(74)90034-7 Hullo, M. F., Moszer, I., Danchin, A., and Martin-Verstraete, I. (2001). CotA of Bacillus subtilis is a copper-dependent laccase. J. Bacteriol. 183, 5426–5430. doi: 10.1128/JB.183.18.5426-5430.2001 Setlow, B., Atluri, S., Kitchel, R., Koziol-Dube, K., and Setlow, P. (2006). Role of dipicolinic acid in resistance and stability of spores of Bacillus subtilis with or without DNA-protective alpha/beta-type small acid-soluble proteins. J. Bacteriol. 188, 3740–3747. doi: 10.1128/JB.00212-06 Imamura, D., Kuwana, R., Takamatsu, H., and Watabe, K. (2010). Localization of proteins to diﬀerent layers and regions of Bacillus subtilis spore coats. J. 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The Bacillus subtilis spore coat protein interaction network. Mol. Microbiol. 59, 487–502. doi: 10.1111/j.1365-2958.2005.04968.x Vizcaíno, J. A., Deutsch, E. W., Wang, R., Csordas, A., Reisinger, F., Rios, D., et al. (2014). ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat. Biotech. 32, 223–226. doi: 10.1038/nbt.2839 Kooiman, W. J., Barker, A. N., Gould, G. W., and Wolf, J. (1973). “The screw cap tube technique: a new and accurate technique for the determination of the wet heat resistance of bacterial spores,” in Spore Research, eds A. N. Barker, G. W. Gould, and J. Wolf (London: Academic Press), 87–92. REFERENCES Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Gould, and J. Wolf (London: Academic Press), 87–92. Little, S., and Driks, A. (2001). Functional analysis of the Bacillus subtilis morphogenetic spore coat protein CotE. Mol. Microbiol. 42, 1107–1120. doi: 10.1046/j.1365-2958.2001.02708.x Copyright © 2016 Abhyankar, Kamphorst, Swarge, van Veen, van der Wel, Brul, de Koster and de Koning. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Mckenney, P. T., Driks, A., and Eichenberger, P. (2013). The Bacillus subtilis endospore: assembly and functions of the multilayered coat. Nat. Rev. Microbiol. 11, 33–44. doi: 10.1038/nrmicro2921 McKenney, P. T., Driks, A., Eskandarian, H. A., Grabowski, P., Guberman, J., Wang, K. H., et al. (2010). A distance-weighted interaction map reveals a October 2016 \| Volume 7 \| Article 1636 Frontiers in Microbiology \| www.frontiersin.org 10
https://openalex.org/W1940355494	https://revistasojs.ucaldas.edu.co/index.php/lunazul/article/download/1736/1652	Spanish; Castilian	null	FUNCIONAMIENTO NORMAL Y AUMENTADO DE LA GLÁNDULA TIROIDES EN EQUINOS	Revista luna azul	2,012	cc-by	7,775	PALABRAS CLAVE Equinos, hormona tiroidea, metabolismo. RESUMEN Objetivo: Describir y analizar el funcionamiento normal de la glándula tiroides en equinos y los factores que aumentan en ellos los niveles de hormonas tiroideas. Materiales y métodos: Mediante la revisión de la literatura disponible de los últimos 50 años en las bases de datos BBCS-LILACS, fuente académica, IB-PsycINFO, IB- SSCI, IB-SciELO, Scopus y Scirus, al igual que artículos históricos, textos y referencias citadas en trabajos publicados. p Resultados: la información obtenida relacionada con los objetivos propuestos en la presente revisión, permite clasificar en 4 secciones a saber: síntesis y secreción de hormonas tiroideas; transporte y metabolismo; funciones; hipertiroidismo en los equinos. Conclusión: La glándula tiroides juega un papel importante, como productora de hormonas tiroideas, las cuales son necesarias para la diferenciación celular y crecimiento del organismo. El buen funcionamiento de las vías metabólicas depende de estas hormonas, las que tienen efectos específicos sobre diferentes órganos, manteniendo la homeostasis en todos los tejidos. Luna Azul ISSN 1909-2474 Luna Azul ISSN 1909-2474 No. 35, julio - diciembre 2012 ABSTRACT Objective: to describe and analyze the normal functioning of the thyroid gland in equines and the factors which increase the thyroid hormone levels. Materials and methods: information from the last 50 years included in the BBCS-LILACS, Fuente Académica, IB-PsycINFO, IB-SSCI, IB-SciELO, Scopus and Scirus, data bases as well as historical articles, texts and references cited in work published to date were analyzed. Results: important information related to the objectives 321 ©Universidad de Caldas Luna Azul ISSN 1909-2474 No. 35, julio - diciembre 2012 proposed in the present review was found and analyzed. It was then divided into two sections as follow: synthesis, liberation and metabolism of thyroid hormones; factors that modify the thyroid hormone levels. Conclusion: the thyroid gland plays an important role producing thyroid hormones which are necessary for cellular differentiation and organic growth. The adequate functioning of metabolic ways depends on these hormones, which have specific effects on different organs maintaining homeostasis between all the tissues. KEY WORDS: Equines, thyroid hormone, metabolism. Equines, thyroid hormone, metabolism. INTRODUCCION La glándula tiroides tiene dos orígenes diferentes embriológicos: Divertículo tiroideo y cuerpo timobraquial (tejido ectópico) (Toribio & Duckett, 2004). La primera se origina del endodermo de la laringe primitiva, luego se mantiene como un conducto estrecho al crecer y se denomina conducto tirogloso el cual finalmente se convierte en un pedúnculo sólido de células foliculares las cuales son responsables de la síntesis de tiroglobulina y de hormonas tiroideas (THs). (Toribio & Duckett, 2004). A partir de los cuartos arcos braquiales se origina el cuerpo timobraquial, donde se desarrollan las células para foliculares o células C, denominadas como fuente de la hormona tirocalcitonina (Sisson & Grossman, 2000). Tan pronto como transcurre la ontogénesis la función tiroidea comienza, pero el metabolismo continúa siendo basal, las estructuras hipotalámicas y el sistema pituitario portal es organizado para mantener el eje hipotálamo-pituitario- tiroides (HPT) funcional (Toribio & Duckett, 2004). La glándula tiroides se encuentra dorsal a la tráquea a nivel del tercer y sexto anillos, justo distal a la laringe, es de contextura lisa y firme, bilobulada donde ambos lóbulos se conectan por un istmo estrecho que contiene tejido fibroso, mide aproximadamente 2.5 cm x 2.5 cm x 5 cm, con un peso aproximado de 0.04g/kg del peso corporal (Blomme et al, 1999). Histológicamente la glándula está compuesta por células cúbicas en el epitelio bajo organizado en folículos. Las células epiteliales producen tiroglobulina, una glicoproteína que contiene residuos múltiples de tiroxina (Brehaus, 2011). En la mayoría de caballos saludables la glándula tiroides no es visible pero puede ser palpable como una revista.luna.azúl. 2012; 35: 321-337 ©Universidad de Caldas Luna Azul ISSN 1909-2474 No. 35, julio - diciembre 2012 estructura firme y movible. Posee una coloración gris rosácea, con una consistencia intermedia. El peso de la glándula en gramos de tejido tisular en comparación a kilogramos de masa corporal es mayor para fetos y potros (es decir 0.28 g/kg, en un rango de 0.12 a 0.67 g/kg), disminuyendo progresivamente con la edad. La relación con la masa corporal para adultos es en promedio 0.08 g/kg con un rango entre 0.01 a 0.15 g/kg. El peso total de una masa glandular normal de un potro recién nacido es alrededor de 15 g (Toribio & Duckett, 2004). SÍNTESIS Y SECRECIÓN DE HORMONAS TIROIDEAS El tejido tiroideo se forma de folículos de células epiteliales alrededor del lumen con un contenido coloide. Estos folículos se agrupan en una glándula discreta, la glándula tiroides. El epitelio celular de la tiroides toma el yodo y secreta la proteína tiroglobulina al interior del lumen de tales folículos, donde se encuentran más de 100 residuos de tirosina por molécula de tiroglobulina, luego de cierto número de residuos son yodados al grupo hidroxil fenólico a la forma 3-yodotirosina (T1) y 3,5-diyodotirosina (T2) (Hulbert, 2000). La mayor función de la tiroides activa es atrapar y conservar el yodo alcanzando un alto gradiente de concentración. El yodo puede ser absorbido en forma soluble, (yodo (I-)), por la mucosa intestinal u otra superficie mucosa o piel descubierta. El yodo activo es transportado y concentrado al interior por un simposio de Na+/I-. (Hulbert, 2000; Toribio & Duckett, 2004). Dicho yodo captado es oxidado por una peroxidasa tiroidea en la presencia de un peróxido de hidrógeno y se incorpora al interior de los residuos de tirosina de la forma tiroglobulina para formar precursores inactivos de la T1 y T2. Posteriormente se arman parejas entre estas dos yodotironinas dando lugar a la tiroxina (T4) y a la triyodotironina (T3) (Gϋrbϋz et al, 2005). Las THs y sus metabolitos son moléculas reconocidas en el organismo por contener yodo y la mayoría de la masa molecular de ambas hormonas tiroideas está constituida por átomos de yodo (Hulbert, 2000.) Existe otra hormona conocida como T3 inversa (rT3) la cual también ejerce efectos tiróideos (Baragli et al, 2011). Mientras las hormonas no son requeridas por el organismo son almacenadas a modo de tiroglobulina, la cual dependiendo de la necesidad del organismo se libera a la sangre como T4 o como T3, después de un proceso de hidrólisis que se encarga de separarla. La T4 posee 4 átomos de yodo por cada molécula, mientras que, la T3 posee 3 átomos de yodo por cada molécula (Dittrich et al, 2011). La mayoría de TH liberada a la circulación es T4, las concentraciones en suero de T4 total son 20 veces mayores que las concentraciones de T3 en suero. La síntesis hormonal y liberación son controladas por la disponibilidad del yodo y por la TSH (Christensen et al, 1997), La síntesis y secreción las THs es regulada por un sistema de retroalimentación negativo basado en el eje Hipotálamo-pituitaria-tiroides (HPT). INTRODUCCION La irrigación de la glándula tiroides está formada de la siguiente manera: 2 arterias tiroideas superiores, que son ramas de la carótida externa, cada una se trifurca en interna, externa y posterior, y 2 arterias tiroideas inferiores, son ramas de la subclavia, Cada una de ellas se trifurca en inferior, posterior, profunda (Sisson & Grossman, 2000). En algunas ocasiones es posible encontrar una arteria tiroidea media o tiroidea de Neubauer, cuyo origen se da en la aorta o en el tronco braquiocefálico, sus ramificaciones tortuosas se dirigen inicialmente por la superficie de la glándula y posteriormente se dirigen hacia el interior de su espesor (Gray H, 2000). La circulación venosa forma el plexo venoso tiroideo alrededor de la glándula, las venas que lo conforman se encuentran clasificadas de la siguiente manera: Venas tiroideas superiores, correspondientes a las arterias del mismo nombre, desembocan en la yugular interna directamente en algunos casos, en otros, lo hace en el tronco tirolinguofacial (Sisson & Grossman, 2000); venas tiroideas inferiores, se originan en el borde inferior de la tiroides desembocan en la yugulares internas y en el tronco braquiocefalico izquierdo (Sisson & Grossman, 2000); y venas tiroideas medias, localizadas entre las superiores y las inferiores desembocan en la yugular interna (Gray H, 2000). Es importante resaltar que las venas tiroideas son avalvulares, probablemente por su ubicación anatómica con respecto al corazón (Sisson & Grossman, 2000). La circulación linfática de la glándula tiroides se constituye por un plexo peritiroideo el cual está dividido en: linfático descendente, que filtra la linfa en ganglios que están ubicados en la cara anterior de la tráquea (Sisson & Grossman, 2000) y linfático ascendente, que filtra en los ganglios prelaríngeos y en los laterales del cuello (Gray H, 2000). La inervación de la glándula se conforma de el nervio simpático cervical porción del ganglio cervical medio y dos nervios laríngeos provenientes del superior recurrente (Gray, 2000). 323 ©Universidad de Caldas Luna Azul ISSN 1909-2474 No. 35, julio - diciembre 2012 SÍNTESIS Y SECRECIÓN DE HORMONAS TIROIDEAS La síntesis y secreción de la hormona tiroidea es incitada por la hormona estimulante de la tiroides tirotropina (TSH), la cual es una glicoproteína compuesta por 2 subunidades (α y β) y es secretada por las tirotropinas de la glándula revista.luna.azúl. 2012; 35: 321-337 ©Universidad de Caldas Luna Azul ISSN 1909-2474 No. 35, julio - diciembre 2012 pituitaria (Brehaus, 2011; Toribio & Duckett, 2004). La subunidad α es compartida por otras hormonas (hormona luteinizante y hormona folículo estimulante). La secreción de TSH de la pituitaria anterior (adenohipófisis) es controlada por los efectos de las neuronas hipotalámicas que desatan un tripéptido liberador de hormona tirotropina (TRH) (Hurcombe, 2011). La síntesis de tirotropina y la secreción es también controlada por retroalimentación negativa (inhibición a largo plazo) debido a la estimulación de las THs. Las concentraciones de TSH dependen de la tasa de dispersión de las THs y los niveles de TSH en la circulación al igual que en la medida que T4 se convierte en T3. El hipotálamo también puede inhibir (inhibición a corto plazo) la secreción de TSH a través de la dopamina y la somatostatina liberada en el medio eminente. Las neuronas que producen TRH positiva están presentes en diferentes áreas del cerebro (Hyypp, 2005). Con el hipotálamo la división parvocelular del núcleo paraventricular contiene la mayoría de las neuronas TRH positivas, y sus proyecciones finales en la zona externa de la eminencia media. (Brehaus, 2011; Hurcombe, 2011) En la glándula pituitaria, T3 disminuye la respuesta tirotropa (secreción de TSH) una vez la glándula es estimulada por la TRH, y en el hipotálamo la T3 disminuye la expresión del ARN mensajero de TRH al igual que la secreción. En la pituitaria, T4 es convertida a T3 por la desyodinasa tipo II (Hurcombe, 2011). Para que la T4 pueda acceder a las neuronas del hipotálamo, debe ser transportada a través del plexo coloide hacia el ventrículo lateral ligada a la transtiretina (proteína de unión a T4), una vez en el cerebro T4 es convertida a T3 por la desyodinasa tipo II. (Toribio, 2011; Hurcombe, 2011) La TRH es liberada en la eminencia media del hipotálamo y a través del sistema pituitario portal alcanza la pituitaria anterior (Johnson, 2002). TRANSPORTE Y METABOLISMO DE LAS HORMONAS TIROIDEAS EN EQUINOS La circulación de las hormonas tiroideas en la sangre, aparece de una manera pulsátil la cual es distinta según la especie (especie específico) con distintas variaciones ontogénicas, lo cual es responsable de la variación morfológica individual y del comportamiento sexual de cada animal (Crockford, 2008). La circulación de las THs es principalmente de T4, T3, y rT3. T4L y T3L son hormonas biológicamente activas, mientras que T3 y rT3 son productos de desyodación de T4. La T4 circulante es derivada directamente de la glándula tiroides. Sólo un 10% a un 20% de la T3 circulante es secretada directamente de la glándula tiroides (Allermeier et al, 2011) En los caballos los porcentajes de T4 circulante ligado a la globulina fijadora de tiroxina equivale a un 61%, ligado a prealbúmina fijadora de tiroxina un 22% y T4 fijada a albúmina un 17% (Buff et al, 1997). La T3 también corre ligada a la globulina fijadora de tiroxina y albúmina pero no se liga a la prealbúmina fijadora de tiroxina. Estas hormonas son unidas de manera reversible a las proteínas transportadoras, quienes actúan como reservorios de TH (Kratzsch & Pulzer, 2008). Las hormonas ya sean ligadas o libres están listas para atravesar el endotelio capilar y ejercer sus efectos biológicos en los tejidos tisulares. La proteína fijadora tiene como propiedad determinar la actividad biológica de cada hormona (Buff et al, 1997). La T3 es mucho más potente que la T4 y tiene una vida media más corta. La tiroxina tiene mayor afinidad por las proteínas transportadoras, sin embargo la T3 se contacta con mayor facilidad a los receptores de los tejidos periféricos, ya que la tiroxina puede actuar algunas veces como pro-hormona (Geor, 2008). La vida media de la T4 en los caballos es de aproximadamente 50 horas. Una larga fracción de THs se encuentra ligada a proteínas plasmáticas, por lo tanto, los cambios en las proteínas fijadoras pueden alterar los resultados de las concentraciones totales de TH (Toribio & Duckett, 2004). La degradación metabólica de la tironina después de la des-yodación incluye la des-aminación, la des-carboxilación y conjugación. Las enzimas responsables de la producción de T4 y rT3 también lo son de su destrucción. SÍNTESIS Y SECRECIÓN DE HORMONAS TIROIDEAS En la pituitaria, la interacción de TRH con los receptores específicos en los tirotropos inducen la liberación de TSH, y también induce la liberación de prolactina (Brehaus, 2011; Toribio & Duckett, 2004).) El eje HPT puede ser inhibido por altas temperaturas y estimulado por bajas temperaturas. El estrés inhibe la producción de TSH a través de la liberación hipotalámica de somatostatina (Ge´rard et al, 1999). La inflamación suprime la secreción de TSH. Las citoquinas (interleuquina 1 y 6) y el factor α de necrosis tumoral inhiben la secreción de TSH y estimulan la secreción de somatostatina (Toribio, 2011). 325 ©Universidad de Caldas Luna Azul ISSN 1909-2474 No. 35, julio - diciembre 2012 Luna Azul ISSN 1909-2474 TRANSPORTE Y METABOLISMO DE LAS HORMONAS TIROIDEAS EN EQUINOS Las tres clases de 5´- desyodinasas son las siguientes: Tipo I: Es encontrada principalmente en tejidos periféricos como la tiroides, el riñón y el hígado, es la responsable de la conversión de T4 a T3 y rT3, y es inhibida de forma no competitiva por el propiltiouracilo, su actividad es incrementada por la TSH y T3 y disminuida en hipotiroidismo. (Toribio & Duckett, revista.luna.azúl. 2012; 35: 321-337 ©Universidad de Caldas Luna Azul ISSN 1909-2474 No. 35, julio - diciembre 2012 2004); Tipo II: Está localizada en el tejido de grasa parda, en el cerebro y en la glándula pituitaria, no se afecta por el propiltiouracilo, y su función principal es convertir T4 a T3 a nivel intracelular (Johnson et al, 2004). Su actividad se incrementa en el hipotiroidismo probablemente por el mantenimiento intracelular de las concentraciones de T3 debido a una baja en las concentraciones de T4 periféricas, primordialmente en el cerebro. (Toribio & Duckett, 2004); Tipo III: Se encuentra en la placenta, desarrollo del cerebro y piel, su función principal es inactivar T4 y T3, convirtiendo T4 en rT3 y a T3 inactiva. (Toribio & Duckett, 2004). Los metabolitos son excretados vía urinaria y algunos son conjugados e introducidos a la circulación enterohepática. La mayoría del yodo regresa a la glándula tiroides (Toribio & Duckett, 2004). FUNCIONES DE LAS HORMONAS TIROIDEAS EN LOS EQUINOS La principal acción de las hormonas tiroideas es estimular el consumo de oxígeno. Las THs estimulan la síntesis y el catabolismo de proteínas, ayudan a regular el metabolismo lipídico, el metabilismo basal y la producción de calor corporal (Hulbert, 2000). Estas hormonas no son esenciales para la vida, pero juegan un papel importante en el crecimiento y maduración del organismo. Las acciones de las THs se dividen en genómicas y no genómicas (Brehaus, 2011). Las acciones genómicas son mediadas por receptores que requieren factores de transcripción nuclear que incrementa o disminuye la expresión de los genes que regulan la función celular (Brehaus, 2011). Las acciones no genómicas pueden ser iniciadas por los receptores de la membrana plasmática, dentro del citoplasma o la mitocondria. Tales acciones incluyen roles en la división celular, angiogénesis, transporte de proteínas, migración celular, agregación plaquetaria, síntesis de óxido nítrico y activación de la bomba de Na/k (sodio-potasio), modulación de calcio ATPasa y cambio de Na/H, modulación de neuronas y Na miocitos, y actividad del receptor del factor de crecimiento epidermal (Cianchetta et al, 2010; Nagy et al, 2000). La glándula tiroides contiene células parafoliculares, comúnmente referidas como células C. Dichas células secretan factores que ayudan a regular la actividad de las células foliculares, también secretan calcitonina, un importante regulador de las concentraciones de calcio extracelular (Breuhaus, 2011; Rudi’s et al, 2005). Los receptores de TH comienzan por la superfamilia de los receptores nucleares que trabajan como factores de transcripción. Los dos tipos de receptores son TR-α y TR- β. La T3 puede ser transportada directamente al interior 327 ©Universidad de Caldas Luna Azul ISSN 1909-2474 No. 35, julio - diciembre 2012 celular o derivada de T4 de manera intercelular (Hubert et al, 2007). Los receptores de tiroides interactúan con secuencias de ADN específicas (T3-específicas) regulando la expresión de los genes. El crecimiento y la termogénesis dependen de la presencia de THs. Las THs disminuyen la expresión de las subunidades α y β de los genes de TSH y de TRH. (Baragli et al., 2011). De estos efectos en la expresión genética, la acción de T3 resulta en termogénesis, incrementando la expresión de proteínas asociadas con la fosforilación oxidativa (Clutterbuck et al, 2010). FUNCIONES DE LAS HORMONAS TIROIDEAS EN LOS EQUINOS Aumenta el consumo de oxígeno, incrementa la síntesis de proteínas, incrementa la tasa metabólica, aumenta la absorción de carbohidratos y el metabolismo de la glucosa, la estimulación del crecimiento, maduración y eritropoyesis, incrementa el metabolismo lipídico y la conversión de colesterol en sales biliares, activa la lipasa lipoproteína, aumenta la sensibilidad del tejido adiposo para la lipólisis, estimula la tasa cardiaca, el gasto cardiaco y el flujo sanguíneo, incrementa la transmisión neural además del desarrollo cerebral y neuronal en los animales jóvenes (Toribio & Duckett, 2004; Baragli et al., 2011). Entre las funciones que cumplen las THs esencialmente se refieren al metabolismo y al desarrollo (Barsnick & Toribio, 2011). Además de las funciones dichas anteriormente también actúan sobre el equilibrio de las mucoproteínas y liquido extracelular, ayudan en la formación de la vitamina A por medio de los carotenos, los cuales son indispensables para el desarrollo del sistema nervioso tanto central como periférico, intervienen en los procesos de contracción muscular y motilidad intestinal; participan en el desarrollo y erupción de las piezas dentales (Tahboub & Arafah, 2009). Controlan la liberación y el uso de las hormonas del crecimiento, que son las encargadas de estimular al hígado a liberar (IGF-I) (Barsnick & Toribio, 2011; Buff et al, 2007). Las hormonas tiroideas son requeridas para la maduración bioquímica, hipertrofia, y penetración capilar del cartílago en crecimiento. La triyodotironina puede actuar de forma indirecta para promover la condrogénesis por estimulación de la pituitaria para secretar y sintetizar la hormona del crecimiento (Clutterbuck et al, 2010). Las THs incrementan el crecimiento del esqueleto fetal por medio de los receptores de la hormona del crecimiento y el factor de crecimiento insulínico tipo I, luego la THs influencia el crecimiento y el desarrollo esquelético por la actividad local del eje somatotrófico (Toribio & Duckett, 2004). Las THs modulan los efectos de los receptores adrenérgicos (Buff et al, 2007). Los tejidos tienen una mayor respuesta a las catecolaminas b-adrenérgicas cuando las concentraciones de THs son altas, e revista.luna.azúl. 2012; 35: 321-337 ©Universidad de Caldas Luna Azul ISSN 1909-2474 No. 35, julio - diciembre 2012 inversamente cuando hay hipotiroidismo. Se ha identificado que cuando hay una disminución en la densidad de los receptores b-adrenérgicos en los animales hipotiroideos disminuyen las fijaciones del sistema Adenin ciclasa y por último la función del receptor es minimizada (Crockford, 2003). FUNCIONES DE LAS HORMONAS TIROIDEAS EN LOS EQUINOS De igual forma las THs son necesarias para la formación normal y la estabilidad del citoesqueleto por medio de la migración y crecimiento neuronal. Las células del citoesqueleto son el principal sitio de acción de las THs, ya que, definen la arquitectura y motilidad celular (Buff et al, 2007). Un análisis por polimerización microtubular del desarrollo del cerebelo revela que la expresión de la familia de microtubulos asociados a proteínas deja 5 formas de tubulina que fueron reguladas por las THs (Rudi’s et al, 2005). Los microfilamentos son grandes componentes de las células del citoesqueleto y están compuestos por fibrillas y haces de fibras de polímeros de la proteína mecano-química actina. Las THs se encargan de regular dinámicamente la polimerización de actina en astrocitos por un proceso no genómico, pues poseen la capacidad de organizar la actina en el citoesqueleto y desarrollar el cerebelo rápidamente una vez que se logra regular la migración neuronal (Fitzgerald, Davison, 1998). Además de las THs el yodo también cumple una función importante a nivel del tejido ovárico, ya que este tejido ovárico no sintetiza yodo proteínas hormonales, por ende su actividad de tomar y almacenar yodo puede ser inhibida por fármacos como el tiocianato, perclorato y dosis excesivas de yodo, situación que es conocida como el efecto Wolff–Chaikoff, lo cual también ocurre en el tejido tiroideo (Slebodzi´nski, 2005). Las presencia de un transportador de Na+/I− symporter (NIS), en los tejidos que no son de la tiroides, permite considerar ésta como una de las opciones más viables a través de la cual los ovarios y otras estructuras del organismo toman y acumulan el yodo; Sin embargo, existen otros aniones que también son transportados por el NIS como el technetio y el pertecnato, los cuales inhiben de manera competitiva la captación del yodo (Ge´rard N et al, 1999). HIPERTIROIDISMO EN LOS EQUINOS La tirotoxicosis se desarrolla como un desorden primario de la glándula tiroides principalmente. El hipertiroidismo primario se caracteriza por síntomas y signos correspondientes al exceso de THs donde las concentraciones de T4L o de T3 o ambas se encuentran elevadas y los niveles de TSH se encuentran suprimidos. Raras veces la tirotoxicosis se produce por una sobreproducción de TSH por parte de la glándula pituitaria 329 ©Universidad de Caldas Luna Azul ISSN 1909-2474 No. 35, julio - diciembre 2012 con un crecimiento secundario de la tiroides e hiperfunción, lo cual se denomina hipertiroidismo central (Frank et al, 2002). Esta patología es de común presentación en animales de edad avanzada (más de 17 años) (Glover et al, 2009; Nicassio et al, 2008). Existen dos causas conocidas de hipertiroidismo central: tumores de la glándula pituitaria con excesiva producción de TSH (TSHomas) (Lucke & Lane, 1984) y síndrome de resistencia de la glándula pituitaria a la hormona tiroidea. Tales desórdenes producen manifestaciones clínicas de tirotoxicosis con elevadas concentraciones en suero de T4L y T3, pero se distingue por los hallazgos críticos de los niveles de TSH en suero, ya sea que se encuentre normal o elevada (Frank et al, 2002). Se cree que los TSHomas exhiben dos características que los diferencian de los tirotropos normales, la primera que son una proliferación celular excesiva; la segunda que poseen una producción autónoma de TSH como poca respuesta de los controles reguladores (McDermott & Ridgway, 1998; Beck- Peccoz et al, 2009). El bocio es definido como no inflamatorio, no neoplásico aumento de la glándula tiroides, puede ser clasificado en hiperplasia y coloide. Pueden ser nodulares o difusos (Geor, 2010). Normalmente los niveles bajos de tiroxina se deben a una producción de TSH desde la pituitaria. La glándula tiroides responde funcionalmente a esta hormona poli-péptida por incrementeo del yodo fijado, así como por la síntesis y secreción de tiroixina (Doige, 1981). Morfológicamente la estimulación de TSH produce una hipertrofia e hiperplasia de las células del epitelio folicular e incrementa la vascularidad de la glándula (Gerlach & Aurich, 2000). Dichos cambios contribuyen al aumento de la glándula tiroides. Si el yodo es insuficiente o si la síntesis de tiroxina es baja, mucho tejido coloide puede estar presente y esta glándula puede continuar creciendo (Doige, 1981). Tal tipo de bocio es conocido como hiperplásico o parenquimatoso. HIPERTIROIDISMO EN LOS EQUINOS El bocio fetal hiperplásico es el resultado de la estimulación de la glándula fetal por TSH. El tamaño del bocio fetal y el desarrollo de la hiperplasia se manifiesta como una variable dependiente del grado y la duración de la estimulación de TSH (Doige, 1981). Se cree que el bocio coloide representa la fase de involución del bocio hiperplásico. En este caso cuando el yodo es necesitado por el animal, los folículos acumulan coloide y crecen (Malinowski et al, 1996). De acuerdo a esta teoría se puede decir que el bocio coloide no es necesariamente asociado con hipotiroidismo (Doige, 1981). El adenoma es la neoplasia más común de la glándula tiroides de los equinos. El fenómeno va ligado a la edad avanzada (mayores de 16 años) (McDermott MT & Ridgway, 1998). Es de carácter beningo, generalmente revista.luna.azúl. 2012; 35: 321-337 ©Universidad de Caldas Luna Azul ISSN 1909-2474 No. 35, julio - diciembre 2012 unilateral y no está asociada con disfunción tiroidea. Ocasionalmente la talla del adenoma puede requerir la escisión quirúrgica. El pronóstico es bueno. La hemitiroidectomía de la glándula afectada restablece las concentraciones de TH (Frank, 2002). Los adenomas provenientes de las células foliculares de la tiroides son clasificados en los de tipo papilar y los de tipo folicular (Toribio & Duckett, 2004; Petersen & Schoot, 2005). La mayoría de los tumores provienen de las células epiteliales. Los tumores de células epiteliales son inmunohistoquimicamente positivos a tiroglobulina mientras que los tumores de células parafoliculares (células tipo c) y los derivados de ellos son positivos a calcitonina, a péptidos relacionados con el gen de la calcitonina (CGRP), cromogranina, enolasa neuro especifico (NSE) y sinaptofisina (Doige, 1981). El estudio confirma que los nódulos blancos de la tiroides ocurren frecuentemente en caballos de 20 o más años; de hecho en algunas ocasiones son hallazgos incidentales al incidir la glándula en las necropsias, los síntomas asociados con la hipersecreción ce calcitonina no se encontraban presentes. Su aparición no se relaciona con el sexo ni la raza de los animales (Ueki et al, 2004). La proliferación de tipo patológico de las células c se divide en hiperplasia, adenoma y carcinoma. HIPERTIROIDISMO EN LOS EQUINOS La hiperplasia es una acumulación focal de un tamaño menor al de un folículo lleno de coloide, su diagnóstico debe darse cuando existe un aumento definitivo en el número de células de tipo c en cada lóbulo de la tiroides al ser comparado con un grupo de control de animales de la misma edad (Frank, 2002). Las células C de los adenomas, bien diferenciadas, poseen un citoplasma abundante que es medianamente eosinofílico o claro en tinciones realizadas con hematoxilina eosina (HE) (Medica et al, 2011). Por su parte los carcinomas poseen más células que los adenomas de células c, además las células que conforman el tumor son más pleomórficas (Toribio & Duckett, 2004). Este tipo de tumores no aparenta ser funcional debido a que los caballos no manifiestan ninguna sintomatología referente a un exceso en la secreción de calcitonina y simplemente parecen ser una anormalidad relacionada con los caballos de edades avanzadas (Ueki et al, 2004). No existen reportes sobre hipertiroidismo en caballos asociado a una condición autoinmune (Messer et al, 1998) Los tumores suelen ocurrir en razas de bajo peso y en caballos de trabajo, son más frecuentes en caballos de edad avanzada que en animales jóvenes. Por otra parte la agregación celular tiene 3 clasificaciones: residuo ultimobraquial incrustado en el tejido tiroidal sin compresión de células adyacentes (Messer et al, 1998); hiperplasia nodular de residuo ultimobraquial que ( , ) No existen reportes sobre hipertiroidismo en caballos asociado a una condición autoinmune (Messer et al, 1998) Los tumores suelen ocurrir en razas de bajo peso y en caballos de trabajo, son más frecuentes en caballos de edad avanzada que en animales jóvenes. Por otra parte la agregación celular tiene 3 clasificaciones: residuo ultimobraquial incrustado en el tejido tiroidal sin compresión de células adyacentes (Messer et al, 1998); hiperplasia nodular de residuo ultimobraquial que 331 ©Universidad de Caldas Luna Azul ISSN 1909-2474 No. 35, julio - diciembre 2012 comprime las células foliculares adyacentes (Toribio & Duckett, 2004); y adenoma del segmento residual ultimobraquial rodeado por una cápsula fibrosa (Toribio & Duckett, 2004). Entre las manifestaciones clínicas más frecuentes se encuentran tremores, excitabilidad, taquicardia, taquipnea, sudoración, pérdida de peso a pesar de presentar un aumento en el apetito, temperamento hiperactivo, alopecia y caquexia (Ahmed et al, 2008). Los caballos que presentan tumores en la glándula pituitaria principalmente desarrollan síntomas como hirsutismo, letargia, laminitis, polidispsia y poliuria. HIPERTIROIDISMO EN LOS EQUINOS Si el caballo tiene hipertiroidismo o si las glándulas comienzan a aumentar de tamaño rápidamente, se debe considerar le remoción quirúrgica y comenzar con una terapia de soporte (Brehaus, 2011; Warner & Beckett, 1998). Si se contempla la cirugía, se debe realizar antes de que las glándulas crezcan lo suficiente para complicar la remoción (Alberts et al, 2001). Si el tamaño de la glándula no es un problema, pero el caballo es hipertiroideo, se puede administrar propiltiouracilo oral para disminuir las concentraciones de THs circulantes. Este fármaco actúa inhibiendo la síntesis de THs por antagonismo de la peroxidasa tiroidea (la cual cataliza la oxidación de yoduro a yodo), inhibiendo la síntesis de tiroglobulina, inhibiendo el crecimiento de las células foliculares de la tiroides e inhibiendo la conversión de T4 a T3. Se administra a una dosis de 8mg/kg/día, pero una vez que disminuyan los niveles séricos de THs se puede suministrar cada 48 horas. Tratamiento. Si la neoplasia es diagnosticada y las concentraciones en suero de THs son normales y no hay todavía un agrandamiento de la glándula suficiente para comprometer la respiración o la ingestión de alimentos no es necesario remover las glándulas tiroides inmediatamente. Si las concentraciones de THs en suero son bajas, el caballo puede suplementarse con uno de los productos severos (Alberts et al, 2001). Si el caballo tiene hipertiroidismo o si las glándulas comienzan a aumentar de tamaño rápidamente, se debe considerar le remoción quirúrgica y comenzar con una terapia de soporte (Brehaus, 2011; Warner & Beckett, 1998). Si se contempla la cirugía, se debe realizar antes de que las glándulas crezcan lo suficiente para complicar la remoción (Alberts et al, 2001). Si el tamaño de la glándula no es un problema, pero el caballo es hipertiroideo, se puede administrar propiltiouracilo oral para disminuir las concentraciones de THs circulantes. Este fármaco actúa inhibiendo la síntesis de THs por antagonismo de la peroxidasa tiroidea (la cual cataliza la oxidación de yoduro a yodo), inhibiendo la síntesis de tiroglobulina, inhibiendo el crecimiento de las células foliculares de la tiroides e inhibiendo la conversión de T4 a T3. Se administra a una dosis de 8mg/kg/día, pero una vez que disminuyan los niveles séricos de THs se puede suministrar cada 48 horas. El manejo del propiltiouracilo debe ser cuidadoso, el personal debe usar siempre guantes y máscara (Brehaus, 2011). HIPERTIROIDISMO EN LOS EQUINOS Otros signos incluyen disminución de la fertilidad, depresión inmunitaria, hiperhidrosis, convulsiones, diabetes insípida, anovulación persistente, endometritis y pseudolactancia. (Durham, 2010; Walsh et al, 2009). Diagnóstico. Los altos niveles de T3 y T3L, durante los 10 a 13 meses de edad se pueden asociar al aumento del uso de T4L o a cambios en la tasa de monodeionización de T4 o de T3 (Fazio et al, 2007). Las concentraciones de THs en plasma se encuentran elevadas, sin embargo éstas pueden aumentar por estados fisiológicos no asociados a signos clínicos anormales, como en la preñez debido a los altos requerimientos por parte del feto para su desarrollo (Allermeier et al, 2011). Se utilizan los métodos tanto directos como indirectos citados en la sección de hipotiroidismo, donde las concentraciones de las hormonas se encuentran por encima de los valores establecidos (Divers, 2008; Ramirez, 2010). La aspiración o biopsia simple puede ayudar a diferenciar los quistes, neoplasias, bocio hiperplásico, bocio coloide e inflamación (Toribio & Duckett, 2004). Para las imágenes diagnósticas se utiliza el pertecnato (tectenium -99m) que es un componente usado para diagnosticar el mecanismo de captación del yodo. La imagen scintigráfica ha sido usada clínicamente para evaluar la glándula tiroides de los caballos. Se ha observado patrones anormales en el carcinoma de tiroides. La evaluación ultrasonográfica de las glándulas puede diferenciar estructuras sólidas quísticas, ha sido utilizada también para evaluar la morfología de la glándula tiroides (Frank, 2002). La scintigrafía es muy usada para la evaluación de la función tiroidea, la detección de enfermedades metastásicas y para medir el volumen de los tumores metabólicos en humanos y en pequeños animales, de igual forma se ha descrito su uso en caballos. Tal técnica es especialmente usada para determinar la función de la glándula y si hay diferencias de función entre los lóbulos, lo cual es de gran importancia al momento de tomar una decisión quirúrgica (hemitiroidectomía) (Breuhaus, 2011; Roser, 2008). revista.luna.azúl. 2012; 35: 321-337 ©Universidad de Caldas Luna Azul ISSN 1909-2474 No. 35, julio - diciembre 2012 Tratamiento. Si la neoplasia es diagnosticada y las concentraciones en suero de THs son normales y no hay todavía un agrandamiento de la glándula suficiente para comprometer la respiración o la ingestión de alimentos no es necesario remover las glándulas tiroides inmediatamente. Si las concentraciones de THs en suero son bajas, el caballo puede suplementarse con uno de los productos severos (Alberts et al, 2001). BIBLIOGRAFÍA  Abraham, G.; Allersmeier, M.; Schusser, G.F.; Ungemach, F. R. (2011). Serum thyroid hormone, insulin, glucose, triglycerides and protein concentrations in normal horses: Association with topical dexamethasone usage. Vet J; 188: 307–312.  Abraham, G.; Allersmeier, M.; Schusser, G.F.; Ungemach, F. R. (2011). Serum thyroid hormone, insulin, glucose, triglycerides and protein concentrations in normal horses: Association with topical dexamethasone usage. Vet J; 188: 307–312.  Ahmed, O.M.; El-Gareib, A.W.; El-bakry, A.M.; Abd El- Tawab, S.M.; Ahmed R.G. (2008). 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https://openalex.org/W3146965191	https://aladabj.uobaghdad.edu.iq/index.php/aladabjournal/article/download/1126/884	Arabic	null	الاصلاحات الاقتصادية في العصر البويهي" في العراق	Al-ādāb	2,021	cc-by	10,619	DOI: 10.31973/aj.v1i136.1126 DOI: 10.31973/aj.v1i136.1126 Economic reforms in the Bohemian era in Iraq from the period (334-447 Ah) M. Dr. Haidar Salem Mohammed Al-Maliki Ministry of Education / General Directorate of Third Rasafa Education halmaiky1982@gmail.com Economic reforms in the Bohemian era in Iraq from the period (334-447 Ah) (334-447 Ah) M. Dr. Haidar Salem Mohammed Al-Maliki Ministry of Education / General Directorate of Third Rasafa Education halmaiky1982@gmail.com 0206 / م6110 هـ 0206 / م6110 هـ جمةل الآداب / العدد631 (آآذار) جمةل الآداب / العدد631 (آآذار) Abstract: د حيـــدر ســـالم محمـــد المالكي وزارة التربية / المديرية العامة لتربية الرصافة الثالثة halmaiky1982@gmail.com )(مُمَخَّصُ البَحث لم تكن الخالفة العباسية منذ بزوغيا عمى الساحة السياحة، قد أخذذ عمذى عاتايذا زمذام األمور من الناحية الدينية، والسياسية، في المشرق االسالمي، فمذم تكذن تمذأل األرض خا ذعة ليا بشكلٍ مستمر كمذا ىذو الحذال فذي ب،ذداد، فبذين الحذين ارخذر تظيذر دذورا عديذد ذدىا لمبعذذد الر،رافذذذي عذذذ ن الخالفذذذة العباسذذذية فذذذي ب،ذذذداد، وفذذي الوقذذذ ن سذذذو بذذذدا ال ذذذع يذذذد فذذذي .الخالفة، وىذا ما حصال في العيد البوييي" الذي ىو مو ع بحدنا ىذا 322 0206 / م6110 هـ جمةل الآداب / العدد631 (آآذار) جمةل الآداب / العدد631 (آآذار) عنذذذدما تذذذولى البويييذذذون اامذذذار فذذذي المشذذذرق، ووصذذذموا الذذذى ب،ذذذداد تذذذدىور األو ذذذاع االقتصادية في عاصمة الخالفة العباسية، ألسبا عديد منيا أنَّ أواخر عيد العباسي األول ،ظير الكدير من الدورا في ب،داد، مما سب الروع والاحط، ف ذالً عذن انتشذار الطذاعون فعطمذذ الحيذذا ، وأىم مذذ الزراعذذة الاذذو األساسذذي لمسذذكان انذذذاأل، وممذذا سذذب فذذي شذذل حركذذة الترار . ف ال عذن ذلذأل أنّأ أمذرال البذويييين لذم يرذدوا األمذوال الكافيذة فذي ب،ذداد، ممذا دفعيذم .الى إعطال األرا ي اقطاعا ، فبدأ ظيور الدورا الراف ة ليم ،الكممات المفتاحية: االصالح، االقتصادي، في العهد.البويهي المقدمة بعذذد ذذع الخالفذذة العباسذذية فذذي ب،ذذداد وخراسذذان ، تمكذذن بنذذي بويذذو" مذذن بسذذط ن ذذوذىم عمييا ، فأصبح مااليد الدولة بيدىم ، سوال اكذان مذن الناحيذة السياسذية أم االقتصذادية فعمذى سب يل المدال ال الحصر تعين الخم ال ، أو األمرال وغيرىم من قبل امرال بني بويذو"، فع نذد قيذام كل دولة تمر بمراحل من ال ع الى الاذو سذوال أكذان مذن الناحيذة السيا سذية أو االقتصذادية أو االرتماعية ، وىذذا مذا شذيدتو الخالفذة العباسذية فذي تمذأل الحابذة ، فكانذ تمذر ب،ذداد بتذدىور االو ذذذاع السياسذذذية واالقتصذذذادية، فعنذذذد دخذذذول بنذذذي بويذذذو" ، كانذذذ ب،ذذذداد تعذذذاني مذذذن أزمذذذا اقتصذذا دية، وصذذو بة المعيشذذة فييذذا، وغذذالل األسذذعار ، ممذذا أدى الذذى شذذل الدولذذة كاممذذة، وسذذر ىذه األزما حتى الخم ال واألمرال وقاد الريش ، فور عمذى أمذرال بنذي بويذو" إيرذاد الحمذول لتمذذذأل األزمذذذا ، فكذذذان البحذذذو يتنذذذاول اارذذذرالا التذذذي اتخذذذذىا بنذذذي بويذذذو" فذذذي معالرذذذة تمذذذأل . Abstract: The Abbasid caliphate, since its emergence on the tourism scene, which took control of the religious and political aspects of the Islamic Orient, was not subject to it continuously, as in Baghdad, among the other Hassan shows several revolutions, against it for the geographical distance from the Abbasid caliphate in Baghdad, or the emergence of princes of the caliphate, but or is it weak as a result of the takeover of weak princes, or not? The emergence of princes of strength in number and number, and at the same time the weakness of the caliphate, and this is what happened in the faith of the Bohemian Covenant, which is the subject of our discussion. When they took over the Bohemian Emirate of the Orient, and arrived in Baghdad, the economic situation deteriorated in the capital of the Abbasid caliphate, for several reasons, including that the late Abbasid era, many revolutions appeared in Baghdad, causing hunger and drought, as well as the spread of the plague, disrupting life, neglecting agriculture the basic forces of the population at that time, and causing the paralysis of the trade movement. Moreover, the Princes of The Boi's Yin did not find enough money in Baghdad, which led them to give the territories interruptions, and the emergence of revolutions rejecting them. Keywords: Hervorming, Ekonomiese, Verbond, Boheemse. االصالحات االقتصادية في العصر "البويهي في العراق في عهد األميرين معز الدولة وعضد الدولة لممدة ( 333 - 334هـ) م. (1) ٗتهفظ انرثٍ انظ٘ ذأكهّ انضٔاب: اؿى يذهح كثٛغج كاَد تثغدضاص يٕعدغ يبداتغ شدغٚق انردٙ قٛٓدا شثدغ يٕؿد ًٕ٘انكاظى ( ػهٛٓى انـالو ) .... ُٚظغ( ٚاشٕخ انذ1995 : 1 306 ) (Ruby Al-Hamwi, 1995:1, 306 . ) Abstract: ودفن ببا التبن 1 ) فذي ماذابر قذريش وكانذ إمارتذو إِحذدى وعشذرين ،سذنة وأحذد عشر .ًشيرا ،ابن الوردي1663 : 1 / 353 ) Seun van Pienk, 1996: 1/282 ). ،وشذذذ الرنذذذد عمذذذى األميذذذر معذذذز الدولذذذة" فذذذي سذذذنة 222 /ه623 )م ،أي ذذذا وأسذذذمعوه المكذذروه بسذذب ،أرزاقيذذم فوعذذدىم إلذذى ، مذذد فا ذذطر إلذذى أخذذذ األمذذوال مذذن غيذذر ،ورييذذا دذذم أقطع الارى رميعيا التي كان ،لمسمطان وأصحا األمالأل. ، النويري1232 :ه33 / 154 ) (Al-Nawiri, 1423H: 26/187. واىذتم معذز الدولذة" فذي سذنة 226 /ه631 م) فذي معالرة مشكمة موعد رباية الخراج وعالريا، وتُنس كل ىذا االصذالح الذى وزيذره أبذي محمذد الكشذذ عذذن أو ذذاع ب،ذذداد فذذي تمذذأل الحابذذة، ومذذدى الريذذود التذذي بذذذليا بنذذو بويذذو" فذذي االصالح االقتصادي سوال أكان من الناحية الترار والزراعية والصناعة، أم مدى تأديره عمى .الحيا االقتصادية وتطورىا في ب،داد :أوال الو ع االقتصادي في عيد معز الدولة " 222 - 223ىذذذذ / 623ذذذ 645م) :دانيا الو ع االقتصادي عيد ع د الدولة " 234 - 243 ه / 656ذذ 662م ) أوال: الوضع االقتصادي في عهد معز الدولة" ( 333 - 353 /هــــ653 ـــ645 )م أوال: الوضع االقتصادي في عهد معز الدولة" ( 333 - 353 /هــــ653 ـــ645 )م ىو أبو الحسين أحمد بن بي شراع بويذو "بذن فناخسذرو بذن تمذام بذن كذوىي بذن شذيرزيل األص،ر بن شيركوه بن شيرزيل األكبر بذن شذيران شذاه بذن شذيرفنو بذن شسذتان شذاه يذن سسذن فرو بن شروذيل بن سناد بن بيرام رور الممأل بن يزد رردين بذن ىرمذز كرمانشذاه بذن سذابور ذي األكتا ، وباية النس معروفة فذي ممذوأل بنذي .ساسذان ابن،خمكذان 1611 : 1 / 142 - 142 ) (Ibn Khalkan, 1900: 1/174-175) وفي سنة سذ وخمسذين ودالدمائذة مذا معز الدولة "بذن بويذو " .بب،ذداد ،ابذن المعبذري1661 : 1 / 136 ) Ibn Al-Amari, 1991: 1/169) ). ودفن ببا التبن 1 ) فذي ماذابر قذريش وكانذ إمارتذو إِحذدى وعشذرين ،سذنة وأحذد عشر .ًشيرا ،ابن الوردي1663 : 1 / 353 ) Seun van Pienk, 1996: 1/282 ). ،وشذذذ الرنذذذد عمذذذى األميذذذر معذذذز الدولذذذة" فذذذي سذذذنة 222 /ه623 )م ،أي ذذذا وأسذذذمعوه المكذذروه بسذذب ،أرزاقيذذم فوعذذدىم إلذذى ، مذذد فا ذذطر إلذذى أخذذذ األمذذوال مذذن غيذذر ،ورييذذا دذذم أقطع الارى رميعيا التي كان ،لمسمطان وأصحا األمالأل. ، النويري1232 :ه33 / 154 ) (Al-Nawiri, 1423H: 26/187. Abstract: األزما وأنَّ ااصذذال حا والخذذدما التذذي قذذدميا بعذذض األمذذرال البذذويييين، وأخذذص بالذذذكر معذذز الدولذذة"، وع ذذد دولذذة" خذذالل تمذذأل الحابذذة التذذي حكمذذوا فييذذا العذذراق، إذ إنّ العذذراق انذذتعش فذذي بعذذذذذض الحذذذذذاال فذذذذذي مختمذذذذذ الحيذذذذذا ،كالزراعذذذذذة ، والترذذذذذار والصذذذذذناعة، عمذذذذذى الذذذذذرغم مذذذذذذن .اال طرابا التي كان تحصل ىنا وىناأل ركذذز المصذذادر التاريخيذذة عمذذى الرانذذ السياسذذي فذذي ب،ذذداد فذذي العيذذد البذذوييي، وفذذي حذذين أنَّيذذا كانذذ معموماتيذذا قميمذذة عذذن االصذذالحا التذذي أرراىذذا بنذذو بويذذو" ،فذذي تمذذأل الحابذذة ف ذذذذال عذذذذن ذلذذذذأل لذذذذم تتحذذذذدو االَّ عذذذذن المشذذذذاكل التذذذذي عصذذذذ بب،ذذذذداد، ومذذذذن خذذذذالل البحذذذذو واالستاصذذذذذال، والبحذذذذذذ و فذذذذذي طيذذذذذذا السذذذذذطور أمذذذذذذا الكتذذذذذ ورذذذذذذد معمومذذذذذا قميمذذذذذذة عذذذذذذن االصذذالحا فذذي المذذد222 - 243 ه)، فذذي حذذين أنَّيذذا أىممذذ الحابذذة المتبايذذة، لذذذلأل لذذذم .أعدر عن أيِّ معموما عنيا، وىذه من الصعوبا التي وارييا الباحو 322 0206 / م6110 هـ 0206 / م6110 هـ جمةل الآداب / العدد631 (آآذار) اهمية البحث:: ترتكز اىمية البحو عمى 1 - تشخيص الواقع الذي مر بو ب،داد إبّان حكم البوييي" .من الناحية االقتصادية 3 - المساعي التي سعى الييا بنو بويو" . في اصالح الزراعي والصناعة والترار 2 - تحديد المعرال التي أرراىا بنو بويو" .لإلصالح االقتصادي 2 - تطور ب،داد في عيد األمرال البويييين" .في ظل الخروج من األزما االقتصادية أهداف ال:بحث اااإ 2 - تطور ب،داد في عيد األمرال البويييين" .في ظل الخروج من األزما االقتصادية أهداف ال:بحث الكشذذ عذذن أو ذذاع ب،ذذداد فذذي تمذذأل الحابذذة، ومذذدى الريذذود التذذي بذذذليا بنذذو بويذذو" فذذي االصالح االقتصادي سوال أكان من الناحية الترار والزراعية والصناعة، أم مدى تأديره عمى .الحيا االقتصادية وتطورىا في ب،داد :قسمت البحث الى :أوال الو ع االقتصادي في عيد معز الدولة " 222 - 223ىذذذذ / 623ذذذ 645م) :دانيا الو ع االقتصادي عيد ع د الدولة " 234 - 243 ه / 656ذذ 662م ) أوال: الوضع االقتصادي في عهد معز الدولة" ( 333 - 353 /هــــ653 ـــ645 )م ىو أبو الحسين أحمد بن بي شراع بويذو "بذن فناخسذرو بذن تمذام بذن كذوىي بذن شذيرزيل األص،ر بن شيركوه بن شيرزيل األكبر بذن شذيران شذاه بذن شذيرفنو بذن شسذتان شذاه يذن سسذن فرو بن شروذيل بن سناد بن بيرام رور الممأل بن يزد رردين بذن ىرمذز كرمانشذاه بذن سذابور ذي األكتا ، وباية النس معروفة فذي ممذوأل بنذي .ساسذان ابن،خمكذان 1611 : 1 / 142 - 142 ) (Ibn Khalkan, 1900: 1/174-175) وفي سنة سذ وخمسذين ودالدمائذة مذا معز الدولة "بذن بويذو " .بب،ذداد ،ابذن المعبذري1661 : 1 / 136 ) Ibn Al-Amari, 1991: 1/169) ). Abstract: واىذتم معذز الدولذة" فذي سذنة 226 /ه631 م) فذي معالرة مشكمة موعد رباية الخراج وعالريا، وتُنس كل ىذا االصذالح الذى وزيذره أبذي محمذد اا ىو أبو الحسين أحمد بن بي شراع بويذو "بذن فناخسذرو بذن تمذام بذن كذوىي بذن شذيرزيل األص،ر بن شيركوه بن شيرزيل األكبر بذن شذيران شذاه بذن شذيرفنو بذن شسذتان شذاه يذن سسذن فرو بن شروذيل بن سناد بن بيرام رور الممأل بن يزد رردين بذن ىرمذز كرمانشذاه بذن سذابور ذي األكتا ، وباية النس معروفة فذي ممذوأل بنذي .ساسذان ابن،خمكذان 1611 : 1 / 142 - 142 ) (Ibn Khalkan, 1900: 1/174-175) وفي سنة سذ وخمسذين ودالدمائذة مذا معز الدولة "بذن بويذو " .بب،ذداد ،ابذن المعبذري1661 : 1 / 136 ) Ibn Al-Amari, 1991: 1/169) ). ودفن ببا التبن 1 ) فذي ماذابر قذريش وكانذ إمارتذو إِحذدى وعشذرين ،سذنة وأحذد عشر.ًشيرا ،ابن الوردي1663 : 1 / 353 ) Seun van Pienk, 1996: 1/282 ). ر ير بن ور ي/ ) , 996 / 8 ) ،وشذذذ الرنذذذد عمذذذى األميذذذر معذذذز الدولذذذة" فذذذي سذذذنة 222 /ه623 )م ،أي ذذذا وأسذذذمعوه المكذذروه بسذذب ،أرزاقيذذم فوعذذدىم إلذذى ، مذذد فا ذذطر إلذذى أخذذذ األمذذوال مذذن غيذذر ،ورييذذا دذذم أقطع الارى رميعيا التي كان ،لمسمطان وأصحا األمالأل. ، النويري1232 :ه33 / 154 ) (Al-Nawiri, 1423H: 26/187. واىذتم معذز الدولذة" فذي سذنة 226 /ه631 م) فذي معالرة مشكمة موعد رباية الخراج وعالريا، وتُنس كل ىذا االصذالح الذى وزيذره أبذي محمذد (1) ٗتهفظ انرثٍ انظ٘ ذأكهّ انضٔاب: اؿى يذهح كثٛغج كاَد تثغدضاص يٕعدغ يبداتغ شدغٚق انردٙ قٛٓدا شثدغ يٕؿد ًٕ٘انكاظى ( ػهٛٓى انـالو ) .... ُٚظغ( ٚاشٕخ انذ1995 : 1 306 ) (Ruby Al-Hamwi, 1995:1, 306 . ) 322 322 0206 / م6110 هـ 0206 / م6110 هـ جمةل الآداب / العدد631 (آآذار) الميمبذذي" 3 )، كمذذا أنَّذذو دخذذل البصذذر "، وتظمذذم إليذذذو أىميذذا "مذذن العبذذر التذذي رعمذذ عمذذييم فذذذي أر ي "الحنطة"، والشعير فوعدىم بكل ما أنسوا بو. دم قرر أمرىم عمى أن يردوا إلى رسذميم الاذذديم فذذي أخذذذ العشذذر حبذذا بعينذذو مذذن غيذذر ،ترييذذع وال تسذذعير، ونظذذر فيمذذا بذذين ذلذذأل وبذذين مذذا يؤخذذذ مذذنيم عمذذى تاريذذ فأشذذار عمذذى أربذذا العشذذر أن يبتذذاعوا ف ذذل مذذا بذذين المعاممذذة عمذذى الظمذذم والمعاممذذة عمذذى اانصذذا بذذدمن يرغذذ فيذذو معذذز الدولذذة "عذذارال فيسذذيل عميذذو مذذا يذذنحط من االرت اع مع ما يتعرل لو من المال دم ي ا إلى ذلأل ما يدمره العدل وموقعو من قمو الناس مع الررال في المستابل لزياد االرت اع. Abstract: ،فاسترابوا وتارر األمر بينيم عمذى أل ذى ألذ درىم ومائتي أل درىم وكتذ ليذم بذذلأل ودياذة دذم حذط مذن الرميذع عذن ال ذع ي مذائتي ألذ درىذذذم وكتذذذ إلذذذى معذذذز الدولذذذة " َّبذذذأن فذذذي ذلذذذأل حظذذذا وصذذذالحا ووفذذذورا فذذذي ارت ذذذاع الناحيذذذة فذذذي المستابل فحسن موقع فعمو من معز الدولة" فأم ا ،مسكويو 3:3111 / 131 ) (Miskoy, 2000:6/160 .) وفي سنة 222 /ه623م) عنذد اشذتداد ،ال،ذالل اشذترى معذز الدولذة"، كذرّا معذذدّال ،حنطذذة بعشذذرين ألذذ درىذذم، كذذان ييذذد فذذي ذلذذأل تخ ذذيض السذذعر بعذذدىا، فمالذذ إليذذو العامذة إلذى أيذام وأحبذوه. التنذوخي " ، 1261 ىذذ : 1 ، 223 ) (Al-Tankhi) , 1391 E:1, 352) . وقذذذام معذذذز الدولذذذة " فذذذي سذذذنة222 /ه624 )م فذذذي سذذذد بدذذذق نيذذذر الرفيذذذل 2 ) وسذذذد بدذذذق بادوريا 2 ) فإنو خرج بن سو حتى سد ىذا البدق وحمل التذرا بن سذو فذي بركذة قبائذو حتذى فعذل رميذذع العسذذكر مدذذل فعمذذو وسذذد ذلذذأل البدذذق دذذم خذذرج إلذذى النيروانذذا 2 ) فسذذد بداابيذذا وكانذذ النيروانذذا قذذد بطمذذ وكذذذلأل بادوريذذا فممذذا سذذد بدوقيذذا عمذذر ب،ذذداد وبيذذع الخبذذز الناذذي رطذذال بذذذذدرىم فمالذذذذ العامذذذذة إلذذذذى أيذذذذام معذذذذز الدولذذذذة " .وأحبذذذذوه ،مسذذذذكويو 3111 : 3 / 312 - 312 ) (Miskoy, 2000:6/203-204) (2) انذـٍ تٍ يذًض تٍ ػثض هللا"تٍ ْاعٌٔ أتٕ يذًدض اندٕػٚغ انًٓهثدٙ" يدٍ ٔندض انًٓهدة" اتدٍ أتدٙ دفغج ٍٛكداٌ كاذددة يؼددؼ انضٔنددح"اتٍ تٕٚددّ ٔنًددا يدداخ انزددٛغيٙ شهددضِ يؼددؼ انضٔنددح"انٕػاعج يكاَددّ ؿددُح ذـددغ ٔشالشدد ٔشهصًائددح ٔشغتددّ ٔأصَددا ِ ٔاسددرب تددّ ٔػظددى ناْددّ ػُددضِ ٔكدداٌ ٚددضتغ أيددغ انددٕػاعج نهً ٛددغ يددٍ ٛددغ ذـددًٛح انددٕػاعج شددى نددضصخ نددّ انشهددغ يددٍ صاع انشالقددح تانـددٕاص ٔانـددٛا ٔانًُ بددح ٔنبثددّ انً ٛددغ تددانٕػاعج ٔصتددغ ٍٛانضٔنر . ٔكاَد ٔقاذّ ؿُح اشُرٍٛ ٔسًـٍٛ ٔشهصًائح ت غٚق ٔاؿظ ٔدًم ئنٗ تغضاص ٍٛٔ ئ ٗ ٔ غٚق ٔ ٍٛ ٔ ٍٛ ٔ ٔ ٔ ُٚظددغ : (اتددٍ كدد "ٙاكغ انكرثدد1993 : 1 / 353 – 359 )( (Ibn Shaker al-Kutbi, 1973:1/353- 357 . (3)أ (3) َٓغ ٚزة قٙ صنهح تثغضاص يأسظِ يٍ َٓغ ػٛـدٗ ْٔدٕ اندظ٘ ػهٛدّ شُ دغج انلدٕي ... ُٚظدغ : (اتدٍ ػثدض "انذق1412 : ْـ3 1402 ) ( Ibn Abdul Haq), 1412 E: 3, 1402 . ) 4ا (3) َٓغ ٚزة قٙ صنهح تثغضاص يأسظِ يٍ َٓغ ػٛـدٗ ْٔدٕ اندظ٘ ػهٛدّ شُ دغج انلدٕي ... ُٚظدغ : (اتدٍ ػثدض "انذق1412 : ْـ3 1402 ) ( Ibn Abdul Haq), 1412 E: 3, 1402 . 323 (2) انذـٍ تٍ يذًض تٍ ػثض هللا"تٍ ْاعٌٔ أتٕ يذًدض اندٕػٚغ انًٓهثدٙ" يدٍ ٔندض انًٓهدة" اتدٍ أتدٙ دفغج ٍٛكداٌ كاذددة يؼددؼ انضٔنددح"اتٍ تٕٚددّ ٔنًددا يدداخ انزددٛغيٙ شهددضِ يؼددؼ انضٔنددح"انٕػاعج يكاَددّ ؿددُح ذـددغ ٔشالشدد ٔشهصًائددح ٔشغتددّ ٔأصَددا ِ ٔاسددرب تددّ ٔػظددى ناْددّ ػُددضِ ٔكدداٌ ٚددضتغ أيددغ انددٕػاعج نهً ٛددغ يددٍ ٛددغ ذـددًٛح انددٕػاعج شددى نددضصخ نددّ انشهددغ يددٍ صاع انشالقددح تانـددٕاص ٔانـددٛا ٔانًُ بددح ٔنبثددّ انً ٛددغ تددانٕػاعج ٔصتددغ ٍٛانضٔنر . ٔكاَد ٔقاذّ ؿُح اشُرٍٛ ٔسًـٍٛ ٔشهصًائح ت غٚق ٔاؿظ ٔدًم ئنٗ تغضاص ُٚظددغ : (اتددٍ كدد "ٙاكغ انكرثدد1993 : 1 / 353 – 359 )( (Ibn Shaker al-Kutbi, 1973:1/353- 357 . (3) َٓغ ٚزة قٙ صنهح تثغضاص يأسظِ يٍ َٓغ ػٛـدٗ ْٔدٕ اندظ٘ ػهٛدّ شُ دغج انلدٕي ... ُٚظدغ : (اتدٍ ػثدض "انذق1412 : ْـ3 1402 ) ( Ibn Abdul Haq), 1412 E: 3, 1402 . ) (4) ٙطـٕض" يٍ كٕعج االؿراٌ تانجاَة انغغتٙ يٍ تغضاص ْٕٔ انٕٛو يذـٕب يٍ كٕعج َٓغ ػٛـدٗ تدٍ ػهد : يُٓا: انُذاؿٛح ٔانذاعشٛح َٔٓغ أعيا ٔقٙ طغقّ تُٙ تؼض تغضاص ... ُٚظغ( "ٕ٘ٚاشٕخ انذًد1995 : 1 319 ) (Ruby Al-Hamwi, 1995:1, 317) . (5) يٛدداِ انُرٓغٔاَدداخ ْددٙ يٛدداِ يددٍ انلددغ ذذددد تغددضاص قدداطا نددأػخ ٔاؿددظ "ٙ... ُٚظددغ : (انًبضؿدد1991 : 124 ( ) Al-Maqdisi), 1991: 124 ) Abstract: ) (4) ٙطـٕض" يٍ كٕعج االؿراٌ تانجاَة انغغتٙ يٍ تغضاص ْٕٔ انٕٛو يذـٕب يٍ كٕعج َٓغ ػٛـدٗ تدٍ ػهد : يُٓا: انُذاؿٛح ٔانذاعشٛح َٔٓغ أعيا ٔقٙ طغقّ تُٙ تؼض تغضاص ... ُٚظغ( "ٕ٘ٚاشٕخ انذًد1995 : 1 319 ) (Ruby Al-Hamwi, 1995:1, 317) . (5) يٛدداِ انُرٓغٔاَدداخ ْددٙ يٛدداِ يددٍ انلددغ ذذددد تغددضاص قدداطا نددأػخ ٔاؿددظ "ٙ... (6) ... ٌذُأ أ٘ أشاو قٙ أعض األػانى ٚبال ذُأ تانثهض ئطا أشاو تّ ٔيُّ ؿًٙ انرُاء ألْم انضٛاع ٔاإلشايح تانثهضا "ُ٘ٚظغ : (انؼـكغ1402 :ِ 1 / 244 ( ) (Militêre), 1402H: 1/244 . (9) يإٚض انضٔنح" أتٕ يُزٕع اتٍ عكٍ انضٔنح كاٌ ٔػٚغِ انزادة" اتٍ ػثاص قضدثظ يًهكردّ ٔأدـدٍ انردضتٛغ ٌٔكاٌ شض ذؼٔض تُد ػًّ ػتٛضج تُد يؼؼ انضٔنح" أَفق قٙ ػغؿّ ػهٛٓا ؿثغ يائح أنا صُٚاع ذٕقٙ قٙ نغنا ّتانشٕاَٛق قٙ شانس ػلغ كؼثاٌ ؿُح شالز ٔؿثؼٍٛ ٔشالز يائح ٔن : شالز ٔأعتؼٌٕ ؿُح ...ُٚظغ "٘(انزفض2000 : 10 / 204 ) ( Safadi, 2000: 10/204 ) (8)انًـُاج: عدفٛغج ذثُدٗ نهـدٛم نردغص انًداء ؿدًٛد يـدُاج ألٌ قٛٓدا يفداذٛخ نهًداء تبدضع يدا ٚذرداض ئنٛدّ يًدا ال ٚغهة "٘.... ُٚظغ : (االػْغ2001 : 13 / 54 ) (Al-Azhari, 2001: 13/54) . Abstract: ُٚظددغ : (انًبضؿدد1991 : 124 ( ) Al-Maqdisi), 1991: 124 ) 323 جمةل الآداب / العدد631 (آآذار) جمةل الآداب / العدد631 (آآذار) وكمذذذذذا قذذذذذام" فذذذذذي سذذذذذنة 222 /ىذذذذذذذذ464م) سذذذذذد بدذذذذذق الرومانيذذذذذة وسذذذذذد البدذذذذذق بذذذذذالنيروان ، المادسي1625 : 162 ) (Al-Maqdisi, 1958: 193) ثانيا : الوضع االقتصادي في عهد عضد الدولة"( 334 - 34٣ /ه656 ــ663 )م وع د الدولة ىو أبوشراع فناخسرو بن ركن الدولة أبي عمي الحسن بن ،"بويذو المماذ ع د الدولة " .الديممي ) ، "ابن خمكان1611 : 2 / 21 ) Ibn Khalkan, 1900: 4/50 ) توفى بعمة الصرع في ،شوال سنة ادنتين، وسبعين ودالدة مائة ،بب،داد ولو دمان وأربعون سنة ودفن بمشيد االمام عمي .) عميو السالم ) ،"الذىبي 3112 : 5 / 243 ) Goue, 2003: 8/376 ) ويعد ع د الدولة" من أف ل أمرال بني بويذو" الذذين أرذروا اصذالحا فذي الرانذ االقتصذادي، والسياسذذي، وغيرىمذذا مذذن خذذالل مسذذاىمتو "فذي ،االقتصذذاد فكذذان لذذو ًنشذذاطا ،ًكبيذذرا وحاذذق نراحذذا ،فائاذذة ممذذا أدى إلذذى ازدىذذار أو ذذاع العذذراق ،االقتصذذادية بعذذد التذذدىور الذذذي عانى منو العراق في أوائل العصر ،"البوييي وأن ذ ع د الدولة "إلى خزائنو ماال كديرا، وديابا، وفرشا رميال من رميع األصنا وعد مذذن الخيذذل، والمراكذذ ، والرقيذذق، وارال ، وقذذرر يذذده فذذي ذذياع الخدمذذة المرسذذومة بالخم ذذال ” ، مسذكويو 3:3111 / 265 ) Miskoy, 2000:6/398 ) وقذد اقططعيذا الخم ذال ممذن سذباوه فرد ع د الدولة "ذلأل كمو إلى .حاو ا ثانيا : الوضع االقتصادي في عهد عضد الدولة"( 334 - 34٣ /ه656 ــ663 )م وع د الدولة ىو أبوشراع فناخسرو بن ركن الدولة أبي عمي الحسن بن ،"بويذو المماذ ع د الدولة " .الديممي ) ، "ابن خمكان1611 : 2 / 21 ) Ibn Khalkan, 1900: 4/50 ) توفى بعمة الصرع في ،شوال سنة ادنتين، وسبعين ودالدة مائة ،بب،داد ولو دمان وأربعون سنة ودفن بمشيد االمام عمي .) عميو السالم ) ،"الذىبي 3112 : 5 / 243 ) Goue, 2003: 8/376 ) ويعد ع د الدولة" من أف ل أمرال بني بويذو" الذذين أرذروا اصذالحا فذي الرانذ االقتصذادي، والسياسذذي، وغيرىمذذا مذذن خذذالل مسذذاىمتو "فذي ،االقتصذذاد فكذذان لذذو ًنشذذاطا ،ًكبيذذرا وحاذذق نراحذذا ،فائاذذة ممذذا أدى إلذذى ازدىذذار أو ذذاع العذذراق ،االقتصذذادية بعذذد التذذدىور الذذذي عانى منو العراق في أوائل العصر ،"البوييي وأن ذ ع د الدولة "إلى خزائنو ماال كديرا، وديابا، وفرشا رميال من رميع األصنا وعد مذذن الخيذذل، والمراكذذ ، والرقيذذق، وارال ، وقذذرر يذذده فذذي ذذياع الخدمذذة المرسذذومة بالخم ذذال ” ، مسذكويو 3:3111 / 265 ) Miskoy, 2000:6/398 ) وقذد اقططعيذا الخم ذال ممذن سذباوه فرد ع د الدولة "ذلأل كمو إلى .حاو أ وبذذال الرنذذد فذذذي برمذذع المذذذال مذذن دون االعتنذذال بتحسذذذين أمذذر العسذذذكر والرذذيش فاتسذذذع الخرق، حتى صار الرسم راريا بذأن يخذر الرنذد إقطاعذاتيم، دذم يردوىذا ويعتا ذوا عنيذا مذن حيو يختذارون ويتوصذمون إلذى حصذول ال ذل وال ذوز ..بذالرب ،مسذكويو 3:3111 / 121 ) (Miskoy, 2000:6/131) وأمذذا انعكاسذذا ىذذذه السياسذذة ااقطاعيذذة عمذذى ال الحذذين، وأصذذحا األرا ذذي، فاذذد أد إلى انصرا موظ ي الري عن أعماليم لخروج تدبير أمور الزراعذة عذن يذد الخمي ذة، واقتصذر عمميذذذذذم عمذذذذذى إن ياذذذذذدر مذذذذذا يحتذذذذذاج إليذذذذذو الذذذذذري، وان تؤخذذذذذذ حصذذذذذة الدولذذذذذة الماليذذذذذة مذذذذذن تمذذذذذأل ااقطاعذذا ، عمذذى شذذكل أقسذذاط، وانصذذر عمذذال .المصذذال ،عنذذو وليسذذ دفعذذة واحذذد ، مذذن خالل كتا عاد بين ،الدولة والماطع لخروج األعمذال عذن يذد السذمطان، ووقذع االقتصذار فذي عمميذذا عمذذى أن ياذذدر مذذا يحتذذاج إليذذو، وياسذذط وكمذذا أد تمذذأل السياسذذة إلذذى أن فسذذد عمذذى الماطعذذين تاسذذيطا يتااعذذدون بيذذا و بأدائيذذا المشذذار ، وبطمذذ أعمذذال الذذري، واتذذ الذذويال عمى ال الحين، و ع أحواليم ،فمن بين ىار ، ليذأمن شذره ،االرذال وبذين مظمذوم صذابر، لا ينص ، وبين .مستري إلى تسذميم ذيعتو إلذى الماطذع ورقذ ال الحذون وفسذد ، المشذار 324 0206 / م6110 هـ جمةل الآداب / العدد631 (آآذار) جمةل الآداب / العدد631 (آآذار) وبطم المصال. Abstract: ،وات .الروائ عمى التنال 3 )، ويوافاو أحواليم، فمذن بذين ىذار رذال، وبذين مظموم صابر ال ينص ، وبين .مستري إلى تسميم يعتو إلى الماطذع، ليذأمن شذره ،و يوافاذو وأعطيذذذ األرا ذذذي التذذذي لذذذم تاطذذذع بال ذذذمان. ،مسذذذكويو 3:3111 / 131 ) Miskoy, 2000:6/160 .) وأم ذذي لمرعيذذة الرسذذوم الصذذحيحة، وحذذذف عنيذذا الزيذذادا ، والتذذأويال ووق عمى مظذالم المتظممذين وحممذوا عمذى التعذديل ورفعذ الربايذة عذن قوافذل ،،الحرذي وزال مذذا كذذان يرذذرى عمذذييم مذذن .الابذذائ و ذذرو العسذذ وأقيمذذ ليذذم السذذواني فذذي مناىذذل الطريذذق وأح ذذذذذر اربذذذذذار واست ي ذذذذذ الدولذذذذذة 4 ). ،مسذذذذذكويو 3111 : 3 / 223 ) (Miskoy, 2000:6/456 يبذل لو الطاعة ،والموافاة فالتااه ع د الدولذة " ،بن سذو ،وأكرمذو وأقطذع أخذاه مؤيد الدولذة ىمذذان ،وغيرىذا وأقذام عنذد ع ذد الدولذة "إلذى أن عذاد إلذى ،ب،ذداد فذرده إلذى مؤيذد ،الدولذذة فأقطعذذو إقطاعذذا ،كديذذرا وسذذير معذذو عسذذكرا يكذذون عنذذد مؤيذذد الدولذذة فذذي .خدمتذذو ابذذن ،االدير1664 : 4 / 243 ) (Ibn al-Atheer, 1997: 7/376) 3 - أراضي :الممك استمرار ىذا النوع في زمن ع د الدولذة"، وذلذأل عنذدما وقعذ أرض أحذد ال الحذذين فذذي إقطذذاع الاائذذد العسذذكري إسذذ ار بذذن كردويذذو، فذذأمر ع ذذد الدولذذة " الاائذذد، بإعذذاد األرض إلى .صاحبيا Abu Shujaa, 2003:34 ) ابو ،"شذراع 3112 : 22 ) وفذي سذنة 236 /ه661م) أمذذر أصذذحا الممكيذذا مذذن األرا ذذي، بعمذذار أرا ذذييم، فمذذن قصذذر يذذده عن ذلأل، ياترض من بي مالو ليرترع منو الميسر ومن لم يودق منو ذلأل أو كان غائبا اقيم عنذذذو وكيذذذل وأطمذذذق لذذذو مذذذا يحتذذذاج اليذذذو فعمذذذر ب،ذذذداد وعذذذاد. كأحسذذذن مذذذا كانذذذ ،مسذذذكويو 3111 : 2 / 222 ) (Miskoy, 2000:5/455 وكان عمى المالكين واربا ،ىامة ف ذال عذن دفذع ال ذرائ فذي عيذد ،"ع ذد الدولذة فكذذذذان عمذذذذييم أن يسذذذذاىموا فذذذذي ن اذذذذا إصذذذذالح الانذذذذوا المائيذذذذة المذذذذار .بأرا ذذذذييم ،مسذذذذكويو 3111 : 2 / 225 ) (Miskoy, 2000:5/448 واما في سنة 236 /ه661م)، قام مذالأل األرا ذذي فذذي السذذواد، بذذذا العمذذل مذذن االصذذالح وأي ذذا أمذذر األغنيذذال بعمذذار مسذذناتيم 5 ). Ibn Al-Jawzi, 1992: 14/291 )ابن ،"الروزي 1663 : 12 / 361 ) وبطم المصال. ،وات .الروائ عمى التنال 3 )، ويوافاو أحواليم، فمذن بذين ىذار رذال، وبذين مظموم صابر ال ينص ، وبين .مستري إلى تسميم يعتو إلى الماطذع، ليذأمن شذره ،و يوافاذو وأعطيذذذ األرا ذذذي التذذذي لذذذم تاطذذذع بال ذذذمان. Abstract: ،مسذذذكويو 3:3111 / 131 ) Miskoy, 2000:6/160 .) وأم ذذي لمرعيذذة الرسذذوم الصذذحيحة، وحذذذف عنيذذا الزيذذادا ، والتذذأويال ووق عمى مظذالم المتظممذين وحممذوا عمذى التعذديل ورفعذ الربايذة عذن قوافذل ،،الحرذي وزال مذذا كذذان يرذذرى عمذذييم مذذن .الابذذائ و ذذرو العسذذ وأقيمذذ ليذذم السذذواني فذذي مناىذذل الطريذذق وأح ذذذذذر اربذذذذذار واست ي ذذذذذ الدولذذذذذة 4 ). ،مسذذذذذكويو 3111 : 3 / 223 ) (Miskoy, 2000:6/456 يبذل لو الطاعة ،والموافاة فالتااه ع د الدولذة " ،بن سذو ،وأكرمذو وأقطذع أخذاه مؤيد الدولذة ىمذذان ،وغيرىذا وأقذام عنذد ع ذد الدولذة "إلذى أن عذاد إلذى ،ب،ذداد فذرده إلذى مؤيذد ،الدولذذة فأقطعذذو إقطاعذذا ،كديذذرا وسذذير معذذو عسذذكرا يكذذون عنذذد مؤيذذد الدولذذة فذذي .خدمتذذو ابذذن االد1664 4 / 243 ) (Ib l Ath 1997 7/376) وكان عمى المالكين واربا ،ىامة ف ذال عذن دفذع ال ذرائ فذي عيذد ،"ع ذد الدولذة فكذذذذان عمذذذذييم أن يسذذذذاىموا فذذذذي ن اذذذذا إصذذذذالح الانذذذذوا المائيذذذذة المذذذذار .بأرا ذذذذييم ،مسذذذذكويو 3111 : 2 / 225 ) (Miskoy, 2000:5/448 واما في سنة 236 /ه661م)، قام مذالأل األرا ذذي فذذي السذذواد، بذذذا العمذذل مذذن االصذذالح وأي ذذا أمذذر األغنيذذال بعمذذار مسذذناتيم 5 ). Ibn Al-Jawzi, 1992: 14/291 )ابن ،"الروزي 1663 : 12 / 361 ) 325 جمةل الآداب / العدد631 (آآذار) 5ـــ الضرائب: 1 - :الخراج ل ظة عرف منذ األيام األولذى لإلسذالم وتعنذي ال ذريبة السذنوية الم رو ذة عمذى األرا ي التي تزرع حبوبا ونخيال ،وفاكية يدفعيا المزارع لمماطع صاح األرض ااقطاعيذة ليؤدييا بدوره إلذى خزانذة الدولذة بعذد اسذتاطاع مختمذ المصذروفا . Abu Yusuf, D.T.3 ) أبو ،" يوس د. Abstract: : 2 ) وقد أسال استعمال طرياة الرباية في المد البوييية" ، إذ أىمم الحكومة مراقبذة ، الربذا ولذذذم تيذذذتم بمعرفذذذة الطذذذرق التذذذي يعامذذذل بيذذذا الذذذزراع مذذذن ،ظمذذذم، ورذذذور، فنذذذت عذذذن ذلذذذأل ظيذذذور رائ رديد لم تكن مورود من قبل، ومصادر أموال المزارعين، وباي الو ع السيل ىذذا حتى رال ع ذد الدولذة"، فبذدأ فذي سذنة 236 /ه661م) سياسذتو ااصذالحية لمخذراج، فكذان أول عمل ياوم بو ىو أخذ ال ريبة الرسمية المارر مذن دون إ ذافا ، أ ذ إلذى ذلذأل، أنذو منذذع الربذذا مذذن ظمذذم الذذزراع، ومذذن يتعذذرض لمظمذذم فإنذذو يحذذق لذذو عذذرض شذذكواه عمذذى ع ذذد الدولة"، فإنو سو .ينص و ،مسذكويو 3111 : 2 / 226 ) (Miskoy, 2000:5/449 وأخذر افتتذذاح الخذذراج الذذى النيذذروز المعت ذذدي 11 )، وكذذان يؤخذذذ سذذم ا قبذذل إدراأل ، ال،ذذال وأم ذذي لمرعيذذذذذذة الرسذذذذذذوم الصذذذذذذحيحة، وحذذذذذذذذف عنيذذذذذذا الزيذذذذذذادا ، والتذذذذذذذأويال ووقذذذذذذ عمذذذذذذى مظذذذذذذذالم .المتظممين Ek is baie", 2003: 15/398 ) ابن ،"كدير 3112 : 12 / 265 ). Abstract: األموال التي رنوىا من الزكا Abu Shujaa, 2003:44 ) ابو ،"شراع 3112 : 22 ) 3 - :االصالحا العمرانية 1ـــــ حفر األنهار ،والترع وتطهيرها كالنير الذي ح ره من نير الخالص المورود فذي الرانذ الشرقي من ب،داد، إلى وسط مدينة .ب،داد Al-Baghdadi, 2002: 1/426 ) الب،دادي"، 3113 : 1 / 233 ) ّوشذق ع ذد الدولذة "نيذرا عظيمذا مذن نيذر األىذواز الذى نيذر درمذة طولذو اربعذذة فراسذذ والطريذذق اليذذوم فيذذو وتأخذذذ مذذن .األىذذواز Al-Maqdisi) , 1991: 419 ) ،"المادسذذي 1661 : 216 ) وكمذذا عمذذل عمذذى ح ذذر المرذذاري التذذي تأخذذذ مذذن األنيذذار الكبيذذر كدرمذذة، وال ذذرا لتوصذذيل المذذال إلذذى األرا ذذي البعيذذد ، كذذالمررى الذذذي ح ذذره بذذين نيذذر طذذابق ودرمة، فاد كان ال ع ال م طرون الى ان يحمموا مياه درمة في المسافة البعيد فامر بح ر عمدانيا كما عمل عمى ررى أمر الرسر" بب،داد ،مسكويو 3111 : 2 / 225 ) (Miskoy, 2000:5/448 )وأمذذذر ع ذذذد الدولذذذة "بح ذذذر النيذذذر مذذذن عمذذذود ،الخذذذالص وسذذذياقة المذذذال إلذذذى بستان ،داره فبدأ في ذلأل وحشر الررال .لعممو Ibn Al-Jawzi, 1992: 14/281 )ابذن ،"الروزي 1663 : 12 / 361 ) وفي سنة 236 ه / 661 )م أعاد ح ر ما ددر من ،األنيار وتنظيذ ،مرارييذا وبعذد أن كانذذذ السذذذب فذذذي انتشذذذار اامذذذراض بذذذين أبنذذذال ب،ذذذداد، ومنيذذذا نيذذذر العبذذذار ، ونيذذذر مسذذذرد االنبذذارين"، ونيذذر البذذزازين"، ونيذذر "الذذدراج 11 )، ونيذذر الااليذذين"، ونيذذر "طذذابق 13 )، وميزابيذذا إلى درمة، والصرا" 12 )، ونير عيسى 12 )، ونير بناحية "الحربية 12 ) يأخذ من ."الدريل (11)ألْم انكغر ٔيا اذزم تّ َٓغ ٚبال نّ َٓغ انضناض ٔئًَدا ؿدًرٙ َٓدغ اندضناض ألٌ أ دذاب اندضناض كدإَا ٙٚبفٌٕ ػُضِ ... ُٚظغ : (انٛؼبٕت1422 : ْـ 43 ) ( Jacobi, 1422 E: 43 .) (12) يذهح تثغضاص يٍ انجاَة انغغتٙ شغب َٓغ انبالئٍٛ" كغشا ٔئًَا ْٕ َٓغ تاتك يُـٕب ئنٗ تاتك تٍ تٓدغاو :ُٚظ تٍ تاتك ْ شضٚ تاتك ْ انظ٘ اذشظ ان بض انظ٘ ػهّٛ ش( "٘ٚاش خ ان1995 : 5 (11)ألْم انكغر ٔيا اذزم تّ َٓغ ٚبال نّ َٓغ انضناض ٔئًَدا ؿدًرٙ َٓدغ اندضناض ألٌ أ دذاب اندضناض كدإَا ٙٚبفٌٕ ػُضِ ... ُٚظغ : (انٛؼبٕت1422 : ْـ 43 ) ( Jacobi, 1422 E: 43 .) 12ا ٙغ () ( , ) (12) يذهح تثغضاص يٍ انجاَة انغغتٙ شغب َٓغ انبالئٍٛ" كغشا ٔئًَا ْٕ َٓغ تاتك يُـٕب ئنٗ تاتك تٍ تٓدغاو : تٍ تاتك ْٕٔ شضٚى ٔتاتك ْٕ انظ٘ اذشظ انؼبض انظ٘ ػهّٛ شزدغ .... Abstract: Seun van die Hysbak", 2009: 5/184 ) ابن ،"الرفعة 3116 : 2 / 152 ) وذكر ابو شراع ما ذكر من صدقاتو ومبراتذو ومذا تذادى ذلأل من ف ل احتياطو ومراعاتو فانو كان يخرج عند افتتاح مذا كذل سذنة شذيئا كديذرا فذي البذر والصذذدقة الذذى العمذذال النذذواحي بتسذذميمو الذذى ق ذذاتيا وورذذوه أىميذذا ليصذذرفيا الذذى ذوي الحارذذة والمسذذكنة، ... بدالدذذ ين ألذذ درىذذم لمصذذدقة ورسذذم وزن ذلذذأل ...)) وأمذذر ع ذذد الدولذذة بتوزيذذع . األموال التي رنوىا من الزكا Abu Shujaa, 2003:44 ) ابو ،"شراع 3112 : 22 ) 3 - :االصالحا العمرانية 1ـــــ حفر األنهار ،والترع وتطهيرها كالنير الذي ح ره من نير الخالص المورود فذي الرانذ الشرقي من ب،داد، إلى وسط مدينة .ب،داد Al-Baghdadi, 2002: 1/426 ) الب،دادي"، 3113 : 1 / 233 ) ّوشذق ع ذد الدولذة "نيذرا عظيمذا مذن نيذر األىذواز الذى نيذر درمذة طولذو اربعذذة فراسذذ والطريذذق اليذذوم فيذذو وتأخذذذ مذذن .األىذذواز Al-Maqdisi) , 1991: 419 ) ،"المادسذذي 1661 : 216 ) وكمذذا عمذذل عمذذى ح ذذر المرذذاري التذذي تأخذذذ مذذن األنيذذار الكبيذذر كدرمذذة، وال ذذرا لتوصذذيل المذذال إلذذى األرا ذذي البعيذذد ، كذذالمررى الذذذي ح ذذره بذذين نيذذر طذذابق ودرمة، فاد كان ال ع ال م طرون الى ان يحمموا مياه درمة في المسافة البعيد فامر بح ر عمدانيا كما عمل عمى ررى أمر الرسر" بب،داد ،مسكويو 3111 : 2 / 225 ) (Miskoy, 2000:5/448 )وأمذذذر ع ذذذد الدولذذذة "بح ذذذر النيذذذر مذذذن عمذذذود ،الخذذذالص وسذذذياقة المذذذال إلذذذى بستان ،داره فبدأ في ذلأل وحشر الررال .لعممو Ibn Al-Jawzi, 1992: 14/281 )ابذن ،"الروزي 1663 : 12 / 361 ) وفي سنة 236 ه / 661 )م أعاد ح ر ما ددر من ،األنيار وتنظيذ ،مرارييذا وبعذد وىذي صذدقة ،ال طذر والكتذا يشذمميما . Seun van die Hysbak", 2009: 5/184 ) ابن ،"الرفعة 3116 : 2 / 152 ) وذكر ابو شراع ما ذكر من صدقاتو ومبراتذو ومذا تذادى ذلأل من ف ل احتياطو ومراعاتو فانو كان يخرج عند افتتاح مذا كذل سذنة شذيئا كديذرا فذي البذر والصذذدقة الذذى العمذذال النذذواحي بتسذذميمو الذذى ق ذذاتيا وورذذوه أىميذذا ليصذذرفيا الذذى ذوي الحارذذة والمسذذكنة، ... بدالدذذ ين ألذذ درىذذم لمصذذدقة ورسذذم وزن ذلذذأل ...)) وأمذذر ع ذذد الدولذذة بتوزيذذع . Abstract: ٣- :الجزية ال تؤخذ الرزية إال من كافر حذر بذال ذكذر قذوي عمذى االكتسذا وال رزيذة عمذى النسذذال وال عمذذى الصذذذبيان وال عمذذى المرذذذانين الم،مذذوبين عمذذذى عاذذوليم وال عمذذذى الرىبذذان أىذذذل الصذوامع وال عمذى شذي فذان وال عمذى .فايذر Ibn Abd al-Bar, 1400H: 1/479 )ابذن عبذد ،"البذر 1211ىذذذ : 1 / 246 ) واذن ع ذد الدولذة "الذذى وزيذره نصذر بذذن ىذارون فذي عمذذار .البيذذذذع والذذذذدير واوطذذذذالق االمذذذذوال ل اذذذذرائيم ،مسذذذذكويو 3111 : 2 / 226 ) (Miskoy, 2000:5/449 2 - :الزكاة عبار عن قدر من المال يخررو المسمم في وق مخصوص ألىذل ،السذيمان مذع ،النية والصدقة تشذرأل الزكذا فذي ىذذا ،المعنذى وىمذا ياعذان عمذى زكذا ،األمذوال وزكذا األبذدان وقد أسال استعمال طرياة الرباية في المد البوييية" ، إذ أىمم الحكومة مراقبذة ، الربذا ولذذذم تيذذذتم بمعرفذذذة الطذذذرق التذذذي يعامذذذل بيذذذا الذذذزراع مذذذن ،ظمذذذم، ورذذذور، فنذذذت عذذذن ذلذذذأل ظيذذذور رائ رديد لم تكن مورود من قبل، ومصادر أموال المزارعين، وباي الو ع السيل ىذذا حتى رال ع ذد الدولذة"، فبذدأ فذي سذنة 236 /ه661م) سياسذتو ااصذالحية لمخذراج، فكذان أول عمل ياوم بو ىو أخذ ال ريبة الرسمية المارر مذن دون إ ذافا ، أ ذ إلذى ذلذأل، أنذو منذذع الربذذا مذذن ظمذذم الذذزراع، ومذذن يتعذذرض لمظمذذم فإنذذو يحذذق لذذو عذذرض شذذكواه عمذذى ع ذذد الدولة"، فإنو سو .ينص و ،مسذكويو 3111 : 2 / 226 ) (Miskoy, 2000:5/449 وأخذر افتتذذاح الخذذراج الذذى النيذذروز المعت ذذدي 11 )، وكذذان يؤخذذذ سذذم ا قبذذل إدراأل ، ال،ذذال وأم ذذي لمرعيذذذذذذة الرسذذذذذذوم الصذذذذذذحيحة، وحذذذذذذذذف عنيذذذذذذا الزيذذذذذذادا ، والتذذذذذذذأويال ووقذذذذذذ عمذذذذذذى مظذذذذذذذالم .المتظممين Ek is baie", 2003: 15/398 ) ابن ،"كدير 3112 : 12 / 265 ). (9)انٕشا انذثؾ نغح ٔٔشا انضٛؼح ْٕ دثـٓا ػٍ ذًهك انٕاشا ٔ ٛدغ انٕاشدا ٔاؿدرغالنٓا نهزدغى ئندٗ يدا ٙؿًٙ يٍ انًزاعى ٔنظا ؿًٙ دثٛـا ... ُٚظغ : (انُفـ1311 : ْـ105 () (Psigo, 1311H: 105 . (10)أيغ انًؼرضض قٙ انًذغو يُٓا تاَلاء انكرة ئنٗ نًٛغ انؼًال قٙ انُٕادٙ ٔاأليزاع ترغي اق رراح انشغاض قٙ انُٛغٔػ انظ٘ ْٕ َٛدغٔػ انؼجدى ٔذدأسٛغ طندك ئندٗ انٛدٕو انذداص٘ ػلدغ يدٍ دؼٚدغاٌ ٔؿدًٙ طندك انُٛدغٔػ "٘انًؼرضض٘ .... ُٚظغ : (ان ثغ1409 :ِ 5 / 610 () (Al-Tabari, 1407H: 5/610 . 326 0206 / م6110 هـ جمةل الآداب / العدد631 (آآذار) جمةل الآداب / العدد631 (آآذار) وىذي صذدقة ،ال طذر والكتذا يشذمميما . 321 (11)ألْم انكغر ٔيا اذزم تّ َٓغ ٚبال نّ َٓغ انضناض ٔئًَدا ؿدًرٙ َٓدغ اندضناض ألٌ أ دذاب اندضناض كدإَا ٙٚبفٌٕ ػُضِ ... ُٚظغ : (انٛؼبٕت1422 : ْـ 43 ) ( Jacobi, 1422 E: 43 .) (12) يذهح تثغضاص يٍ انجاَة انغغتٙ شغب َٓغ انبالئٍٛ" كغشا ٔئًَا ْٕ َٓغ تاتك يُـٕب ئنٗ تاتك تٍ تٓدغاو : تٍ تاتك ْٕٔ شضٚى ٔتاتك ْٕ انظ٘ اذشظ انؼبض انظ٘ ػهّٛ شزدغ .... ُٚظدغ( "ٕ٘ٚداشٕخ انذًد1995 : 5 321 ) (Ruby Al-Hamwi, 1995:5, 321) . (13) :َ٘ٓغ ُٚلؼة يٍ انفغاخ ٔٚجغ٘ ئنٗ تغضاص ٔٚبال: انزغا تال ْاء أٚضاً ألَّ دغ٘ ػدٍ انفدغاخ أ "٘ش غ. .. ُٚظغ : (انذًٛغ1980 : 359 () Al-Hamri, 1980: 357 ) . (14)اتٍ ػهٗ تٍ ػثض هللا انٓاكًٗ تٍ ػثاؽ شال: كٕعج كثٛغج ٔشغٖ كصٛغج ٔػًم ٔاؿغ قٗ غتدٗ تغدضاص ٚأسظ يٍ انفغاخ ػُض شُ غج صيًا شى ًٚغ قٛـبٗ طـٕض" قٛغٔػ ؿاتٕع دردٗ ُٚرٓدٗ ئندٗ انًذدٕل شدى ٚرفدغع ٌيٍ أَٓاع ذرشغ ئنٗ يضُٚح انـالو شى ًٚغ تانٛاؿغٚح شى شُ غج انغٔيٛح ٔشُ غج انؼٚداذٍٛ ٔشُ دغج األكدُا ٔشُ غ ج انلٕي ٔشُ غج انغياٌ ٔشُ غج انًغٛض ػُض األعداء شدى شُ دغج انثـدراٌ. شدى شُ دغج انًؼثدض٘ شدى : شُ غج تُٗ ػعٚق شى ٚزة قٙ صنهح ػُض شزغ ػٛـٗ تٍ ػهٗ. ُٚظغ ( Ibn Abdul Haq), 1412 E: 3, 1402 ") . (اتٍ ػثض انذق1412 : ْـ3 1402 ) Abstract: ُٚظدغ( "ٕ٘ٚداشٕخ انذًد1995 : 5 321 ) (Ruby Al-Hamwi, 1995:5, 321) . 13أ ًا ) ( y , , ) (13) :َ٘ٓغ ُٚلؼة يٍ انفغاخ ٔٚجغ٘ ئنٗ تغضاص ٔٚبال: انزغا تال ْاء أٚضاً ألَّ دغ٘ ػدٍ انفدغاخ أ "٘ش غ. .. ُٚظغ : (انذًٛغ1980 : 359 () Al-Hamri, 1980: 357 ) . 1لله (14)اتٍ ػهٗ تٍ ػثض هللا انٓاكًٗ تٍ ػثاؽ شال: كٕعج كثٛغج ٔشغٖ كصٛغج ٔػًم ٔاؿغ قٗ غتدٗ تغدضاص ٚأسظ يٍ انفغاخ ػُض شُ غج صيًا شى ًٚغ قٛـبٗ طـٕض" قٛغٔػ ؿاتٕع دردٗ ُٚرٓدٗ ئندٗ انًذدٕل شدى ٚرفدغع ٌيٍ أَٓاع ذرشغ ئنٗ يضُٚح انـالو شى ًٚغ تانٛاؿغٚح شى شُ غج انغٔيٛح ٔشُ غج انؼٚداذٍٛ ٔشُ دغج األكدُا ٔشُ غ ج انلٕي ٔشُ غج انغياٌ ٔشُ غج انًغٛض ػُض األعداء شدى شُ دغج انثـدراٌ. شدى شُ دغج انًؼثدض٘ شدى : شُ غج تُٗ ػعٚق شى ٚزة قٙ صنهح ػُض شزغ ػٛـٗ تٍ ػهٗ. Abstract: ُٚظغ 321 0206 / م6110 هـ جمةل الآداب / العدد631 (آآذار) جمةل الآداب / العدد631 (آآذار) ،مسكويو 3111 : 2 / 225 ) Miskoy, 2000:5/448 وكان لسده بدذق السذيمية فذي النيروان، قر ب،داد أىمية خاصة، وو ع عميذو إبذراىيم .األغذر لحمايتذو Abu Shujaa, 2003:45 ) ابذذو ،"شذذراع 3112 : 22 ) وكذذذلأل أعذذاد بنذذال الكديذذر مذذن قنذذاطر األنيذذار التذذي أصذذابيا الخذذرا فذذي السذذواد، ورمذذع كذذل مذذا يحتذذاج إلذذى ذلذذأل مذذن .اررذذر، والرذذص ،مسذذكويو 3111 : 2 / 225 ) Miskoy, 2000:5/448 أ 2003:45 ) ابذذو ،"شذذراع 3112 : 22 ) وكذذذلأل أعذذاد بنذذال الكديذذر مذذن قنذذاطر األنيذذار التذذي أصذذابيا الخذذرا فذذي السذذواد، ورمذذع كذذل مذذا يحتذذاج إلذذى ذلذذأل مذذن .اررذذر، والرذذص ،مسذذكويو 3111 : 2 / 225 ) Miskoy, 2000:5/448 وأعاد بنال الكدير من السذدود، وو ذع عمييذا الحذراس سذنة 236 /ه661 )م وفذي ذلذأل عمل الرسور، وأن ا فييا األمذوال، وأعذد عمييذا ارال ، ووكَّذل بيذا الررذال ياذول أبذو شذراع وألذذذذزميم ح ظيذذذذا بالميذذذذل والنيذذذذار، وراعذذذذى ذلذذذذأل مذذذذنيم أتذذذذم مراعذذذذا ، فذذذذي اونذذذذة المذذذذدود أوقذذذذا ال ي انا الروار السيول وأزمنة، وأوقا ال،يوو األمطار اليواطل وأوقذا الريذاح وطالذ الرعية بالعمار مطالبة .رفياة Abu Shujaa, 2003:45 ) ابو ،"شراع 3112 : 22 ) ٣- :أمَّا وسائل اإلرواء فاد بُني الانذاطر عمذى كديذر مذن أفذواه الانذوا لتنظذيم توزيذع ،المذال ولرفع منسوبيا في الانوا ال رعية، لتسييل الساي سيحا 13 )، وكان ىذه الاناطر تبنى عذاد بذالرص والنذور .واررذر ،مسذكويو 3111 : 2 / 225 ) Miskoy, 2000:5/448 )ونتيرذة إصالحا ع د الدولة "لنظام الرباية، ونظام ااروال فشرع النذاس عمذى الزراعذة، ف ذي سذنة 236 /ه661م) أمر ع د الدولذة " أصذحا األرا ذي، بإعذاد زراعذة أرا ذييم التذي كانذ خرا ، فعاد كأحسن ما كان ، ومن قصر يده عن ذلأل، اقترض من بيذ المذال، ليرترذع منو عند الميسر ، فعمر .ب،داد ،مسكويو 3111 : 2 / 224 ) Miskoy, 2000:5/447 ) وبم من اىتمامو في ،الزراعة أنَّو رم ال،روس من ،فذارس وسذائر الذبالد إلذى ،العذراق ومذذن أنذذواع ال،ذذروس المسذذتورد إلذذى العذذراق فذذي عيذذده التذذاري وحذذ النيمذذو وبمذذ مذذن تشذذريعو عمى الزراعة وأمر األغنيال حول من البادية، فزرعوا وعمروا البرية أن ي،رسوا في كل . Abstract: خرا Ibn Al-Jawzi, 1992: 14/281 )ابن ،"الروزي 1663 : 12 / 361 ) 3 - تشــــييد "المبــــاني لمذذذذا دخذذذذل ع ذذذذد الدولذذذذة "إلذذذذى ب،ذذذذداد وىذذذذو معذذذذو مذذذذن خاصذذذذتو رذذذذدد البيمارسذذذتان"" 14 )، وصذذذار يأخذذذذ رزقذذذين وىمذذذا برسذذذم خذذذاص دالدمائذذذ ة درىذذذم شذذذراعية وبرسذذذم البيمارسذتان ""دالدمائذ ة درىذم شذراعية سذوى .الررايذة 211): Ibn Abi A'a'a" D.T ،)ابذن أبي أصيبعة " ، د – : 311 ) 321 (15)يُـٕتح يذهح كثٛغج يلٕٓعج تثغضا ص ػُض تاب دغب شغب يبثغج تلدغ انذداقٙ ٔأدًدض تدٍ دُثدم ٔ ٛغًْدا ذُـة ئنٗ دغب تٍ ػثض هللا انثهشٙ ٔٚؼغى تانغأَض٘ أددض شدٕاص أتدٙ نؼفدغ انًُزدٕع ٔكداٌ ٚردٕنٗ كدغطح :تغضاص....ُٚظغ( "ًٕ٘ٚاشٕخ انذ1995 : 2 239 ) (Ruby Al-Hamwi, 1995:2, 237) . (16)انًاء ؿٛذا: ئطا نغٖ ػهٗ ٔن : ّ األعض.... ُٚظغ "ٌ(َلٕا1999 : 5 / 3301 ) (Nashwan, 1999: 5/ 3301) . (14)دار المر ى وىو . معر ينظر : "الرواىري ، 1654 : 2 / 645 ) (Al-Jawahiri, 1987: 3/978 . 321 (15)يُـٕتح يذهح كثٛغج يلٕٓعج تثغضا ص ػُض تاب دغب شغب يبثغج تلدغ انذداقٙ ٔأدًدض تدٍ دُثدم ٔ ٛغًْدا ذُـة ئنٗ دغب تٍ ػثض هللا انثهشٙ ٔٚؼغى تانغأَض٘ أددض شدٕاص أتدٙ نؼفدغ انًُزدٕع ٔكداٌ ٚردٕنٗ كدغطح :تغضاص....ُٚظغ( "ًٕ٘ٚاشٕخ انذ1995 : 2 239 ) (Ruby Al-Hamwi, 1995:2, 237) . (16)انًاء ؿٛذا: ئطا نغٖ ػهٗ ٔن : ّ األعض.... ُٚظغ "ٌ(َلٕا1999 : 5 / 3301 ) (Nashwan, 1999: 5/ 3301) . (14)دار المر ى وىو . معر ينظر : "الرواىري ، 1654 : 2 / 645 ) (Al-Jawahiri, 1987: 3/978 . Abstract: تٛغٔخ أ خا ى  ("٘اتٍ انجٕػ1992) ػثض انغدًٍ تٍ ػهٙ تدٍ يذًدض (خ599 /ْدـ1219 و) انًُدرظى قدٙ ذداعٚز األيدى .ٔانًهٕي ذخ: يذًض ػثض انباصع ػ ا يز فٗ ػثض انباصع ػ ا صاع انكرة انؼهًٛح تٛغٔخ  ("اتٍ ػثض انذق1412ِ) ػثض انًإيٍ تٍ ػثض ا نذق تٍ كدًائم (خ939 /ْدـ1361 و) يغا دض االطدالع .ػهٗ أؿًاء األيكُح ٔانثباع صاع انجٛم تٛغٔخ هلل عأ  ( "٘انذًٛغ1980 ) يذًض تٍ ػثض هللا تٍ ػثض انًدُؼى (خ900 / ِ 1522 و) اندغٔض انًؼ داع قدٙ سثدغ األش اع ذخ: ئدـاٌ ػثاؽ يإؿـح َا غ نهصباقح ط2 . تٛغٔخ أ خأ  ("ٌاتددٍ سهكددا1900) أدًددض تدد ٍ يذًددض تددٍ ئتددغاْٛى (خ681 /ْددـ1301 و) ٔقٛدداخ األػٛدداٌ ٔأَثدداء أتُدداء .انؼياٌ ذخ: ئدـاٌ ػثاؽ صاع اصع تٛغٔخ إا خ  ( "ٙانظْث2003 ) يذًض تٍ أدًدض تدٍ ػصًداٌ (خ948 /ْدـ1390 و) ذداعٚز اإلؿدالو ََٔٔقٛداخ انًلداْٛغ َٔاألػالو ذخ: تلاع ػٕراص يؼغٔى صاع انغغب اإلؿاليٙ ص– .و  ( ٙانظْث2003 ) يذًض تٍ أدًدض تدٍ ػصًداٌ (خ948 /ْدـ1390 و) ذداعٚز اإلؿدالو ٔٔقٛداخ انًلداْٛغ َٔاألػالو ذخ: تلاع ػٕراص يؼغٔى صاع انغغب اإلؿاليٙ ص– .و  ٍات( "انغقؼح2009 ) أدًض تٍ يذًض تٍ ػهٙ (خ910 / ِ 1332 :و) كفاٚح انُثّٛ قدٙ كدغح انرُثٛدّ ذدخ يجض٘ يذًض ؿغٔع صاع انكرة انؼهًٛح ص– .و ان ػرا (خ ( خ) انؼ12 5 / 1829ا ان ) ذ ا ٙاإ خ واأ و  ٍات( "انغقؼح2009 ) أدًض تٍ يذًض تٍ ػهٙ (خ910 / ِ 1332 :و) كفاٚح انُثّٛ قدٙ كدغح انرُثٛدّ ذدخ يجض٘ يذًض ؿغٔع صاع انكرة انؼهًٛح ص– .و انبايٕؽ ذخ: يجًٕػح يٍ انًذببٍٛ صاع انٓضاٚح ص و . Abstract: تٛغٔخ  ("ٌاتددٍ سهكددا1900) أدًددض تدد ٍ يذًددض تددٍ ئتددغاْٛى (خ681 /ْددـ1301 و) ٔقٛدداخ األػٛدداٌ ٔأَثدداء أتُدداء .انؼياٌ ذخ: ئدـاٌ ػثاؽ صاع اصع تٛغٔخ  ( "ٙانظْث2003 ) يذًض تٍ أدًدض تدٍ ػصًداٌ (خ948 /ْدـ1390 و) ذداعٚز اإلؿدالو ََٔٔقٛداخ انًلداْٛغ َٔاألػالو ذخ: تلاع ػٕراص يؼغٔى صاع انغغب اإلؿاليٙ ص– .و  ٍات( "انغقؼح2009 ) أدًض تٍ يذًض تٍ ػهٙ (خ910 / ِ 1332 :و) كفاٚح انُثّٛ قدٙ كدغح انرُثٛدّ ذدخ يجض٘ يذًض ؿغٔع صاع انكرة انؼهًٛح ص– .و  انؼتٛددض٘ (ص.خ) يذًددض تددٍ يذًددض تددٍ ػثددض انددغػرا (خ1205 /ْددـ1829 و) ذدداض انؼددغٔؽ يددٍ نددٕاْغ انبايٕؽ ذخ: يجًٕػح يٍ انًذببٍٛ صاع انٓضاٚح ص – و– .  ("ٙاتٍ كاكغ انكرث1993 ) يذًض تٍ كاكغ تٍ أدًض (خ964 /ْـ1386 ٌو) قٕاخ انٕقٛداخ ذدخ: ئدـدا .ػثاؽ صاع اصع تٛغٔخ  ("اتٕ كجاع2003 ) يذًض تٍ انذـٍٛ تٍ يذًض تٍ ػثض هللا (خ488 /ِ 1110 :و) طٚم ذجاعب االيى ذخ ؿٛض كـغٔ٘ صاع انؼهًٛح ص– و– .  "٘انزددفض( 2000 ) سهٛددم تددٍ أٚثددك تددٍ ػثددض هللا (خ964 /ْددـ1386 و) انددٕاقٙ تانٕقٛدداخ ذددخ: أدًددض .األعَاؤٔط ٔذغكٙ يز فٗ صاع ئدٛاء انرغاز تٛغٔخ  ان ثددغ٘" يذًددض تددٍ نغٚددغ تددٍ ٚؼٚددض (خ310 /ْددـ932 و) ذدداعٚز األيددى ٔانًهددٕي صاع انكرددة انؼهًٛددح (تٛغٔخ- 1409 )ِ  ( ٌٕاتٍ طٕن1998 ّٚٔ) يذًض تٍ ػهٙ تٍ سًاع (خ953 /ْـ1595 و) ئَثداء األيدغاء تأَثداء اندٕػعاء .ذخ: يُٓرا دًض انًُٓرا صاع انثلائغ االؿاليٛح تٛغٔخ  ("٘اتٍ انؼثغ1992 ) غٚغٕعٕٚؽ تٍ ْداعٌٔ تدٍ ذٕيدا (خ685 /ْدـ1309 ) ذداعٚز يشرزدغ اندضٔل ذخ: أَ ٌٕ انذاَٙ انٛـٕػٙ صاع انلغ ط3 . تٛغٔخ  ( "٘انؼـددكغ1402 ِ) ٍ انذـدد تددٍ ػثددض هللا تددٍ ؿددؼٛض (خ382 /ْددـ1004 :و) ذزددذٛفاخ انًذددضشٍٛ ذددخ .يذًٕص أدًض يٛغج انً ثؼح انؼغتٛح انذضٚصح انباْغج  أتددٕ انفددضاء"(ص.خ) ئؿددًاػٛم تددٍ ػهددٙ تددٍ يذًددٕص (خ932 / ْددـ134 و) انًشرزددغ قددٙ أسثدداع انثلددغ انً ثؼح انذـُٛٛح انًزغٚح ص– .و  ("ٙ انفب2005 ) ػهٙ تدٍ ٕٚؿدا تدٍ ئتدغاْٛى (خ646 /ْدـ1268 و) ئسثداع انؼهًداء تأسثداع انذكًداء .ذخ: ئتغاْٛى كًؾ انضٍٚ صاع انكرة انؼهًٛح تٛغٔخ و  ( "٘انجٕاْغ1989 ) ئؿًاػٛم تٍ دًاص (خ393 / ْـ1015 و) انزذاح ذداض انهغدح ٔ دذاح انؼغتٛدح ذخ: أدًض ػ ثض انغفٕع ػ اع صاع انؼهى نهًالٍٚٛ ط4 . Abstract: 0206 / م6110 هـ 0206 / م6110 هـ جمةل الآداب / العدد631 (آآذار) جمةل الآداب / العدد631 (آآذار) وقذذد أمذذر ع ذذد الدولذذة " سذذنة 243 /ه662م) بنذذال البيمارسذذتان ""الع ذذدي " المنسذذو إليذذو قصذذد أن يكذذون فيذذو رماعذذة مذذن أفا ذذل األطبذذال، وأعيذذانيم فذذأمر أن يح ذذروا لذذو ذكذذر األطبذذال المشذذيورين حينئذذذ بب،ذذداد، وأعماليذذا فكذذانوا متذذوافرين عمذذى المائذذة فاختذذار مذذنيم نحذذو خمسذذين بحسذذ مذذا عمذذم مذذن رذذود .أحذذواليم:215) Ibn Abi A'a'a" D.T ،)ابذذن أبذذي أصذذيبعة " ، د – : 212 ) و رمذذع األطبذذال مذذن ارفذذاق فذذارتمع فيذذو أربعذذة وعشذذرون طبيبذذا وأن ق عميو أمواال ،عظيمة وىو بيمارستان "عظيم ليس في الدنيا مدل .ترتيبذو Al-Sabye) , 2005:181 ) ال اطذذذذي،" 3112 : 151) وخصذذذص لذذذذو أوقافذذذا كديذذذذر ، وزوده ، بذذذذارال ،واألدوية، واالشربة، وال رش ورت ،فيو األطبال، والوكالل، والبوابين، والنذاظرين، والمعذالرين .والخزان Ibn Al-Jawzi, 1992: 14/289 )ابن ال،"روزي 1663 : 12 / 356 ) وامر بابتدال بالمسارد الرامعة أي ا، وكان أي ا في نياية الخرا فأن ق عمييذا مذاال عظيمذذذا وىذذذدم مذذذا كذذذان متيذذذدما مذذذن بنيانيذذذا ،وأعادىذذذا عمذذذى إحكذذذام وشذذذيدىا وأعالىذذذا وفرشذذذيا وكسذذاىا وتاذذذدم بذذإدرار أرزاق قواميذذذا ،،ومؤذنييذذا ،واالئمذذذة والاذذرال فييذذذا ،واوقامذذة الررايذذذا لمذذذن يذذأوي إلييذذا مذذن ال،ربذذال ،وال ذذع ال وكذذان ذلذذأل كمذذو ميمذذال ال ي كذذر فيذذو ، وىذذذا ال يعنذذي أنَّذذو اختص المسممين عن غيرىم بل ان ق أمواالً لعمار الدير ، وبسط ًرسذوما لم اذرال وال ايذال والم سرين والمتكممين والمحددين والنسذابين ،والشذعرال ، والنحذويين ،والعرو ذين ،، واألطبذال ،والمنرمذذذين ، والحسذذذا .والميندسذذذين ،مسذذذكويو 3111 : 2 / 224 - 226 ) Miskoy, 2000:5/447-449 :ــــ التجارة ولما سذيطر البويييذون "عمذى ب،ذداد، حذاولوا تنشذيط ، الترذار فمراقبذوا الترذار مراقبذة دقياذذذة وحذذذدوا مذذذن رشذذذع الترذذذار ولذذذم .تسذذذم الحكومذذذة ألحذذذد أن يحتكذذذر بيذذذع أي صذذذن مذذذن الب ائع، سذوى المنسذورا الرسذمية، أو ،الطذراز فكذان ىذذا احتكذار لمحكومذة، وقذد زاد ع ذد الدولة "أصنافا تتولى الحكومة احتكارىا، وىي صناعة ،،الاز، والدم .والحر Abu Shujaa, 2003:137 ) ابو ،"شراع 3112 : 124 ) وتميز مد ع د الدولة "بتوافر األمن الداخمي فإنو حمى البالد من كل .م سد Abu Shujaa, 2003:36 ) ابو ،"شراع 3112 : 23 ) فكان لو األدر البال في ازدىار النشاط الترذاري مذا لذم يبم،ذو أحذد مذن بنذى بويذو"، ودانذ لذو الذبالد .والعبذاد Ibn Al-TaghreBardi, D-T: 4/142 ) ابذن الت،ذري بذردي ،" د– : 2 / 123 ) 1 ذذذذ :األسواق تعر أسواق ب،داد في عيذد ع ذد الدولذة "إلذى حريذق سذنة 241 /ه663 ) السذذذيما منطاذذذة الكذذذرخ، إذ كذذذان الحريذذذق يمتذذذد مذذذن در الاذذذراطيس، إلذذذى بعذذذض البذذذزازين مذذذن الرانبين، وأتى عمى االساك ة، والحذائين، واحترق فيو رماعة من الناس وباي ليبو .أسبوعا Ibn Al-Jawzi, 1992: 14/289 )ابن ،"الروزي 1663 : 12 / 356 ) 323 جمةل الآداب / العدد631 (آآذار) 0206 / م6110 هـ وأمذذر ع ذذد الدولذذة" ًبعمذذار منذذازل ب،ذذداد واسذذواقيا وكانذذ مختم ذذة احذذرق بع ذذيا بع ذذا .فأن ق عمييا ماال عظيما ،مسكويو 3111 : 2 / 224 ) Miskoy, 2000:5/447 وكان ييد الى اصالح أحوال الناس االقتصادية بسب ما مروا بو من تذدىور أحذواليم السذذيما فذذي ا.لكذذرخ، فعمذذل راىذذدا فذذي اصذذالح تمذذأل األو ذذاع االقتصذذادية وشذذمم إصذذالحاتو العمرانيذذة، الطذذرق التراريذذة، ومذذن أرذذل تشذذريع .الترذذار ، أصذذم الرسذذور التذذي كانذذ ، ذذياة وتتزاحم الناس عمييا، فوسعيا حتى أصبح كالشوارع ال سيحة، وحصنيا بالدرابزينا، ووكذل بيا الح ظة .والحراس ،مسكويو 3111 : 2 / 225 ) Miskoy, 2000:5/448 الخاتمة جمةل الآداب / العدد631 (آآذار) جمةل الآداب / العدد631 (آآذار) وأمذذر ع ذذد الدولذذة" ًبعمذذار منذذازل ب،ذذداد واسذذواقيا وكانذذ مختم ذذة احذذرق بع ذذيا بع ذذا .فأن ق عمييا ماال عظيما ،مسكويو 3111 : 2 / 224 ) Miskoy, 2000:5/447 وكان ييد الى اصالح أحوال الناس االقتصادية بسب ما مروا بو من تذدىور أحذواليم السذذيما فذذي ا.لكذذرخ، فعمذذل راىذذدا فذذي اصذذالح تمذذأل األو ذذاع االقتصذذادية وشذذمم إصذذالحاتو العمرانيذذة، الطذذرق التراريذذة، ومذذن أرذذل تشذذريع .الترذذار ، أصذذم الرسذذور التذذي كانذذ ، ذذياة وتتزاحم الناس عمييا، فوسعيا حتى أصبح كالشوارع ال سيحة، وحصنيا بالدرابزينا، ووكذل بيا الح ظة .والحراس ،مسكويو 3111 : 2 / 225 ) Miskoy, 2000:5/448 الخاتمة :،توصل البحو الى اىم النتائ 1 - .تحول الدولة البوييية بعد استيالئيا عمى ب،داد الى أمار تابعة لمخالفة العباسية 3 - عندما استمم بنو بويو زمام األمور في ب،داد كان تعاني مذن تذردي األو ذاع االقتصذادية .الى حد اافالس 2 - عان االمار البوييية من قمة االيذرادا الدولذة العباسذية، بسذب مذا تعر ذ لذو األرا ذي .الزراعية، وقو ظيور النظام ااقطاعي 2 - عمذذذذل األميذذذذر معذذذذز الدولذذذذة عمذذذذى ااصذذذذالح النظذذذذام االقتصذذذذادية، فذذذذأمر بااقطذذذذاع، فذذذذوزع االقطاعا عمى قاد الريش، بسب قمة الواردا الدولة، ولذم نذر أيَّ إصذالح بعذده االَّ مذا .ذكرناه :،توصل البحو الى اىم النتائ 1 - .تحول الدولة البوييية بعد استيالئيا عمى ب،داد الى أمار تابعة لمخالفة العباسية 3 - عندما استمم بنو بويو زمام األمور في ب،داد كان تعاني مذن تذردي األو ذاع االقتصذادية .الى حد اافالس 2 - عان االمار البوييية من قمة االيذرادا الدولذة العباسذية، بسذب مذا تعر ذ لذو األرا ذي .الزراعية، وقو ظيور النظام ااقطاعي ااأ 2 - عمذذذذل األميذذذذر معذذذذز الدولذذذذة عمذذذذى ااصذذذذالح النظذذذذام االقتصذذذذادية، فذذذذأمر بااقطذذذذاع، فذذذذوزع االقطاعا عمى قاد الريش، بسب قمة الواردا الدولة، ولذم نذر أيَّ إصذالح بعذده االَّ مذا .ذكرناه 2 - عمذذذذل األميذذذذر معذذذذز الدولذذذذة عمذذذذى ااصذذذذالح النظذذذذام االقتصذذذذادية، فذذذذأمر بااقطذذذذاع، فذذذذوزع االقطاعا عمى قاد الريش، بسب قمة الواردا الدولة، ولذم نذر أيَّ إصذالح بعذده االَّ مذا .ذكرناه 2 - يعُّذذذد ع ذذذد الدولذذذة مذذذن أف ذذذل األمذذذرال الذذذذين سذذذعوا الذذذى إصذذذالح النظذذذام االقتصذذذادي فذذذي الخالفذذذذة العباسذذذذذية فذذذذي ب،ذذذذذداد، بعذذذذذد أن شذذذذيدىا، الذذذذذدمار، والخذذذذذرا ، الذذذذذي خم ذذذذذو النظذذذذذام .االقطاعي، وما تعر لو من في انا وغيرىما 3 - أنرذذز مشذذاريع إصذذالحية عديذذد منيذذا اصذذالحو الن ظذذام االقتصذذادي، والمذذالي، ف ذذالً عذذن .العمراني، إذ ال نبال أن ناول بأنَّو كان من أف ل االمرال الذين حكموا في ب،داد 322 جمةل الآداب / العدد631 (آآذار) 0206 / م6110 هـ  اتٍ انرغغ٘ تغص٘" (ص.خ) ٕٚؿا تٍ ذغدغ٘ تدغص٘ تدٍ ػثدض هللا (خ894 /ْدـ1496 و) انُجدٕو انؼاْدغج ٙق .يهٕي يزغ ٔانباْغج ٔػاعج انصباقح ٔاإلعكاص انبٕيٙ صاع انكرة يزغ  (ٙانرُدددٕس1391 ِ) انًذـدددٍ تدددٍ ػهدددٙ تدددٍ يذًدددض (خ384 /ْدددـ1006 و) َلدددٕاع انًذاعدددغج ٔأسثددداع انًظاكغج ص– .و  ( "٘انجٕاْغ1989 ) ئؿًاػٛم تٍ دًاص (خ393 / ْـ1015 و) انزذاح ذداض انهغدح ٔ دذاح انؼغتٛدح ذخ: أدًض ػ ثض انغفٕع ػ اع صاع انؼهى نهًالٍٚٛ ط4 . Abstract: تٛغٔخ  ("٘اتٍ انجٕػ1992) ػثض انغدًٍ تٍ ػهٙ تدٍ يذًدض (خ599 /ْدـ1219 و) انًُدرظى قدٙ ذداعٚز األيدى .ٔانًهٕي ذخ: يذًض ػثض انباصع ػ ا يز فٗ ػثض انباصع ػ ا صاع انكرة انؼهًٛح تٛغٔخ  ("اتٍ ػثض انذق1412ِ) ػثض انًإيٍ تٍ ػثض ا نذق تٍ كدًائم (خ939 /ْدـ1361 و) يغا دض االطدالع .ػهٗ أؿًاء األيكُح ٔانثباع صاع انجٛم تٛغٔخ  ( "٘انذًٛغ1980 ) يذًض تٍ ػثض هللا تٍ ػثض انًدُؼى (خ900 / ِ 1522 و) اندغٔض انًؼ داع قدٙ سثدغ األش اع ذخ: ئدـاٌ ػثاؽ يإؿـح َا غ نهصباقح ط2 . تٛغٔخ  ("ٌاتددٍ سهكددا1900) أدًددض تدد ٍ يذًددض تددٍ ئتددغاْٛى (خ681 /ْددـ1301 و) ٔقٛدداخ األػٛدداٌ ٔأَثدداء أتُدداء .انؼياٌ ذخ: ئدـاٌ ػثاؽ صاع اصع تٛغٔخ  ( "ٙانظْث2003 ) يذًض تٍ أدًدض تدٍ ػصًداٌ (خ948 /ْدـ1390 و) ذداعٚز اإلؿدالو ََٔٔقٛداخ انًلداْٛغ َٔاألػالو ذخ: تلاع ػٕراص يؼغٔى صاع انغغب اإلؿاليٙ ص– .و  ٍات( "انغقؼح2009 ) أدًض تٍ يذًض تٍ ػهٙ (خ910 / ِ 1332 :و) كفاٚح انُثّٛ قدٙ كدغح انرُثٛدّ ذدخ يجض٘ يذًض ؿغٔع صاع انكرة انؼهًٛح ص– .و  انؼتٛددض٘ (ص.خ) يذًددض تددٍ يذًددض تددٍ ػثددض انددغػرا (خ1205 /ْددـ1829 و) ذدداض انؼددغٔؽ يددٍ نددٕاْغ انبايٕؽ ذخ: يجًٕػح يٍ انًذببٍٛ صاع انٓضاٚح ص – و– .  ("ٙاتٍ كاكغ انكرث1993 ) يذًض تٍ كاكغ تٍ أدًض (خ964 /ْـ1386 ٌو) قٕاخ انٕقٛداخ ذدخ: ئدـدا .ػثاؽ صاع اصع تٛغٔخ  ("اتٕ كجاع2003 ) يذًض تٍ انذـٍٛ تٍ يذًض تٍ ػثض هللا (خ488 /ِ 1110 :و) طٚم ذجاعب االيى ذخ ؿٛض كـغٔ٘ صاع انؼهًٛح ص– و– .  "٘انزددفض( 2000 ) سهٛددم تددٍ أٚثددك تددٍ ػثددض هللا (خ964 /ْددـ1386 و) انددٕاقٙ تانٕقٛدداخ ذددخ: أدًددض .األعَاؤٔط ٔذغكٙ يز فٗ صاع ئدٛاء انرغاز تٛغٔخ  ان ثددغ٘" يذًددض تددٍ نغٚددغ تددٍ ٚؼٚددض (خ310 /ْددـ932 و) ذدداعٚز األيددى ٔانًهددٕي صاع انكرددة انؼهًٛددح (تٛغٔخ- 1409 )ِ  ( ٌٕاتٍ طٕن1998 ّٚٔ) يذًض تٍ ػهٙ تٍ سًاع (خ953 /ْـ1595 و) ئَثداء األيدغاء تأَثداء اندٕػعاء .ذخ: يُٓرا دًض انًُٓرا صاع انثلائغ االؿاليٛح تٛغٔخ  ("٘اتٍ انؼثغ1992 ) غٚغٕعٕٚؽ تٍ ْداعٌٔ تدٍ ذٕيدا (خ685 /ْدـ1309 ) ذداعٚز يشرزدغ اندضٔل ذخ: أَ ٌٕ انذاَٙ انٛـٕػٙ صاع انلغ ط3 . Abstract: تٛغٔخ  ( "٘انؼـددكغ1402 ِ) ٍ انذـدد تددٍ ػثددض هللا تددٍ ؿددؼٛض (خ382 /ْددـ1004 :و) ذزددذٛفاخ انًذددضشٍٛ ذددخ .يذًٕص أدًض يٛغج انً ثؼح انؼغتٛح انذضٚصح انباْغج  أتددٕ انفددضاء"(ص.خ) ئؿددًاػٛم تددٍ ػهددٙ تددٍ يذًددٕص (خ932 / ْددـ134 و) انًشرزددغ قددٙ أسثدداع انثلددغ انً ثؼح انذـُٛٛح انًزغٚح ص– .و  ("ٙ انفب2005 ) ػهٙ تدٍ ٕٚؿدا تدٍ ئتدغاْٛى (خ646 /ْدـ1268 و) ئسثداع انؼهًداء تأسثداع انذكًداء .ذخ: ئتغاْٛى كًؾ انضٍٚ صاع انكرة انؼهًٛح تٛغٔخ  انًـؼٕص٘"(ص.خ) ػهٗ تٍ انذـٍٛ تٍ ػهٗ (خ346 /ْدـ968 و) انرُثٛدّ ٔاإلكدغاى ذزدذٛخ: ػثدض هللا .ئؿًاػٛم انزأ٘ صاع انزأ٘ انباْغج  ( "ّٕٚيـك2000) أدًض ت ٍ يذًض تٍ ٚؼبٕب (خ421 /ْـ1043 :و) ذجاعب األيدى ٔذؼاشدة انًٓدى ذدخ أتٕ انباؿى ئيايٙ ؿغٔف ط2 .ٌ طٓغا  ("ٙانًبضؿ1991 ٕ) يذًض تٍ أدًض (خ َذ380 /ِ 1002 و) أدـٍ انرباؿدٛى قدٙ يؼغقدح األشدانٛى يكرثدح يضتٕنٙ ط3 . انباْغج  ("ٙانًبضؿ1991 ٕ) يذًض تٍ أدًض (خ َذ380 /ِ 1002 و) أدـٍ انرباؿٛى قٙ يؼغقدح األشدانٛى يكرثدح يضتٕنٙ انباْغج- .  ( "ان بض1958(خ اْ ض ٍ ػ ض ان هك ٍ ئ )521 / 1143ذ ) ذك هح ذا ز ان جمةل الآداب / العدد631 (آآذار) جمةل الآداب / العدد631 (آآذار)  اتٍ انرغغ٘ تغص٘" (ص.خ) ٕٚؿا تٍ ذغدغ٘ تدغص٘ تدٍ ػثدض هللا (خ894 /ْدـ1496 و) انُجدٕو انؼاْدغج ٙق .يهٕي يزغ ٔانباْغج ٔػاعج انصباقح ٔاإلعكاص انبٕيٙ صاع انكرة يزغ  (ٙانرُدددٕس1391 ِ) انًذـدددٍ تدددٍ ػهدددٙ تدددٍ يذًدددض (خ384 /ْدددـ1006 و) َلدددٕاع انًذاعدددغج ٔأسثددداع انًظاكغج ص– .و  ( "٘انجٕاْغ1989 ) ئؿًاػٛم تٍ دًاص (خ393 / ْـ1015 و) انزذاح ذداض انهغدح ٔ دذاح انؼغتٛدح ذخ: أدًض ػ ثض انغفٕع ػ اع صاع انؼهى نهًالٍٚٛ ط4 . تٛغٔخ  ("٘اتٍ انجٕػ1992) ػثض انغدًٍ تٍ ػهٙ تدٍ يذًدض (خ599 /ْدـ1219 و) انًُدرظى قدٙ ذداعٚز األيدى .ٔانًهٕي ذخ: يذًض ػثض انباصع ػ ا يز فٗ ػثض انباصع ػ ا صاع انكرة انؼهًٛح تٛغٔخ  ("اتٍ ػثض انذق1412ِ) ػثض انًإيٍ تٍ ػثض ا نذق تٍ كدًائم (خ939 /ْدـ1361 و) يغا دض االطدالع .ػهٗ أؿًاء األيكُح ٔانثباع صاع انجٛم تٛغٔخ  ( "٘انذًٛغ1980 ) يذًض تٍ ػثض هللا تٍ ػثض انًدُؼى (خ900 / ِ 1522 و) اندغٔض انًؼ داع قدٙ سثدغ األش اع ذخ: ئدـاٌ ػثاؽ يإؿـح َا غ نهصباقح ط2 . 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Geskiedenis van Nasies en Konings, Wetenskaplike Boeke Huis (Beirut-1407H)  Al-Tankhi (1391H), Mohsen Bin Ali bin Mohammed (T 384 Ah/ 1006 A.D).  Al-Wasibi (2005), Ali ibn Yusuf bin Ibrahim (t. 646 Ah/ 1268 A.D.) vertel geleerdes die nuus van die wyse, t. Ibrahim Shamseddine, Wetenskaplike Boeke Huis, Beirut.  Al-Wasibi (2005), Ali ibn Yusuf bin Ibrahim (t. References:  Abu al-Fida" (D.T.), Ismail bin Ali bin Mahmoud (t. 732 Ah / 134 A.D.) kort in The News of The Humans, The Egyptian Husseini Press, D-M.  Abu al-Fida" (D.T.), Ismail bin Ali bin Mahmoud (t. 732 Ah / 134 A.D.) kort in The News of The Humans, The Egyptian Husseini Press, D-M.  Abu Shujaa" (2003), Mohammed bin Al-Hussein bin Mohammed bin Abdullah (t 488 Ah/1110 A.D.) die stert van die ervarings van nasies, t: Sayyid Kassraoui, Dar al-Sinaa, D-M.  Abu Shujaa" (2003), Mohammed bin Al-Hussein bin Mohammed bin Abdullah (t 488 Ah/1110 A.D.) die stert van die ervarings van nasies, t: Sayyid Kassraoui, Dar al-Sinaa, D-M.  Abu Yusuf" (D.T.), Yaacoub bin Ibrahim bin Habib (t 182 Ah / 804 A.D.), Al- Kharj, Tah: Taha Abdul Rauf Saad, Hartseer Hassan Mohammed, Al-Azhar Erfenis Biskoperie, Salafi Druk Pers, Kaïro.  Abu Yusuf" (D.T.), Yaacoub bin Ibrahim bin Habib (t 182 Ah / 804 A.D.), Al- Kharj, Tah: Taha Abdul Rauf Saad, Hartseer Hassan Mohammed, Al-Azhar Erfenis Biskoperie, Salafi Druk Pers, Kaïro.  Al-Askari" (1402Ah), Al-Hassan bin Abdullah bin Saeed (T. 382Ah/1004 n.C).  Al-Azhari (2001), Mohammed bin Ahmed (T370Ah/992 n.C.) Taalverfyning, T: Mohammed Awad Mareeb, Arabiese Erfenis Herlewingshuis, Beirut.  Al-Azhari (2001), Mohammed bin Ahmed (T370Ah/992 n.C.) Taalverfyning, T: Mohammed Awad Mareeb, Arabiese Erfenis Herlewingshuis, Beirut.  Al-Baali (2003), Mohammed bin Abi al-Fath (t. 709 Ah/ 1331 A.D.), wat vertroud is met die woorde van die gemaskerde, t. Mahmoud Al-Arnaout en Yasin Mahmoud al-Khatib, Al-Swasdi Biblioteek, D-M. Al-Baghdadi (2002), Ahmad bin Ali bin Thabet (T. 463 Ah / 1085 A.D.), Geskiedenis van Bagdad, T: Bashar Awad Marouf, Dar al-Gharbia, Beirut.  Al-Baali (2003), Mohammed bin Abi al-Fath (t. 709 Ah/ 1331 A.D.), wat vertroud is met die woorde van die gemaskerde, t. Mahmoud Al-Arnaout en Yasin Mahmoud al-Khatib, Al-Swasdi Biblioteek, D-M. Al-Baghdadi (2002), Ahmad bin Ali bin Thabet (T. 463 Ah / 1085 A.D.), Geskiedenis van Bagdad, T: Bashar Awad Marouf, Dar al-Gharbia, Beirut.  Al-Dhahabi" (2003), Mohammed bin Ahmed bin Othman (t. 748 Ah/ 1370 A.D.) Geskiedenis van Islam en die dood van bekendes en vlae, T.: Bashar Awad Marouf, Dar al-Gharbia, D-M.  Al-Dhahabi" (2003), Mohammed bin Ahmed bin Othman (t. 748 Ah/ 1370 A.D.) Geskiedenis van Islam en die dood van bekendes en vlae, T.: Bashar Awad Marouf, Dar al-Gharbia, D-M. Abstract:  ( "ٙانًبضؿ1958 ) يذًض تٍ ػثدض انًهدك تدٍ ئتدغاْٛى (خ521 /ِ 1143 :يدـ) ذكًهدح ذداعٚز ان ثدغ٘ ذدخ أنثغخ ٕٚؿا كُؼاٌ انً ثؼح انكاشٕنٛكٛح (تٛغٔخ- 1958 )  ( "انًُجى1408 ِ) ئؿذا تٍ انذـٍٛ (خ4 / ِ 10 ٙو) آكاو انًغنداٌ قدٙ طكدغ انًدضائٍ انًلدٕٓعج قد كم يكاٌ ػانى انكرة تٛغٔخ 322 جمةل الآداب / العدد631 (آآذار) 0206 / م6110 هـ  اتٍ انًٓاو"(ص.خ) يذًض تٍ ػثض انٕادض (خ861 /ْـ1483 )و قرخ انبضٚغ صاع انفكغ (ص– و– ص– )خ  ( "٘اتٍ انٕعص1996 ) ػًغ تٍ يظفغ (خ949 /ْـ1391 و) ذاعٚز اتٍ اندٕعص٘" صاع انكردة انؼهًٛدح .تٛغٔخ  ("ًٕ٘ٚاشٕخ انذ1995 ) ٚاشٕخ تدٍ ػثدض هللا (خ626 / ْدـ1248 و) يؼجدى انثهدضاٌ صاع داصع ط2 .تٛغٔخ  ( "ٙانٛؼبٕت1422 ِ) ٍ أدًض ت ئؿذا (خ تؼض292 / ْـ914 .و) انثهضاٌ صاع انكرة انؼهًٛح تٛغٔخ  أتٕ ٕٚؿا" (ص.خ) ٚؼبٕب تدٍ ئتدغاْٛى تدٍ دثٛدة (خ182 / ِ 804 و) انشدغاض ذدخ: طدّ ػثدض اندغؤٔى ؿؼض ؿؼض دـٍ يذًض انًكرثح األػْغٚح نهرغاز انً ثؼح انـهفٛح انب.ِاْغ جمةل الآداب / العدد631 (آآذار) 0206 / م6110 هـ  اتٍ انًٓاو"(ص.خ) يذًض تٍ ػثض انٕادض (خ861 /ْـ1483 )و قرخ انبضٚغ صاع انفكغ (ص– و– ص– )خ  ( "٘اتٍ انٕعص1996 ) ػًغ تٍ يظفغ (خ949 /ْـ1391 و) ذاعٚز اتٍ اندٕعص٘" صاع انكردة انؼهًٛدح .تٛغٔخ  ("ًٕ٘ٚاشٕخ انذ1995 ) ٚاشٕخ تدٍ ػثدض هللا (خ626 / ْدـ1248 و) يؼجدى انثهدضاٌ صاع داصع ط2 .تٛغٔخ  ( "ٙانٛؼبٕت1422 ِ) ٍ أدًض ت ئؿذا (خ تؼض292 / ْـ914 .و) انثهضاٌ صاع انكرة انؼهًٛح تٛغٔخ  أتٕ ٕٚؿا" (ص.خ) ٚؼبٕب تدٍ ئتدغاْٛى تدٍ دثٛدة (خ182 / ِ 804 و) انشدغاض ذدخ: طدّ ػثدض اندغؤٔى ؿؼض ؿؼض دـٍ يذًض انًكرثح األػْغٚح نهرغاز انً ثؼح انـهفٛح انب.ِاْغ Abstract:  ("ٙاتٍ كاكغ انكرث1993 ) يذًض تٍ كاكغ تٍ أدًض (خ964 /ْـ1386 ٌو) قٕاخ انٕقٛداخ ذدخ: ئدـدا .ػثاؽ صاع اصع تٛغٔخ اهلل ٙ ٙ خ  ( "٘انؼـددكغ1402 ِ) ٍ انذـدد تددٍ ػثددض هللا تددٍ ؿددؼٛض (خ382 /ْددـ1004 :و) ذزددذٛفاخ انًذددضشٍٛ ذددخ .يذًٕص أدًض يٛغج انً ثؼح انؼغتٛح انذضٚصح انباْغج غ غ غ  أتددٕ انفددضاء"(ص.خ) ئؿددًاػٛم تددٍ ػهددٙ تددٍ يذًددٕص (خ932 / ْددـ134 و) انًشرزددغ قددٙ أسثدداع انثلددغ انً ثؼح انذـُٛٛح انًزغٚح ص– .و و  ("ٙ انفب2005 ) ػهٙ تدٍ ٕٚؿدا تدٍ ئتدغاْٛى (خ646 /ْدـ1268 و) ئسثداع انؼهًداء تأسثداع انذكًداء .ذخ: ئتغاْٛى كًؾ انضٍٚ صاع انكرة انؼهًٛح تٛغٔخ إهلل ى خ  انًـؼٕص٘"(ص.خ) ػهٗ تٍ انذـٍٛ تٍ ػهٗ (خ346 /ْدـ968 و) انرُثٛدّ ٔاإلكدغاى ذزدذٛخ: ػثدض هللا .ئؿًاػٛم انزأ٘ صاع انزأ٘ انباْغج أ ٘ ٘  ( "ّٕٚيـك2000) أدًض ت ٍ يذًض تٍ ٚؼبٕب (خ421 /ْـ1043 :و) ذجاعب األيدى ٔذؼاشدة انًٓدى ذدخ أتٕ انباؿى ئيايٙ ؿغٔف ط2 .ٌ طٓغا أ لأ أ  ("ٙانًبضؿ1991 ٕ) يذًض تٍ أدًض (خ َذ380 /ِ 1002 و) أدـٍ انرباؿدٛى قدٙ يؼغقدح األشدانٛى يكرثدح يضتٕنٙ ط3 . انباْغج أ ٙ  ("ٙانًبضؿ1991 ٕ) يذًض تٍ أدًض (خ َذ380 /ِ 1002 و) أدـٍ انرباؿٛى قٙ يؼغقدح األشدانٛى يكرثدح يضتٕنٙ انباْغج- . 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Mohamed Abdelkader Atta, Mustafa Abdel Kader Atta, Die Huis van Wetenskaplike Boeke, Beut  Ibn al-Rifa'a" (2009), Ahmed bin Mohammed bin Ali (T. 710Ah / 1332 A.D.)  Ibn al-Rifa'a" (2009), Ahmed bin Mohammed bin Ali (T. 710Ah / 1332 A.D.)  Ibn al-Taghrebari Bardi" (D.T.), Youssef bin Taghreeb Bardi bin Abdullah (T. 874 Ah/ 1496 N.D.) die helder sterre van die konings van Egipte en Kaïro, die Ministerie van Kultuur en Nasionale Leiding, Dar al-Books, Egipte.  Ibn al-Taghrebari Bardi" (D.T.), Youssef bin Taghreeb Bardi bin Abdullah (T. 874 Ah/ 1496 N.D.) die helder sterre van die konings van Egipte en Kaïro, die Ministerie van Kultuur en Nasionale Leiding, Dar al-Books, Egipte.  Ibn al-Wardi" (1996), Omar Bin Muzaffar (T. 749 H/1371 n.C.) Geskiedenis van Ibn al-Wardi, Wetenskaplike Boeke Huis, Beirut.  Ibn al-Wardi" (1996), Omar Bin Muzaffar (T. 749 H/1371 n.C.) Geskiedenis van Ibn al-Wardi, Wetenskaplike Boeke Huis, Beirut.  Ibn Khalkan (1900), Ahmed bin Mohammed bin Ibrahim (T. 681 Ah/ 1301 A.D.), Die dood van hoogwaardigheidsbekleërs en die nuus van die Seuns van Tyd, T: Ihsan Abbas, Dar Sader, Beirut.  Ibn Khalkan (1900), Ahmed bin Mohammed bin Ibrahim (T. 681 Ah/ 1301 A.D.), Die dood van hoogwaardigheidsbekleërs en die nuus van die Seuns van Tyd, T: Ihsan Abbas, Dar Sader, Beirut.  Ibn Shaker al-Kutbi (1973), Mohammed ibn Shaker bin Ahmed (t. 764 Ah/ 1386 A.D).  Ibn Shaker al-Kutbi (1973), Mohammed ibn Shaker bin Ahmed (t. 764 Ah/ 1386 A.D).  Ibn Toulon (1998), Muhammad bin Ali bin Khamarueh (T. 953Ah/ 1575 A.D.) Die Prins se nuus van predikante, T. Mahna Hamad Al-Muhanna, Dar al-Bashir al-Islamiya, Beirut. References: 646 Ah/ 1268 A.D.) vertel geleerdes die nuus van die wyse, t. Ibrahim Shamseddine, Wetenskaplike Boeke Huis, Beirut. 322 جمةل الآداب / العدد631 (آآذار) 0206 / م6110 هـ 0206 / م6110 هـ  Al-Yacoubi" (1422 Ah), Ahmed Ibn Ishaq (292 Ah / 914 n.C. Lande, Wetenskaplike Boeke Huis, Beirut.  Al-Yacoubi" (1422 Ah), Ahmed Ibn Ishaq (292 Ah / 914 n.C. Lande, Wetenskaplike Boeke Huis, Beirut.  Al-Yacoubi" (1422 Ah), Ahmed Ibn Ishaq (292 Ah / 914 n.C. Lande, Wetenskaplike Boeke Huis, Beirut.  Die myn" (1408H), Isak bin Al Hussein (T.S. 4H / 10 n.C. Akam Al-Marjan in die noem van die beroemde stede oral, Die Wêreld van Boeke, Beirut.  Die myn" (1408H), Isak bin Al Hussein (T.S. 4H / 10 n.C. Akam Al-Marjan in die noem van die beroemde stede oral, Die Wêreld van Boeke, Beirut.  Ibn Abd al-Bar (1400Ah), Yusuf bin Abdullah bin Mohammed (t. 463 Ah/ 1085 A.D).  Ibn Abd al-Bar (1400Ah), Yusuf bin Abdullah bin Mohammed (t. 463 Ah/ 1085 A.D).  Ibn Abd al-Haq"(1412Ah), Abd al-Momen bin Abdul Haq bin Shimal (T. 739 Ah/ 1361 A.D.) Sterrewagte vir toegang tot die name van plekke en plekke, Dar al-Jil, Beirut.  Ibn Abd al-Haq"(1412Ah), Abd al-Momen bin Abdul Haq bin Shimal (T. 739 Ah/ 1361 A.D.) Sterrewagte vir toegang tot die name van plekke en plekke, Dar al-Jil, Beirut.  Ibn Abi A'a'a" (D.T.), Ahmed bin Al Qasim bin Khalifa (t. 668 Ah/ 1290 A.D.) Oë van nuus in die lae dokters, T: Nizar Reda, Biblioteek van die Lewe, Beirut.  Ibn Abi A'a'a" (D.T.), Ahmed bin Al Qasim bin Khalifa (t. 668 Ah/ 1290 A.D.) 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https://openalex.org/W3196384633	https://europepmc.org/articles/pmc8557899?pdf=render	English	null	The Reemergence of Seasonal Respiratory Viruses in Houston, Texas, after Relaxing COVID-19 Restrictions	Microbiology spectrum	2,021	cc-by	2,395	Volume 9 Issue 2 e00430-21 MicrobiolSpectrum.asm.org 1 Citation Hodjat P, Christensen PA, Subedi S, Bernard DW, Olsen RJ, Long SW. 2021. The reemergence of seasonal respiratory viruses in Houston, Texas, after relaxing COVID-19 restrictions. Microbiol Spectr 9:e00430-21. https://doi.org/10.1128/Spectrum.00430-21. Editor Heba H. Mostafa, Johns Hopkins Hospital Copyright © 2021 Hodjat et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to S. Wesley Long, swlong@houstonmethodist.org. As COVID-19 restrictions ease and mask mandates end, @ParsaHodjatMD @paulchstmd @drswlong and colleagues @MethodistHosp show the rapid return of non-COVID respiratory illness in Houston. Received 27 May 2021 Accepted 13 August 2021 Published 8 September 2021 OBSERVATION OBSERVATION The Reemergence of Seasonal Respiratory Viruses in Houston, Texas, after Relaxing COVID-19 Restrictions Parsa Hodjat,a Paul A. Christensen,a Sishir Subedi,a David W. Bernard,a Randall J. Olsen,a S. Wesley Longa Parsa Hodjat,a Paul A. Christensen,a Sishir Subedi,a David W. Bernard,a Randall J. Olsen,a S. Wesley Longa aCenter for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas, USA ABSTRACT Measures intended to limit the spread of the severe acute respiratory syn- drome coronavirus 2 (SARS-CoV-2) virus at the start of the coronavirus disease 2019 (COVID-19) pandemic resulted in a rapid decrease in other respiratory pathogens. Herein, we describe the trends of respiratory pathogens in a major metropolitan health care sys- tem central microbiology reference laboratory before and during the COVID-19 pandemic, with attention to when COVID-19 mitigation measures were implemented and relaxed. During the initial lockdown period, COVID-19 was the primary respiratory pathogen detected by multiplex respiratory panels. As COVID-19 containment measures were relaxed, the ﬁrst non-COVID respiratory viruses to return to prepandemic levels were members of the rhinovirus/enterovirus family. After the complete removal of COVID-19 precautions at the state level, including an end to mask mandates, we observed the robust return of seasonal coronaviruses, parainﬂuenza virus, and respiratory syncytial virus. Inasmuch as COVID-19 has dominated the landscape of respiratory infections since early 2020, it is important for clinicians to recognize that the return of non-COVID respiratory pathogens may be rapid and signiﬁcant when COVID-19 containment measures are removed. IMPORTANCE We describe the return of non-COVID respiratory viruses after the removal of COVID-19 mitigation measures. It is important for the public and physicians to recognize that, after months of COVID-19 being the primary driver of respiratory infection, more typical seasonal respiratory illnesses have returned, and this return is out of the normal season for some of these pathogens. Thus, clinicians and the public must now consider both COVID-19 and other respiratory illnesses when a patient presents with symptomatic respiratory illness. Citation Hodjat P, Christensen PA, Subedi S, Bernard DW, Olsen RJ, Long SW. 2021. The reemergence of seasonal respiratory viruses in Houston, Texas, after relaxing COVID-19 restrictions. Microbiol Spectr 9:e00430-21. https://doi.org/10.1128/Spectrum.00430-21. Editor Heba H. Mostafa, Johns Hopkins Hospital Copyright © 2021 Hodjat et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to S. Wesley Long, swlong@houstonmethodist.org. The Reemergence of Seasonal Respiratory Viruses in Houston, Texas, after Relaxing COVID-19 Restrictions As COVID-19 restrictions ease and mask mandates end, @ParsaHodjatMD @paulchstmd @drswlong and colleagues @MethodistHosp show the rapid return of non-COVID respiratory illness in Houston. Received 27 May 2021 Accepted 13 August 2021 Published 8 September 2021 Citation Hodjat P, Christensen PA, Subedi S, Bernard DW, Olsen RJ, Long SW. 2021. The reemergence of seasonal respiratory viruses in Houston, Texas, after relaxing COVID-19 restrictions. Microbiol Spectr 9:e00430-21. https://doi.org/10.1128/Spectrum.00430-21. Editor Heba H. Mostafa, Johns Hopkins Hospital Copyright © 2021 Hodjat et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to S. Wesley Long, swlong@houstonmethodist.org. As COVID-19 restrictions ease and mask mandates end, @ParsaHodjatMD @paulchstmd @drswlong and colleagues @MethodistHosp show the rapid return of non-COVID respiratory illness in Houston. Received 27 May 2021 Accepted 13 August 2021 Published 8 September 2021 KEYWORDS COVID-19, coronavirus, inﬂuenza, masking, parainﬂuenza virus, respiratory syncytial virus I mplementation of measures to limit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission during the coronavirus disease 2019 (COVID-19) pandemic coincided with a marked decrease in infections caused by other respiratory viruses (1). The decrease was due, in part, to masking, social distancing, closure of schools and busi- nesses, and other efforts targeted at limiting disease spread (2). Houston Methodist Hospital System is a large multihospital system in one of the most diverse cities in the United States. The centralized reference microbiology laboratory performs a variety of testing, including the BioFire respiratory pathogen panel, which tests for 22 differ- ent pathogen targets. Our testing methods for COVID-19, including genome-sequencing efforts, have been published previously (3–5). Early in the public health response to COVID-19 in Houston, the Houston Livestock Show and Rodeo was cancelled on 11 March 2020 and a citywide stay-at-home order was implemented on 25 March 2020 (https://abc13.com/houston-rodeo-coronavirus-update-texas -cancellation-livestock-show-and/6003475/ and https://www.houstontx.gov/mayor/press/ 2020/stay-home-work-safe-order.html, last accessed 25 May 2021). Subsequently, rates MicrobiolSpectrum.asm.org 1 FIG 1 Histograms of respiratory virus tests from 1 January 2019 to 25 May 2021. (A) Rhinovirus/enterovirus was the ﬁrst virus to rebound to prepandemic levels. (B) Coronaviruses 229E, HKU1, NL63, and OC43. OC43 has been prevalent since March 2021. (C) Parainﬂuenza viruses 1 to 4. Parainﬂuenza 3 has been prevalent since March 2021. (D) Respiratory syncytial virus has also increased in prevalence since March 2021. (E) SARS-CoV-2 testing volume and positivity from March 2020 through June 2021. MicrobiolSpectrum.asm.org 2 The Reemergence of Seasonal Respiratory Viruses in Houston, Texas, after Relaxing COVID-19 Restrictions (F) Respiratory panel test volume from January 2020 through June 2021. Symbols indicate the implementation and relaxation of COVID-19 precautions. , March 2020, start of pandemic lockdown measures; †, May 2020, phase one reopening of Texas; ‡, October 2020, additional reopening measures; §, March 2021, removal of all COVID restrictions, including elimination of mask mandates. All tests were performed on the Bioﬁre respiratory pathogen panel, with the exception of (E) which represents all SARS-CoV-2 PCR testing performed using multiple test vendors. Hodjat et al. Hodjat et al. FIG 1 Histograms of respiratory virus tests from 1 January 2019 to 25 May 2021. (A) Rhinovirus/enterovirus was the ﬁrst virus to rebound to prepandemic levels. (B) Coronaviruses 229E, HKU1, NL63, and OC43. OC43 has been prevalent since March 2021. (C) Parainﬂuenza viruses 1 to 4. Parainﬂuenza 3 has been prevalent since March 2021. (D) Respiratory syncytial virus has also increased in prevalence since March 2021. (E) SARS-CoV-2 testing volume and positivity from March 2020 through June 2021. (F) Respiratory panel test volume from January 2020 through June 2021. Symbols indicate the implementation and relaxation of COVID-19 precautions. , March 2020, start of pandemic lockdown measures; †, May 2020, phase one reopening of Texas; ‡, October 2020, additional reopening measures; §, March 2021, removal of all COVID restrictions, including elimination of mask mandates. All tests were performed on the Bioﬁre respiratory pathogen panel, with the exception of (E) which represents all SARS-CoV-2 PCR testing performed using multiple test vendors. of inﬂuenza virus, respiratory syncytial virus (RSV), rhinovirus/enterovirus, and seasonal coronavirus infections, as diagnosed by the respiratory pathogen panel in the Houston Methodist Hospital centralized microbiology laboratory, declined rapidly (Fig. 1) (https://ﬂu .houstonmethodist.org, last accessed 25 May 2021). Similar declines can be observed both in inﬂuenza surveillance data and respiratory pathogen panel data from the United States Volume 9 Issue 2 e00430-21 Respiratory Viruses Post-COVID Restrictions (https://www.cdc.gov/ﬂu/weekly/index.htm and https://syndromictrends.com, last accessed 1 July 2021) and other countries (6, 7). As COVID-19 measures were gradually relaxed starting in May 2020, very low levels of non- SARS-CoV-2 respiratory pathogens were detected even as cases of COVID-19 began to rise (https://gov.texas.gov/uploads/ﬁles/press/EO-GA-23_phase_two_expanding_opening_COVID -19.pdf, last accessed 25 May 2021) (8). Levels of non-COVID respiratory infections remained exceedingly low through the summer (10 to 28 rhinovirus/enterovirus cases per week [cpw], 0 to 4 cpw of inﬂuenza A and inﬂuenza B virus, and 0 to 1 cpw of RSV). 1. Olsen SJ, Azziz-Baumgartner E, Budd AP, Brammer L, Sullivan S, Pineda RF, Cohen C, Fry AM. 2020. Decreased inﬂuenza activity during the COVID-19 pan- demic—United States, Australia, Chile, and South Africa, 2020. MMWR Morb Mor- tal Wkly Rep 69:1305–1309. https://doi.org/10.15585/mmwr.mm6937a6. 5. Long SW, Olsen RJ, Christensen PA, Bernard DW, Davis JR, Shukla M, Nguyen M, Ojeda Saavedra M, Cantu CC, Yerramilli P, Pruitt L, Subedi S, Hendrickson H, Eskandari G, Kumaraswami M, McLellan, Musser JS. 2020. Molecular architecture of early dissemination and evolution of the SARS-CoV-2 virus in metropolitan Houston, Texas. bioRxiv. https://doi.org/10.1101/2020.05.01.072652. The Reemergence of Seasonal Respiratory Viruses in Houston, Texas, after Relaxing COVID-19 Restrictions In September 2020, rhinovirus/enterovirus cases began increasing to prepandemic levels as schools reopened and many remaining measures were relaxed in October 2020 (97 to 156 rhinovirus/enter- ovirus cases per week, October through December 2020) (Fig. 1A) (https://gov.texas.gov/ uploads/ﬁles/press/EO-GA-32_continued_response_to_COVID-19_IMAGE_10-07-2020.pdf, last accessed 25 May 2021). The ﬁrst week of March 2021, the Texas governor announced that the remaining measures were being eliminated and face masks could no longer be mandated by state or local government (https://open.texas.gov/uploads/ﬁles/organization/opentexas/EO-GA-34 -opening-Texas-response-to-COVID-disaster-IMAGE-03-02-2021.pdf, last accessed 25 May 2021). That same month, we began to observe a marked increase in rhinovirus/enterovirus, parainﬂuenza virus, and seasonal coronavirus infections (Fig. 1B to D). In Houston, seasonal coronaviruses typically peak during the winter months, with very low levels observed during the summer. We are now observing a month-over-month increase in parainﬂuenza and sea- sonal coronaviruses which would be considered out of season compared to their typical sea- sonality. Out-of-season increases in RSV have been reported elsewhere when COVID-19 meas- ures were relaxed (https://www.abc.net.au/news/2021-02-24/rsv-cases-surging-in-south-east -queensland/13186788, last accessed 25 May 2021). We have also seen a recent increase in RSV cases in May 2021 (0 to 1 cpw between 1 June 2020 and 1 March 2021, now increased to 66 cpw in June 2021), although our patient population is primarily comprised of adults. Rhinovirus/enterovirus cases have also increased from 82 to 106 cpw in January and February 2021 to 143 to 156 cpw from the end of March through April 2021, decreasing to just over 100 cpw in June 2021. Inasmuch as the incremental relaxation of COVID-19 prevention measures affected respiratory infection rates over time, the recent discontinuation of mask mandates in March 2021 has coincided with a rapid increase in a variety of non-COVID respiratory pathogens. These observations are important for clinicians to consider as they evaluate patients with respiratory infections in the coming months. They also underscore the high effectiveness of nonpharmacologic preventative measures like masking and social distancing in preventing the spread of respiratory pathogens. 6. Redlberger-Fritz M, Kundi M, Aberle SW, Puchhammer-Stöckl E. 2021. Signiﬁ- cant impact of nationwide SARS-CoV-2 lockdown measures on the circulation of other respiratory virus infections in Austria. J Clin Virol 137:104795. https:// doi.org/10.1016/j.jcv.2021.104795. p g 3. Long SW, Olsen RJ, Christensen PA, Subedi S, Olson R, Davis JJ, Saavedra MO, Yerramilli P, Pruitt L, Reppond K, Shyer MN, Cambric J, Finkelstein IJ, Gollihar J, Musser JM. 2021. Sequence analysis of 20,453 severe acute respiratory syndrome coronavirus 2 genomes from the Houston Metropolitan Area identiﬁes the emer- gence and widespread distribution of multiple isolates of all major variants of concern. Am J Pathol 191:983–992. https://doi.org/10.1016/j.ajpath.2021.03.004. y 2. Chu DK, Akl EA, Duda S, Solo K, Yaacoub S, Schünemann HJ, COVID-19 Sys- tematic Urgent Review Group Effort (SURGE) study authors. 2020. Physical distancing, face masks, and eye protection to prevent person-to-person transmis- sion of SARS-CoV-2 and COVID-19: a systematic review and meta-analysis. Lancet 395:1973–1987. https://doi.org/10.1016/S0140-6736(20)31142-9. 7. Kuitunen I, Artama M, Mäkelä L, Backman K, Heiskanen-Kosma T, Renko M. 2020. Effect of social distancing due to the COVID-19 pandemic on the incidence of vi- ral respiratory tract infections in children in Finland during early 2020. Pediatr Infect Dis J 39:e423–e427. https://doi.org/10.1097/INF.0000000000002845. MicrobiolSpectrum.asm.org 3 8. Long SW, Olsen RJ, Christensen PA, Bernard DW, Davis JJ, Shukla M, Nguyen M, Saavedra MO, Yerramilli P, Pruitt L, Subedi S, Kuo HC, Hendrickson H, Eskandari G, Nguyen HAT, Long JH, Kumaraswami M, Goike J, Boutz D, Gollihar J, McLellan JS, Chou CW, Javanmardi K, Finkelstein IJ, Musser JM. 2020. Molecular architecture of early dissemination and massive second wave of the SARS-CoV-2 Virus in a major metropolitan area. mBio 11:e02707-20. https://doi.org/10.1128/mBio.02707-20. 4. Musser JM, Olsen RJ, Christensen PA, Long SW, Subedi S, Davis JJ, Gollihar J. 2021. Rapid, widespread, and preferential increase of SARS-CoV-2 B.1.1.7 variant in Houston, TX, revealed by 8,857 genome sequences. medRxiv. https://doi.org/10.1101/2021.03.16.21253753. 5. Long SW, Olsen RJ, Christensen PA, Bernard DW, Davis JR, Shukla M, Nguyen M, Ojeda Saavedra M, Cantu CC, Yerramilli P, Pruitt L, Subedi S, Hendrickson H, Eskandari G, Kumaraswami M, McLellan, Musser JS. 2020. Molecular architecture of early dissemination and evolution of the SARS-CoV-2 virus in metropolitan Houston, Texas. bioRxiv. https://doi.org/10.1101/2020.05.01.072652. 6. Redlberger-Fritz M, Kundi M, Aberle SW, Puchhammer-Stöckl E. 2021. Signiﬁ- cant impact of nationwide SARS-CoV-2 lockdown measures on the circulation of other respiratory virus infections in Austria. J Clin Virol 137:104795. https:// doi.org/10.1016/j.jcv.2021.104795. 7. Kuitunen I, Artama M, Mäkelä L, Backman K, Heiskanen-Kosma T, Renko M. 2020. Effect of social distancing due to the COVID-19 pandemic on the incidence of vi- ral respiratory tract infections in children in Finland during early 2020. Pediatr Infect Dis J 39:e423–e427. https://doi.org/10.1097/INF.0000000000002845. 8. Long SW, Olsen RJ, Christensen PA, Bernard DW, Davis JJ, Shukla M, Nguyen M, Saavedra MO, Yerramilli P, Pruitt L, Subedi S, Kuo HC, Hendrickson H, Eskandari G, Nguyen HAT, Long JH, Kumaraswami M, Goike J, Boutz D, Gollihar J, McLellan JS, Chou CW, Javanmardi K, Finkelstein IJ, Musser JM. 2020. Molecular architecture of early dissemination and massive second wave of the SARS-CoV-2 Virus in a major metropolitan area. mBio 11:e02707-20. https://doi.org/10.1128/mBio.02707-20. REFERENCES 8. Long SW, Olsen RJ, Christensen PA, Bernard DW, Davis JJ, Shukla M, Nguyen M, Saavedra MO, Yerramilli P, Pruitt L, Subedi S, Kuo HC, Hendrickson H, Eskandari G, Nguyen HAT, Long JH, Kumaraswami M, Goike J, Boutz D, Gollihar J, McLellan JS, Chou CW, Javanmardi K, Finkelstein IJ, Musser JM. 2020. Molecular architecture of early dissemination and massive second wave of the SARS-CoV-2 Virus in a major metropolitan area. mBio 11:e02707-20. https://doi.org/10.1128/mBio.02707-20. 4. Musser JM, Olsen RJ, Christensen PA, Long SW, Subedi S, Davis JJ, Gollihar J. 2021. Rapid, widespread, and preferential increase of SARS-CoV-2 B.1.1.7 variant in Houston, TX, revealed by 8,857 genome sequences. medRxiv. https://doi.org/10.1101/2021.03.16.21253753. Volume 9 Issue 2 e00430-21 MicrobiolSpectrum.asm.org 3
https://openalex.org/W2883071827	http://www.jaspe.ac.me/clanci/JASPE_July_2018_Bjelica_15-20.pdf	Bosnian	null	Attitudes of Consumers from University of Novi Sad toward Advertising through Sport among the Question how Often they Participate in Sports Activities	Journal of Anthropology of Sport and Physical Education	2,018	cc-by	5,773	DOI 10.26773/jaspe.180703 ORIGINAL SCIENTIFIC PAPER Abstract This research was aimed at gaining relevant knowledge about the attitudes of University of Novi Sad consumers toward advertising through sport among the question how often they participate in sports activities. The sample included 168 students from Faculti of Sporth and Physical Education in Novi Sad, divided into six subsample groups: consumers who do not participate in sport activities at all, then consumers who participate in sport activities less than ones a month, next 1–4 a month, 5–10 a month, 11–20 a month, as well as consumers participate in sport activities more than 20 times a month. The sample of variables contained the system of three general attitudes which were modelled by seven-point Likert scale. The results of the measuring were analyzed by multivariate analysis (MANOVA) and univariate analysis (ANOVA) and Post Hoc test. Based on the statistical analyses it was found that signifi cant diff erences occur at multivariate level, as well as between all three variables at a signifi cance level of p=.000. Hence, it is interesting to highlight that it was found there are signifi cant diff erences showed up between the consumers who participate in various sports activities. The signifi cant diff erences were found in one of three variables, while the consumers who participate less than 4 times a moths had much more negative attitudes toward advertising though sport. Key words: Attitudes, Advertising, Sports Events, Novi Sad Key words: Attitudes, Advertising, Sports Events, Novi Sad Attitudes of Consumers from University of Novi Sad toward Advertising through Sport among the Question how Often they Participate in Sports Activities Dusko Bjelica1, Marko Gusic2, Nebojsa Maksimovic2 1University of Montenegro, Faculty for Sport and Physical Education, Niksic, Montenegro, 2University of Novi Sad, Faculti of Sporth and Physical Education, Novi Sad, Serbia ATTITUDES TOWARD ADVERTISING THROUGH SPORT AND PARTICIPATING IN SPORTS ACTIVITIES RATION \| D. BJELICA ET AL. Samim tim se postavilo i pitanje, kako bavljenje sportskim ak- tivnostima utiče na stavove potrošača prema reklamiranju u sportu kao jedan od niza problema sa kojim se nije susrijeta- lo mnogo autora do sada (Bjelica, Popović, & Akpinar, 2014a; Bjelica, Popović, & Akpinar, 2015c; Bjelica, Popović, & Ak- pinar, 2016b; Bjelica, Popović, & Akpinar, 2017; Gardaševic, Zorić, & Mašanović, 2017; Zorić, Mašanović, & Gardašević 2018; Bajramović, Zorić, & Mašanović, 2018; Gardašević, Baj- ramović, & Mašanović, 2018; Zorić, Gardašević, & Bajramo- vić, 2018), a to upravo i jeste cilj ove studije. stavove ispitanika u istom periodu (Popović, 2011). Ove ne- dosljednosti su navele na sumnju da reklamne kampanje koje koriste elemente iz specifi čnih medijuma utiču na stavove po- trošača prema reklamiranju u skladu sa tim (Popović, 2011). Neki autori su vjerovali da je jedan od najrelevantnijih medija, upravo sport, odnosno sportisti i njihovi timovi, sportski do- gađaji i sportski objekti (Bjelica i Popović, 2011; Mašanović, Zorić, & Gardašević, 2017; Milovic, Corluka, & Masanovic, 2018; Djurisic, Perovic, & Masanovic, 2018; Kovacevic, Milo- sevic, & Masanovic, 2018; Molnar, Masanovic, & Bjelica, 2018; Masanovic, Georgiev, & Sekulic, 2018; Stupar, Gardasevic, & Masanovic, 2018), uglavnom iz razloga što ovi elementi mogu da utiču na stavove potrošača i naprave značajne oscilacije kod stavova u poređenju sa ostalim elementima (Bjelica, Popović, Jakšić, Hadžić, & Akpinar, 2014b; Bjelica i Popović, 2015a; Bje- lica & Popović, 2015b; Bjelica, Gardašević, Vasiljević, & Popo- vić, 2016a; Bjelica, Gardašević, Vasiljević, & Popović, 2016c; Zorić, Mašanović, & Gardašević, 2017; Mašanović, Zorić, & Gardašević, 2018; Gardašević, Zorić, & Mašanović, 2018). Iz tog razloga, mnogi istraživači priznaju sport kao nezavisnu re- klamnu platformu koju poslovni subjekti mogu koristiti kako bi prevazišli negativne stavove potrošača prema reklamiranju. Samim tim se postavilo i pitanje, kako bavljenje sportskim ak- tivnostima utiče na stavove potrošača prema reklamiranju u sportu kao jedan od niza problema sa kojim se nije susrijeta- lo mnogo autora do sada (Bjelica, Popović, & Akpinar, 2014a; Bjelica, Popović, & Akpinar, 2015c; Bjelica, Popović, & Ak- pinar, 2016b; Bjelica, Popović, & Akpinar, 2017; Gardaševic, Zorić, & Mašanović, 2017; Zorić, Mašanović, & Gardašević 2018; Bajramović, Zorić, & Mašanović, 2018; Gardašević, Baj- ramović, & Mašanović, 2018; Zorić, Gardašević, & Bajramo- vić, 2018), a to upravo i jeste cilj ove studije. ATTITUDES TOWARD ADVERTISING THROUGH SPORT AND PARTICIPATING IN SPORTS ACTIVITIES RATION \| D. BJELICA ET AL. ATTITUDES TOWARD ADVERTISING THROUGH SPORT AND PARTICIPATING IN SPORTS ACTIVITIES RATION \| D. BJELICA ET AL. dva dijela, generalnih stavova prema reklamiranju u sportu i socio-demografskih karakteristika ispitanika kada je uče stalost bavljenja sportskim aktivnostima tokom mjeseca u pitanju. Si- stem promjenljivih u ovom upitniku je sadržao tri tvrdnje koje su ispitanici trebali da ocijene prema sedmo-stepenoj Likerto- voj skali vrijednosti, kao i šest socio-demografskih karakteri- stika ispitanika (uopšte se ne bave, manje od jednom mjesečno, 1–4 puta mjesečno, 5–10 puta mjesečno, 11–20 puta mjesečno, i više od 20 puta mjesečno). Popunjavanje upitnika nije trajalo u prosjeku više od 10 minuta, a ispitanici su u anketi učestvo- vali na dobrovoljnoj osnovi. Važno je naglasiti da je anketa bila anonimna i da su svi odgovori bili strogo poverljivi. Vrijedno je napomenuti i da su ispitanici, i pored svega navedenog, imali mogućnost da u svakom momentu opozovu svoje učešće u an- keti, ali se niko od njih nije na tako nešto odlučio. stavove ispitanika u istom periodu (Popović, 2011). Ove ne- dosljednosti su navele na sumnju da reklamne kampanje koje koriste elemente iz specifi čnih medijuma utiču na stavove po- trošača prema reklamiranju u skladu sa tim (Popović, 2011). Neki autori su vjerovali da je jedan od najrelevantnijih medija, upravo sport, odnosno sportisti i njihovi timovi, sportski do- gađaji i sportski objekti (Bjelica i Popović, 2011; Mašanović, Zorić, & Gardašević, 2017; Milovic, Corluka, & Masanovic, 2018; Djurisic, Perovic, & Masanovic, 2018; Kovacevic, Milo- sevic, & Masanovic, 2018; Molnar, Masanovic, & Bjelica, 2018; Masanovic, Georgiev, & Sekulic, 2018; Stupar, Gardasevic, & Masanovic, 2018), uglavnom iz razloga što ovi elementi mogu da utiču na stavove potrošača i naprave značajne oscilacije kod stavova u poređenju sa ostalim elementima (Bjelica, Popović, Jakšić, Hadžić, & Akpinar, 2014b; Bjelica i Popović, 2015a; Bje- lica & Popović, 2015b; Bjelica, Gardašević, Vasiljević, & Popo- vić, 2016a; Bjelica, Gardašević, Vasiljević, & Popović, 2016c; Zorić, Mašanović, & Gardašević, 2017; Mašanović, Zorić, & Gardašević, 2018; Gardašević, Zorić, & Mašanović, 2018). Iz tog razloga, mnogi istraživači priznaju sport kao nezavisnu re- klamnu platformu koju poslovni subjekti mogu koristiti kako bi prevazišli negativne stavove potrošača prema reklamiranju. Me tod Populaciju u ovoj studiji su činili studenti Fakulteta sporta i fi zičkog vaspitanja u Novom Sadu koji su u vrijeme anketira- nja, imali boravište na teritoriji Srbije, dok je uzorak ispitanika organizovan putem kombinovanja ili raslojavanja, tako da su obrađena različita svojstva navedene populacije i različiti pro- stori na kojima je ona egzistirala. Upitnici su distribuirani studentima osnovnih studija u štampanom i elektronskom obliku. Ukupno je prikupljeno 174 upitnika, ali je 6 upitnika bilo isključeno iz analize, budući da nisu bili adekvatno popunjeni, tako da je u istraživanju uče- stvovalo, ukupno 168 ispitanika (Fakulteta sporta i fi zičkog vaspitanja u Novom Sadu). Instrument istraživanja je predsta- vljao standardizovani upitnik (Popović, 2011) i sastojao se iz Tabela 1. Generalni stavovi prema reklamiranju u sportu Tabela 1. Generalni stavovi prema reklamiranju u sportu Mean S.D. Skewness Kurtosis Statistic S.E. Statistic S.E. GSS1 5.09 1.643 -.656 .199 -.069 .396 GSS2 5.36 1.587 -.867 .199 .137 .396 GSS3 4.78 1.483 -.243 .199 -.103 .396 Legenda: Mean – Aritmetička sredina; S.D. – Standardna devijacija; Skewness – Mjera asimetrije; Kurtosis – Mjera spljoštenosti; Statistic – Statistička vrijednost; S.E. – Standardna greška; GSS1 – Moje opšte mišljenje je naklonjeno reklamiranju u sportu; GSS2 – Sveobuhvatno, smatram da je reklamiranje u sportu dobra stvar; GSS3 – Sveobuhvatno, da li ne volite ili volite reklamiranje u sportu Legenda: Mean – Aritmetička sredina; S.D. – Standardna devijacija; Skewness – Mjera asimetrije; Kurtosis – Mjera spljoštenosti; Statistic – Statistička vrijednost; S.E. – Standardna greška; GSS1 – Moje opšte mišljenje je naklonjeno reklamiranju u sportu; GSS2 – Sveobuhvatno, smatram da je reklamiranje u sportu dobra stvar; GSS3 – Sveobuhvatno, da li ne volite ili volite reklamiranje u sportu Legenda: Mean – Aritmetička sredina; S.D. – Standardna devijacija; Skewness – Mjera asimetrije; Kurtosis – Mjera spljoštenosti; Statistic – Statistička vrijednost; S.E. ATTITUDES TOWARD ADVERTISING THROUGH SPORT AND PARTICIPATING IN SPORTS ACTIVITIES RATION \| D. BJELICA ET AL. Empirijski podaci su analizirani putem statističkog paketa za društvene nauke (SPSS 20.0), a kao prvi korak, bila je primi- jenjena deskriptivna statistika kojom su izračunati, prije svega frekvencija, zatim aritmetička sredina, standardna devijacija, kao i mjere asimetrije (Skewness) i spljoštenosti (Kurtosis) za svaku od tvrdnji. Budući da su se promjenljive u ovoj studiji nalazile na neparametrijskim skalama, radi detaljnijih anali- za koje su slijedile, bilo je neophodno da se one, primjenom Blomovog postupka transformišu u skale višeg reda. Zatim su primjenom multivarijatne analize varijanse (MANOVA), univarijatne analize varijanse (ANOVA) i LSD Post Hoc te- sta, utvrđene razlike u generalnim stavovima ispitanika prema reklamiranju u sportu u odnosu na učestalost bavljenja sport- skim aktivnostima tokom mjeseca. Rezultati U prvoj tabeli su prikazani deskriptivni statistički podaci za sve tri tvrdnje koje su se odnosile na generalne stavove ispi- tanika prema reklamiranju u sportu. Prije svega, prikazana je aritmeticka sredina koja oslikava pozitivne vrijednosti stavova kada su sve tri tvrdnje u pitanju, dok vrijednosti standardne devijacije pokazuju da elementi skupa, u prosjeku ne odstu- paju znacajno od aritmeticke sredine. Kada se govori o mjera- ma asimetrije (Skewness) i spljoštenosti (Kurtosis), negativne vrijednosti asimetrije kod svih promjenljivih pokazuju da je većina rezultata desno od srednje vrijednosti, među većim vri- jednostima, dok pozitivne vrijednosti spljoštenosti kod jedne promjenljive (GSS2) pokazuju da je raspodjela šiljatija od nor- male tj. da ima više rezultata nagomilanih oko centra a nega- tivne vrijednosti spljoštenosti kod dvije promjenljive (GSS1 i GSS3) pokazuju da je raspodjela pljosnatija od normalne tj. da ima više rezultata nagomilanih na repovima raspodjele. J. Anthr. Sport Phys. Educ. 2 (2018) 3 Uvod nje brojnih studija koje su istraživali stavove potrošača prema reklamiranju, ali već ina njih smatra stavove potrošača prema reklamiranju samo uopšteno (Bjelica i Popović, 2011; Popo- vić, 2011b; Popović, Molnar, i Radovanović, 2011a; Popović, Matić, Milašinović, Jakšić, & Bjelica, 2015a; Popović, Matić, Milašinović, Hadžić, Milošević, i Bjelica, 2015b; Popović, Ma- tić, Milašinović, Vujović, Milošević, i Bjelica, 2015c). U sva- kom slučaju, neki od njih su zaključili da su stavovi prema re- klamiranju pokazali negativan trend tokom 1960-ih i 1970-ih (Popović, Bjelica, Jakšić, & Georgiev, 2013; Popovic, 2015; Po- pović, & Milašinović, 2016), dok su drugi pronašli povoljnije Zaključci pionirskih studija iz navedene oblasti koji su ob- javljeni od strane Bauer-a i Greiser-a (Popovic, 2011) i Lutz-a (Klačar i Popović, 2010) pokrenuli su ideje koje se odnose na činjenicu da stavovi potrošača prema reklamiranju predsta- vljaju jedan od značajnih faktora koji mogu ojačati efi kasnost reklamnih kampanja, uglavnom zbog razloga da kognitivna sposobnost potrošača prema reklamiranju jeste sadržana u njihovim osjeć anjima i mislima (Muratović, Bjelica, & Po- pović , 2014). Ako se nastavi sa istraživanjem ovog pitanja i ode se mnogo dublje, zanimljivo je da se primjećuje postoja- M. Gusic University of Novi Sad, Faculty of Sporth and Physical Education, Lovcenska 16, Novi Sad, Serbia E-mail: gusicmarko@yahoo.com M. Gusic University of Novi Sad, Faculty of Sporth and Physical Education, Lovcenska 16, Novi Sad, Serbia E-mail: gusicmarko@yahoo.com J. Anthr. Sport Phys. Educ. 2 (2018) 3: 15–20 15 ATTITUDES TOWARD ADVERTISING THROUGH SPORT AND PARTICIPATING IN SPORTS ACTIVITIES RATION \| D. BJELICA ET AL. TTITUDES TOWARD ADVERTISING THROUGH SPORT AND PARTICIPATING IN SPORTS ACTIVITIES RATION \| D. BJELICA ET AL. lost bavljenja sportskim aktivnostima. lost bavljenja sportskim aktivnostima. upoređivanih parametara postoji statistički značajna razlika u generalnim stavovima prema reklamiranju u sportu u odnosu na učestalost bavljenja sportskim aktivnostima (p=.028). Inspekcijom druge tabele u kojoj su prikazani rezultati multivarijatne analize, jasno se uočava da u cijelom sistemu Tabela 2. Multivarijatna značajnost razlika u sistemu generalnih stavova prema reklamiranju u sportu kod ispitanika sa različitim navikama kada je bavljenje sportskim aktivnostima u pitanju p j N Mean S.D. GSS1 Ne bave se 4 3.75 3.202 < 1 3 4.67 2.517 1 – 4 5 4.40 2.191 5 – 10 12 4.25 1.960 11 – 20 37 5.46 1.169 > 20 87 5.17 1.579 Ukupno 148 5.09 1.634 GSS2 Ne bave se 4 4.25 2.754 < 1 3 2.67 1.528 1 – 4 5 5.00 1.225 5 – 10 12 4.67 1.826 11 – 20 37 5.59 1.404 > 20 87 5.53 1.493 Ukupno 148 5.36 1.587 GSS3 Ne bave se 4 4.00 2.160 < 1 3 3.33 1.528 1 – 4 5 3.80 .837 5 – 10 12 4.25 1.712 11 – 20 37 5.16 1.236 > 20 87 4.84 1.493 Ukupno 148 4.78 1.483 F=1.839; p=.028 Inspekcijom treće tabele u kojoj su prikazani rezultati uni- varijatne analize, jasno se uočava da je došlo do statistički zna- čajnih razlika u generalnim stavovima prema reklamiranju u sportu u odnosu na učestalost bavljenja sportskim aktivnosti- ma kod jedne promjenljive (GSS2), dok kod druge dvije vari- jable (GSS1 i GSS3) navedena diskriminacija nije utvrdjena. sportu u odnosu na učestalost bavljenja sportskim aktivnosti- ma kod jedne promjenljive (GSS2), dok kod druge dvije vari- jable (GSS1 i GSS3) navedena diskriminacija nije utvrdjena. Inspekcijom treće tabele u kojoj su prikazani rezultati uni- varijatne analize, jasno se uočava da je došlo do statistički zna- čajnih razlika u generalnim stavovima prema reklamiranju u Tabela 3. Univarijatna značajnost razlika u sistemu generalnih stavova prema reklamiranju u sportu kod ispitanika sa različitim navikama kada je Tabela 3. Univarijatna značajnost razlika u sistemu generalnih stavova prema reklamiranju u sportu kod ispitanika sa različitim navikama kada je bavljenje sportskim aktivnostima u pitanju F p GSS1 1.866 .104 GSS2 3.211 .009 GSS3 2.135 .065 budući na rezultate koji su se pojavili na univarijatnom nivou, uočava se da postoje statisticki značajne razlike pojedinih pa- rametara za dvije promjenljive. J. Anthr. Sport Phys. Educ. 2 (2018) 3 Me tod – Standardna greška; GSS1 – Moje opšte mišljenje je naklonjeno reklamiranju u sportu; GSS2 – Sveobuhvatno, smatram da je reklamiranje u sportu dobra stvar; GSS3 – Sveobuhvatno, da li ne volite ili volite reklamiranje u sportu NOVA), univarijatne analize varijanse (ANOVA) i LSD Post Hoc testa, a u cilju utvrđivanja razlike u generalnim stavovima ispitanika prema reklamiranju u sportu u odnosu na učesta- NOVA), univarijatne analize varijanse (ANOVA) i LSD Post Hoc testa, a u cilju utvrđivanja razlike u generalnim stavovima ispitanika prema reklamiranju u sportu u odnosu na učesta- U nastavku ove studije su prikazani komparativni statistič- ki podaci generalnih stavova prema reklamiranju u sportu koji su dobijeni primjenom multivarijatne analize varijanse (MA- J. Anthr. Sport Phys. Educ. 2 (2018) 3 16 ATTITUDES TOWARD ADVERTISING THROUGH SPORT AND PARTICIPATING IN SPORTS ACTIVITIES RATION \| D. BJELICA ET AL. Tabela 6. Utvrđivanje značajnih razlika u sistemu generalnih stavova prema reklamiranju u sportu primjenom Post Hoc testa između pojedinačnih entiteta sa različitim navikama kada je bavljenje sportskim aktivnostima u pitanju kod tvrdnje Tabela 6. Utvrđivanje značajnih razlika u sistemu generalnih stavova prema reklamiranju u sportu primjenom Post Hoc testa između pojedinačnih entiteta sa različitim navikama kada je bavljenje sportskim aktivnostima u pitanju kod tvrdnje „sveobuhvatno, da li ne volite ili volite reklamiranje u sportu“ vs Ne bave se < 1 1 – 4 5 – 10 11 – 20 < 1 .549 1 – 4 .838 .661 5 – 10 .766 .331 .562 11 – 20 .131 .038 .051 .061 > 20 .261 .080 .123 .191 .260 Došlo se do zaključka da se kod treće tvrdnje „sveobuhvat- no, da li ne volite ili volite reklamiranje u sportu“ pojavljuju razlike između ispitanika koji se bave sportskim aktivnostima manje od jedmom mjesečno i onih ispitanika koji se sportom bave 11-20 puta mjesečno. Možemo uočiti da pripadnici dvije grupe koje se najviše bave sportom imaju najviše pozitivnih rezultata, dok najmanje pozitivnih rezultata uočavamo kod grupe koja se bavi sportskim aktivnostima manje od jednom mjesečno. na, imaju pozitivne stavove prema reklamiranju u sportu, dok je ipak, poređenja radi, vrijedno navesti da prema Mittal-u (Popovic, 2011), različite studije ukazuju na negativne sta- vove kada je reklamiranje proizvoda u tradicionalnim indu- strijama u pitanju. Prema tome, više je nego evidentno da je primjena sporta u savremenoj poslovnoj komunikaciji uticala da se generalni stavovi potrošača značajno promijene kada je reklamiranje u pitanju, a prepoznavanje privlačnosti sporta je omogućilo poslovnim organizacijama da se približe sportskim potrošačima i na mnogo bezbolniji način utiču na njihovo po- našanje. J. Anthr. Sport Phys. Educ. 2 (2018) 3 ATTITUDES TOWARD ADVERTISING THROUGH SPORT AND PARTICIPATING IN SPORTS ACTIVITIES RATION \| D. BJELICA ET AL. ATTITUDES TOWARD ADVERTISING THROUGH SPORT AND PARTICIPATING IN SPORTS ACTIVITIES RATION \| D. BJELICA ET AL. Došlo se do zaključka da se kod prve tvrdnje „moje opšte mišljenje je naklonjeno reklamiranju u sportu“ javlj aju razlike između ispitanika koji se bave sportskim aktivnostima 11-20 puta mjesečno i onih grupa koje se ne bave sportskim aktiv- nostima, odnosno koji se bave sportskim aktivnostima 5-10 puta mjesečno. Može mo uočiti da pripadnici dvije grupe koje se najviše bave sportom imaju najviše pozitivnih rezultata, dok najmanje pozitivnih rezultata uočavamo kod grupe koja se ne bavi sportskim aktivnostima. Tabela 5. Utvrđivanje značajnih razlika u sistemu generalnih stavova prema reklamiranju u sportu primjenom Post Hoc testa između pojedinačnih entiteta sa različitim navikama kada je bavljenje sportskim aktivnostima u pitanju kod tvrdnje „sveobuhvatno, smatram da je reklamiranje u sportu dobra stvar“ Tabela 5. Utvrđivanje značajnih razlika u sistemu generalnih stavova prema reklamiranju u sportu primjenom Post Hoc testa između pojedinačnih entiteta sa različitim navikama kada je bavljenje sportskim aktivnostima u pitanju kod tvrdnje „sveobuhvatno, smatram da je reklamiranje u sportu dobra stvar“ Tabela 5. Utvrđivanje značajnih razlika u sistemu generalnih stavova prema reklamiranju u sportu primjenom Post Hoc testa između pojedinačnih entiteta sa različitim navikama kada je bavljenje sportskim aktivnostima u pitanju kod tvrdnje „sveobuhvatno, smatram da je reklamiranje u sportu dobra stvar“ j j p vs Ne bave se < 1 1 – 4 5 – 10 11 – 20 < 1 .178 1 – 4 .466 .039 5 – 10 .638 .045 .683 11 – 20 .097 .002 .416 .070 > 20 .105 .022 .454 .070 .827 Može mo uočiti da pripadnici dvije grupe koje se najviše bave sportom imaju najviše pozitivnih rezultata, dok najmanje po- zitivnih rezultata uočavamo kod grupe koja se bavi sportskim aktivnostima manje od jednom mjesečno. Može mo uočiti da pripadnici dvije grupe koje se najviše bave sportom imaju najviše pozitivnih rezultata, dok najmanje po- zitivnih rezultata uočavamo kod grupe koja se bavi sportskim aktivnostima manje od jednom mjesečno. Može mo uočiti da pripadnici dvije grupe koje se najviše bave sportom imaju najviše pozitivnih rezultata, dok najmanje po- zitivnih rezultata uočavamo kod grupe koja se bavi sportskim aktivnostima manje od jednom mjesečno. Došlo se do zaključka da se kod druge tvrdnje „sveobu- hvatno, smatram da je reklamiranje u sportu dobra stvar“ javljaju razlike između ispitanika koji se bave sportskim ak- tivnostima manje od jednom mjesečno i svih ostalih entiteta. ATTITUDES TOWARD ADVERTISING THROUGH SPORT AND PARTICIPATING IN SPORTS ACTIVITIES RATION \| D. BJELICA ET AL. budući na rezultate koji su se pojavili na univarijatnom nivou, uočava se da postoje statisticki značajne razlike pojedinih pa- rametara za dvije promjenljive. Inspekcijom naredne tri tabele gdje su prikazani rezultati Post Hoc testa koji ukazuju na značajnost razlika između paro- va pojedinacnih entiteta sa razlicitim navikama kada je bavlje- nje sportskim aktivnostima u pitanju za svaku promjenljivu, Tabela 4. Utvrđivanje značajnih razlika u sistemu generalnih stavova prema reklamiranju u sportu primjenom Post Hoc testa između pojedinačnih entiteta sa različitim navikama kada je bavljenje sportskim aktivnostima u pitanju kod tvrdnje „moje opšte mišljenje je naklonjeno reklamiranju u sportu“ Tabela 4. Utvrđivanje značajnih razlika u sistemu generalnih stavova prema reklamiranju u sportu primjenom Post Hoc testa između pojedinačnih entiteta sa različitim navikama kada je bavljenje sportskim aktivnostima u pitanju kod tvrdnje „moje opšte mišljenje je naklonjeno reklamiranju u sportu“ Tabela 4. Utvrđivanje značajnih razlika u sistemu generalnih stavova prema reklamiranju u sportu primjenom Post Hoc testa između pojedinačnih entiteta sa različitim navikama kada je bavljenje sportskim aktivnostima u pitanju kod tvrdnje „moje opšte mišljenje je naklonjeno reklamiranju u sportu“ j p j j j j j p vs Ne bave se < 1 1 – 4 5 – 10 11 – 20 < 1 .457 1 – 4 .548 .821 5 – 10 .592 .689 .861 11 – 20 .046 .414 .170 .025 > 20 .086 .594 .299 .065 .365 J. Anthr. Sport Phys. Educ. 2 (2018) 3 17 17 ATTITUDES TOWARD ADVERTISING THROUGH SPORT AND PARTICIPATING IN SPORTS ACTIVITIES RATION \| D. BJELICA ET AL. ATTITUDES TOWARD ADVERTISING THROUGH SPORT AND PARTICIPATING IN SPORTS ACTIVITIES RATION \| D. BJELICA ET AL. Mostar Canton in Bosnia and Herzegovina toward Advertising through Sport among the Question how Often they Participate in Sports Activi- ties. Journal of Anthropology of Sport and Physical Education, 2(2), 9-13. Mostar Canton in Bosnia and Herzegovina toward Advertising through Sport among the Question how Often they Participate in Sports Activi- ties. Journal of Anthropology of Sport and Physical Education, 2(2), 9-13. lošću u bavljenju sportskim aktivnostima, slažu se sa prethod- nim studijama (Bjelica i sar., 2014b; Bjelica i Popović, 2015a; Bjelica & Popović, 2015b; Bjelica i sar., 2016a; Bjelica i sar., 2016c; Gardasevic, Zoric, & Masanovic, 2017; Bjelica, Garda- šević, & Ćorluka, 2018; Ćorluka, Bjelica, & Vukotić, 2018; Vu- kotić, Ćorluka, & Mašanović, 2018). Ovi rezultati su veoma važni za poslovne subjekte u Srbiji i za naučnu javnost, uglav- nom iz razloga što ne mogu spojiti sve potencijalne potrošače koji učestvuju u raznim sportskim aktivnostima u jednu ho- mogenu grupu, o čemu govore i prethodna istraživanja (Bje- lica i sar., 2016a; Bjelica i sar., 2016c). Takođe, interesantno je istaći da se velika većina ispitanika identifi kovala sa dva ponu- đena entiteta, njih 124 (od 148), prije svega sa potrošačima ko- ji sportske proizvode kupuju 11-20 puta mjesečno i sa potroša- čima koji sportske proizvode kupuju preko 20 puta mjesečno. Djurisic, V., Perovic, D., & Masanovic, B. (2018). Attitudes of consumers from Podgorica toward advertising through sport among the question how often consumers purchase sporting goods. Journal of Anthropology of Sport and Physical Education, 2(2), 55-60. doi: 10.26773/jaspe.180410 y port and Physical Education, 2(2), 55-60. doi: 10.26773/jaspe.180410 Gardasevic, J., Bajramovic, I., & Masanovic, B. (2018). Attitudes of Consumers from the Sarajevo Canton in Bosnia and Herzegovina toward Adverti- sing through Sport among the Question how Often they Participate in Sports Activities. Journal of Anthropology of Sport and Physical Educa- tion, 2(2), 37-41. Gardašević, J., Zorić, G., & Mašanović, B. (2017). Attitudes of Turkish consu- mers toward advertising through sport among the question how often they participate in sports activities. Journal of Anthropology of Sport and Physical Education, 1(1), 23-7. Gardašević, J., Zorić, G., & Mašanović, B. (2018). Attitudes of Montenegrin consumers toward advertising through sport among the question how often they participate in sports activities. Journal of Anthropology of Sport and Physical Education, 2(1), 15-9. References Bajramović, I., Zorić, G., & Mašanović, B. (2018). Attitudes of Consumers from the Sarajevo Canton in Bosnia and Herzegovina toward Advertising through Sport among the Frequency of Watching Sports Events. Jour- nal of Anthropology of Sport and Physical Education, 2(2), 43-7.i Milovic, N., Corluka, M., & Masanovic, B. (2018). Attitudes of consumers from Podgorica toward advertising through sport among the frequency of watching sports events. Journal of Anthropology of Sport and Physical Education, 2(2), 71-6. doi: 10.26773/jaspe.180413 Bjelica, D., & Popović, S. (2015b). Evolution of Advertising with a Specifi c Re- trospection at Sport Advertising. In Book of Abstracts of the 12th Inter- national Scientifi c Conference on Transformation Process in Sport “Sport Performance” (60-61), Podgorica: Montenegrin Sports Academy. Molnar, S., Lilić, Lj., Popović, S., Akpinar, S., & Jakšić, D. (2011). Attitudes of various demographic groups toward advertising through sport at ME- TU. Facta Universitatis, Series: Physical Education and Sport, 9(3), 255-63. Performance” (60-61), Podgorica: Montenegrin Sports Academy Bjelica, D., Gardašević, J., & Ćorluka, M. (2018). Attitudes of Consumers from the Mostrar Canton in Bosnia and Herzegovina toward Advertising through Sport among the Question how often Consumers purchase Sporting Goods. Journal of Anthropology of Sport and Physical Educa- tion, 2(2), 3-7. Molnar, S., Masanovic, B., & Bjelica, D. (2018). Attitudes of Consumers from University of Novi Sad toward Advertising through Sport among the Frequency of Watching Sports Events. Journal of Anthropology of Sport and Physical Education, 2(3). Muratović, A., Bjelica, D., & Popović, S. (2014). Examining beliefs and attitu- des toward advertising through sport among montenegrin consumers. Facta Universitatis, Series: Physical Education and Sport, 12(2), 95-104. Bjelica, D., Gardašević, J., Vasiljević, I., & Popović, S. (2016a). Ethical Dilemmas of Sport Advertising. Sport Mont, 14(3), 41–3. Bjelica, D., Gardašević, J., Vasiljević, I., & Popović, S. (2016c). Ethical dilemmas of sport advertising. In Book of Abstracts of the 13th International Scien- tifi c Conference on Transformation Process in Sport “Sport Performance” (41), Podgorica: Montenegrin Sports Academy. Popovic, S. (2011). Reklamiranje u sportu kao efektivno sredstvo savremene poslovne komunikacije. Neobjavljena doktoska disertacija, Novi Sad: Univerzitet u Novom Sadu. Popović, S. & Milašinović, R. (2016). Model of Advertising Communication in Sport. Sport Mont, 14(1), 33–8. Bjelica, D., i Popović, S. (2011). Stavovi potrošača prema reklamiranju u spor- tu u odnosu na učestalost posmatranja sportskih događaja. Sportske nauke i zdravlje, 1(2), 114-9. Popović, S. (2011b). ATTITUDES TOWARD ADVERTISING THROUGH SPORT AND PARTICIPATING IN SPORTS ACTIVITIES RATION \| D. BJELICA ET AL. Buduća istraživanja bi trebalo usmjeriti na veći broj ispita- nika, budući da se značajan broj ispitanika grupiše u kategoriji sa manjim brojem kupovine sportskih proizvoda tokom mje- seca, posebno iz razloga što se očekuje da bi neke razlike mo- gle biti interesantne i korisne kako za teoriju tako i za praksu. Klačar, M. & Popović, S. (2010). Reklamiranje u sportu kao efektivno sredstvo savremene komunikacije. Teme, 4, 1219-30. Kovacevic, D., Milosevic, Z., & Masanovic, B. (2018). 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Received: 12 April 2018 \| Accepted: 02 June 2018 \| Published: 13 July 2018 Received: 12 April 2018 \| Accepted: 02 June 2018 \| Published: 13 July 2018 Mašanović, B., Zorić, G., & Gardašević, J. (2018). Attitudes of Montenegrin consumers toward advertising through sport among the frequency of watching sports events. Journal of Anthropology of Sport and Physical Education, 2(1), 9-13. Diskusija Utvrđivanjem razlike u generalnim stavovima ispitani- ka prema reklamiranju u sportu u odnosu na učestvovanje u sportskim aktivnostima, u ovoj studiji su pro nađene razlike u stavovima između ispitanika koji imaju različite navike kada je u pitanju učestvovanje u sportskim aktivnostima. Ove razlike su se pojavile kod jedne od tri promjenljive na univarijatnom nivou. Kod sve tri promjenjive „moje opšte mišljenje je naklo- njeno reklamiranju u sportu“, „sveobuhvatno, smatram da je reklamiranje u sportu dobra stvar“ i „sveobuhvatno, da li ne volite ili volite reklamiranje u sportu“ uočavamo najviše pozi- tivnih rezultata kod dvije grupe koja se najviše bave sportskim aktivnostima, dok se manje pozitivnih rezultata uočava kod grupa koje se najmanje bave sportskim aktivnostima. S obzirom da su rezultati pokazali da ispitanici imaju veo- ma pozitivan stav prema reklamiranju u sportu što potvrđuje visoka vrijednost aritmetičke sredine za sve tri promjenljive, kao i da skoro dvije trećine ispitanika imaju pozitivan stav prema reklamiranju u sportu što se ogleda u izrazito negativ- nim vrijednostima mjera asimetrije, trebalo bi naglasiti da su ovi rezultate usaglašeni sa rezultatima prethodnih istraživanja (Molnar, Lilić, Popović, Akpinar, & Jakšić, 2011; Popović, Jak- šić, Matić, Bjelica, & Maksimović, 2014; Popović, Bjelica, Ge- orgiev, & Akpinar, 2011b; Popović, Matić, Milašinović, Jakšić, & Bjelica, 2015d; Popović, Matić, Milašinović, Hadžić, Milo- šević, & Bjelica, 2015e; Popović, Matić, Milašinović, Vujović, Milošević, & Bjelica, 2015f; Popović, Jakšić, Matić, Bjelica, & Maksimović, 2015g), i da ne postoje značajne razlike koje bi trebalo pomenuti. Dobijeni rezultati, takođe jasno ukazuju da ispitanici koji žive na različitim lokacijama, kao što su Sjedi- njene države, Turska, Crna Gora, Srbija, Bosna i Hercegovi- Na osnovu statističkih analiza, utvrđeno je da se značajne razlike javljaju na multivarijatnom nivou, kao i između nekih varijabli na univarijatnom nivou značajnosti od p=.01. Prona- đene značajne razlike između ispitanika sa različitom učesta- J. Anthr. Sport Phys. Educ. 2 (2018) 3 18 References Stavovi potrošača prema reklamiranju u sportu u odno- su na učestalost kupovine sportskih proizvoda. Sport Mont, 9(28,29,30), 140-7. j Bjelica, D., i Popović, S. (2015a). Evolucija reklamiranja sa posebnim osvrtom na reklamiranje u sportu. Sport Mont, 13(43,44,45), 35-41. Popović, S. (2015). Sport Nowadays (In Montenegrin). In D. Bjelica (Ed.), Advertising Role of Sponsorship in Modern Sport (pp. 111-6). Podgorica: Montenegrin Sports Academy. Bjelica, D., Popović, S., & Akpinar, S. (2014a). Book of Abstracts of the 11th International Scientifi c Conference on Transformation Process in Sport “Sport Performance”. Podgorica: Montenegrin Sports Academy. Popović, S., Bjelica, D., Georgiev, G., & Akpinar, S. (2011b). Comparison of at- titudes toward advertising through sport between Montenegrin and Turkish stakeholders. In Proceedings book of the 6th International Scien- tifi c Conference on Kinesiology “Integrative Power of Kinesiology” (612). Opatija: University of Zegreb, Faculty of Kinesiology. Bjelica, D., Popović, S., & Akpinar, S. (2015c). Book of Abstracts of the 12th International Scientifi c Conference on Transformation Process in Sport “Sport Performance”. Podgorica: Montenegrin Sports Academy. Bjelica, D., Popović, S., & Akpinar, S. (2016b). Book of Abstracts of the 13th International Scientifi c Conference on Transformation Process in Sport “Sport Performance”. Podgorica: Montenegrin Sports Academy. Opatija: University of Zegreb, Faculty of Kinesiology. Popović, S., Bjelica, D., Jakšić, D, & Georgiev, G. (2013). How does Advertising Through Sport Work? Evidence from Montenegro. In Book of Abstracts of the 18thAnnual Congress of the European College of Sport Science (444- 5), Barcelona: National Institute of Physical Education of Catalonia. Bjelica, D., Popović, S., & Akpinar, S. (2017). Book of Abstracts of the 14th Inter- national Scientifi c Conference on Transformation Process in Sport “Sport Performance”. Podgorica: Montenegrin Sports Academy. ), Barcelona: National Institute of Physical Education of Catalonia. Popović, S., Jakšić, D., Matić, R., Bjelica, D., & Maksimović, N. (2014). Exami- ning Beliefs and Attitudes toward Advertising through Sport among Serbian Consumers. In Book of abstracts of International conference “Economics and Management of Sports 2014” (pp. 11-2). Brno: Faculty of Sports Studies. Bjelica, D., Popović, S., Jakšić, D., Hadžić, R., & Akpinar, S. (2014b). How Does Advertising through Sport Work? Evidence from Turkey. In Proceedings book of the 7th International Scientifi c Conference on Kinesiology “Funda- mental and Applied Kinesiology – Steps Forward” (477). Opatija: Univer- sity of Zegreb, Faculty of Kinesiology. Popović, S., Jakšić, D., Matić, R., Bjelica, D., & Maksimović, N. (2015g). ATTITUDES TOWARD ADVERTISING THROUGH SPORT AND PARTICIPATING IN SPORTS ACTIVITIES RATION \| D. BJELICA ET AL. Abstracts of the 12th International Scientifi c Conference on Transforma- tion Process in Sport “Sport Performance” (60), Podgorica: Montenegrin Sports Academy. Abstracts of the 12th International Scientifi c Conference on Transforma- tion Process in Sport “Sport Performance” (60), Podgorica: Montenegrin Sports Academy. mining Beliefs and Attitudes toward Advertising through Sport among Serbian Consumers. Studia Sportiva, 9(1), 225-31. mining Beliefs and Attitudes toward Advertising through Sport among Serbian Consumers. Studia Sportiva, 9(1), 225-31. Popović, S., Matić, R., Milašinović, R., Hadžić, R., Milošević, Z., & Bjelica, D. (2015b). Stavovi srbijanskih potrošača prema reklamiranju u spor- tu u odnosu na učestalost kupovine sportskih proizvoda. Sport Mont, 13(43,44,45), 262-9. y Popović, S., Molnar, S., & Radovanović, D. (2011a). Stavovi potrošača prema reklamiranju u sportu u odnosu na učestalost bavljenja sportskim aktiv- nostima. Sport Mont, 9(28,29,30), 148-55. Popović, S., Matić, R., Milašinović, R., Hadžić, R., Milošević, Z., & Bjelica, D. (2015e). Attitudes of Serbian Consumers toward Advertising through Sport among the Question How Often Consumers Purchase Sporting Goods. In Book of Abstracts of the 12th International Scientifi c Conference on Transformation Process in Sport “Sport Performance” (59-60), Podgori- ca: Montenegrin Sports Academy. Stupar, D., Gardasevic, J., & Masanovic, B. (2018). Attitudes of Consumers from Educons University toward Advertising through Sport among the Frequency of Watching Sports Events. Journal of Anthropology of Sport and Physical Education, 2(3). Vukotić, M., Ćorluka, M., & Mašanović, B. (2018). Attitudes of Consumers from the Mostar Canton in Bosnia and Herzegovina toward Advertising thro- ugh Sport among the Frequency of Watching Sports Events. Journal of Anthropology of Sport and Physical Education, 2(2), 19-23. Popović, S., Matić, R., Milašinović, R., Jakšić, D., & Bjelica, D. (2015a). Stavovi srbijanskih potrošača prema reklamiranju u sportu u odnosu na uče- stalost bavljenja sportskim aktivnostima. Sport Mont, 13(43,44,45), 50-6. Zorić, G., Gardašević, J., & Bajramović, I. (2018). Attitudes of Consumers from the Sarajevo Canton in Bosnia and Herzegovina toward Advertising through Sport among the Question how often Consumers purchase Sporting Goods. Journal of Anthropology of Sport and Physical Educa- tion, 2(2), 25-9. Popović, S., Matić, R., Milašinović, R., Jakšić, D., & Bjelica, D. (2015d). Attitu- des of Serbian Consumers toward Advertising through Sport among the Question How Often They Participate in Sports Activities. In Book of Abstracts of the 12th International Scientifi c Conference on Transforma- tion Process in Sport “Sport Performance” (58-9), Podgorica: Montenegrin Sports Academy. Zorić, G., Mašanović, B., & Gardašević, J. (2017). References Exa- Ćorluka, M., Bjelica, D., & Vukotić, M. (2018). Attitudes of Consumers from the 19 J. Anthr. Sport Phys. Educ. 2 (2018) 3 J. Anthr. Sport Phys. Educ. 2 (2018) 3 ATTITUDES TOWARD ADVERTISING THROUGH SPORT AND PARTICIPATING IN SPORTS ACTIVITIES RATION \| D. BJELICA ET AL. Attitudes of Turskish consu- mers toward advertising through sport among the question how often consumers purchase sporting goods. Journal of Anthropology of Sport and Physical Education, 1(1), 17-21. doi: 10.26773/jaspe.171003 Popović, S., Matić, R., Milašinović, R., Vujović, D., Milošević, Z. & Bjelica, D. (2015c). Stavovi srbijanskih potrošača prema reklamiranju u sportu u odnosu na učestalost posmatranja sportskih događaja. Sport Mont, 13(43,44,45), 270-6. Zorić, G., Mašanović, B., & Gardašević, J. (2018). Attitudes of Montenegrin consumers toward advertising through sport among the question how often consumers purchase sporting goods. Journal of Anthropology of Sport and Physical Education, 2(1), 21-5. Popović, S., Matić, R., Milašinović, R., Vujović, D., Milošević, Z., & Bjelica, D. (2015f). Attitudes of Serbian Consumers toward Advertising through Sport among the Frequency of Watching Sports Events. In Book of 20 J. Anthr. Sport Phys. Educ. 2 (2018) 3
https://openalex.org/W4388297077	https://www.nature.com/articles/s43246-023-00416-w.pdf	English	null	Ring-originated anisotropy of local structural ordering in amorphous and crystalline silicon dioxide	Communications materials	2,023	cc-by	12,120	1 Unprecedented-scale Data Analytics Center, Tohoku University, 468-1 Aoba, Aramaki-Aza, Aoba-ku, Sendai 980-8578, Japan. 2 Graduate School of Information Science, Tohoku University, 6-3-09 Aoba, Aramaki-aza Aoba-ku, Sendai 980-8579, Japan. 3 RIKEN Center for Advanced Intelligence Project, 1- 4-1 Nihonbashi, Chuo-ku, Tokyo 103-0027, Japan. 4 Department of Materials Science, Waseda University, 3-4-1 Ohkubo, Shinjuku, Tokyo 169-8555, Japan. 5 Kagami Memorial Research Institute for Materials Science and Technology, Waseda University, 2-8-26 Nishiwaseda, Shinjuku, Tokyo 169-0051, Japan. 6 Center for Basic Research on Materials, National Institute for Materials Science, 1-2-1, Sengen, Tsukuba, Ibaraki 305-0047, Japan. 7 Institute for Integrated Radiation and Nuclear Science, Kyoto University, 2-1010 Asashiro-nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494, Japan. 8 National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan. ✉email: motoki.shiga.b4@tohoku.ac.jp COMMUNICATIONS MATERIALS \| (2023) 4:91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat Ring-originated anisotropy of local structural ordering in amorphous and crystalline silicon dioxide Motoki Shiga 1,2,3✉, Akihiko Hirata 4,5,6, Yohei Onodera 6,7 & Hirokazu Masai 8 Motoki Shiga 1,2,3✉, Akihiko Hirata 4,5,6, Yohei Onodera 6,7 & Hirokazu Masai Rings comprising chemically bonded atoms are essential topological motifs for the structural ordering of network-forming materials. Quantiﬁcation of such larger motifs beyond short- range pair correlation is essential for understanding the linkages between the orderings and macroscopic behaviors. Here, we propose two quantitative analysis methods based on rings. The ﬁrst method quantiﬁes rings by two geometric indicators: roundness and roughness. These indicators reveal the linkages between highly symmetric rings and crystal symmetry in silica and that the structure of amorphous silica mainly consists of distorted rings. The second method quantiﬁes a spatial correlation function that describes three-dimensional atomic densities around rings. A comparative analysis among the functions for different degrees of ring symmetries reveals that symmetric rings contribute to the local structural order in amorphous silica. Another analysis of amorphous models with different orderings reveals anisotropy of the local structural ordering around rings; this contributes to building the intermediate-range ordering. 1 ARTICLE COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w A t i A morphous materials exhibit a disordered structure owing to the lack of translational periodicity. Unlike crystals, which exhibit translational periodicity, amorphous materials do not have a long-range structural order. The forma- tion of a short-range order, whose deﬁnition is based on the distances between two nearest neighbor atoms, can be observed in the pair distribution function (PDF) obtained via X-ray diffrac- tion (XRD) and neutron diffraction (ND) measurements for both crystals and amorphous materials. For example, the short-range order of amorphous and crystalline silica (SiO2) under ambient conditions is based on a regular SiO4 tetrahedron. The inter- connection of tetrahedra with corner-sharing oxygen atoms forms a network structure. Meanwhile, for some amorphous materials, such as SiO2, these diffraction experiments also provide the evidence of a structural order on a length scale larger than the atomic bond length, i.e., the intermediate range. The formation of intermediate-range order of amorphous SiO2 is often discussed in terms of the ﬁrst sharp diffraction peak (FSDP) observed in the diffraction data1–4. However, the origin of FSDP3,5–8 in amor- phous SiO2 has long been debated. Results To reveal the contribution of the network topological order to the intermediate-range structural order, we propose two analysis methods based on rings: (1) ring shape characterization (Fig. 1) and (2) spatial correlation analysis around rings (Fig. 2). g g Structural models of glass, based on a network structure con- sisting of corner-sharing tetrahedral motifs, as found in SiO2, help to investigate the intermediate-range structural orders in amor- phous materials. The ﬁrst structural model for glass was the crystallite model proposed by Frankenheim10 in 1835, which described glass as an aggregate of small crystallites. The crystallite model, also known as the discrete crystalline model, presumes that crystalline clusters are embedded in a glass matrix, owing to the relatively sharper feature of FSDP in the diffraction data. However, this model has been found inappropriate because the estimated lattice constants of the crystallites are larger than those of β- cristobalite, thereby leading to a discrepancy between the observed and calculated densities. Subsequently, continuous crystal11 and quasi-crystal models7 have been proposed. Meanwhile, a con- tinuous random network model was ﬁrst proposed by Zachariasen12 in 1932, wherein short-range structural units in glass were connected randomly. Zachariasen also addressed the relationship between the structure and glass-forming ability and proposed four rules for glass formation. Although Zachariasen’s models are only based on the short-range-order characteristics, such as chemical bonds and the coordination number, such models have been widely used. Several structural models such as the layer model13 and periodic boundaries of void (or cage) models8,14, which assume larger structural units, have been developed further. To validate such model assumptions on the structural order for various materials, statistical analysis methods using geometric and/or chemical structure information are useful. A major approach for characterizing the structural order is ring (closed-path) analysis in a chemically bonded network generated from a structural model. In the conventional analysis, rings are exhaustively enumerated by the shortest-path algorithms, and ring size distributions are analyzed15–19. Recently, Onodera et al.20 Ring shape characterization. The proposed procedure char- acterizes the shape of a ring through computation of the eigen- vector and eigenvalue of the covariance matrix of atomic coordinates (so-called point cloud) in the ring, as shown in Fig. 1. This procedure ﬁrst selects a ring enumerated from a network consisting of atoms and chemical bonds (Fig. 1a) and then obtains a point cloud composed of atoms in the ring (Fig. 1b). Ring-originated anisotropy of local structural ordering in amorphous and crystalline silicon dioxide It is currently established that the length scale of FSDP is approximately 4 Å ¼ 2π qFSDP , q ¼ 4π λ sin θ with the scattering angle 2θ and X-ray or neutron wavelength λ and that the periodicity with the coherence length is approximately 10 Å ¼ 2π ΔqFSDP , where ΔqFSDP is the width of FSDP9. Structural order analysis based on PDF has limitations caused by a rapid decrease in the atomic pair correlation peak intensity on the intermediate-range scale. Moreover, structural units of intermediate-range orders are larger complexes than chemical bonds, and they cannot be identiﬁed using pairwise correlation analysis. Hence, revealing the structural orders hidden in many-body correlations in amorphous materials remains challenging. reported structural orderings related to ring transformation in densiﬁed silica glass. However, since there is no analytical method for ring geometry, the discussion is limited to the number of atoms constituting the ring. For deeply understanding the intermediate-range order in amorphous materials, various analysis methods for the geometric information of rings and their linkages to structural order are necessary. This progress on understanding intermediate-range structural orders entails uncovering the rela- tionship between the orders and material properties using quan- titative analysis methods21,22, which leads to a new path for designing novel functional materials. Furthermore, such methods contribute to building data-driven material design using large- scale structural models of disordered materials23,24 generated by computational simulations using machine learning potentials24–28, which can realize much faster implementation without compro- mising the theoretical accuracy compared to exact computations based on the density functional theory. Here, we propose extensions from conventional ring analysis for structural order analysis: (1) quantitative characterization of ring shapes and (2) a spatial correlation function around rings to visualize local structural orders. The advantage of our approach is that it enables a direct combination of the size, shape, and spatial distribution of rings based on both atomic conﬁguration and network topology. The direct approach is effective for deeply understanding intermediate-range structural orders because cru- cial topological structures formed by chemical bonds are embedded in the atomic conﬁguration. Our methods are applied to analyze amorphous and crystalline silica for a deeper under- standing of the structural orders around rings. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w Fig. 1 Computational procedure of ring shape characterizations. a Rings observed from top and side views. b The point cloud of atoms in the ring. c The ﬁrst, second, and third eigenvectors and eigenvalues of the variance-covariance matrix of the point cloud. d Ring shape measurements: roundness and roughness, both of which are computed from the eigenvalues. Roundness and roughness are deﬁned as Eqs. (1) and (2), respectively. e Examples of ring characterizations by ring size, roundness, and roughness for various rings along with observation from the top (the third eigenvector) and that from the side (the second eigenvector). Fig. 1 Computational procedure of ring shape characterizations. a Rings observed from top and side views. b The point cloud of atoms in the ring. c The ﬁrst, second, and third eigenvectors and eigenvalues of the variance-covariance matrix of the point cloud. d Ring shape measurements: roundness and roughness, both of which are computed from the eigenvalues. Roundness and roughness are deﬁned as Eqs. (1) and (2), respectively. e Examples of ring characterizations by ring size, roundness, and roughness for various rings along with observation from the top (the third eigenvector) and that from the side (the second eigenvector). Fig. 1 Computational procedure of ring shape characterizations. a Rings observed from top and side views. b The point cloud of atoms in the ring. c The ﬁrst, second, and third eigenvectors and eigenvalues of the variance-covariance matrix of the point cloud. d Ring shape measurements: roundness and roughness, both of which are computed from the eigenvalues. Roundness and roughness are deﬁned as Eqs. (1) and (2), respectively. e Examples of ring characterizations by ring size, roundness, and roughness for various rings along with observation from the top (the third eigenvector) and that from the side (the second eigenvector). Fig. 2 Computational procedure of spatial correlation function around rings. a Illustration of a network topology of amorphous SiO2. Blue and red spheres represent Si and O atoms, respectively. Only 500 enumerated rings chosen from Nring ¼ 45; 423 ð Þ rings at random are visualized by highlighting their edges in green. b Eigenvectors of the variance-covariance matrix from atomic coordinates in each ring are computed. c For each ring, the new coordination system v1; v2; v3 is deﬁned by eigenvectors. The value of vd indicates the relative position from the ring center along the d-th eigenvector. Results Subsequently, the eigenvectors and eigenvalues of the variance- covariance matrix of the coordinates are computed (Fig. 1c). The ﬁrst eigenvector is computed as the direction with the largest variance of the point cloud. Under the restriction that the ﬁrst and second eigenvectors are orthogonal with each other, the second eigenvector is computed by maximizing the variance of the point cloud in the vector. Furthermore, the third eigenvector is computed as the vector orthogonal to the ﬁrst and second eigenvectors. As their eigenvalues are proportional to the var- iances of the point cloud along these vectors, they can be used to measure the ring shape. This approach uses eigenvectors, which provide second-order information. This is equivalent to approx- imating a ring by an ellipsoid, wherein the ﬁrst and second eigenvectors correspond to the directions of the major and minor axes of the ellipse approximating the ring, respectively, whereas the third eigenvector is the normal vector of the plane of the ellipse. We proposed two shape indicators for a ring: roundness and roughness, as illustrated in Fig. 1d, both of which are com- puted from eigenvalues. Assuming a square root of the three eigenvalues, s1, s2, and s3, the ﬁrst indicator “roundness” is COMMUNICATIONS MATERIALS \| (2023) 4:91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat 2 ARTICLE COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w Atomic coordinates in the system are computed by Eqs. (3) and (4). Next, for each ring r, spatial histograms for Si or O atom hSi;r v1; v2; v3 , hO;r v1; v2; v3 are computed using atomic coordinates in the new system. d Spatial correlation functions of Si or O atoms around rings hSi v1; v2; v3 , hO v1; v2; v3 are computed by averaging the spatial histograms, as expressed in Eq. (5). Fig. 2 Computational procedure of spatial correlation function around rings. a Illustration of a network topology of amorphous SiO2. Blue and red spheres represent Si and O atoms, respectively. Only 500 enumerated rings chosen from Nring ¼ 45; 423 ð Þ rings at random are visualized by highlighting their edges in green. b Eigenvectors of the variance-covariance matrix from atomic coordinates in each ring are computed. c For each ring, the new coordination system v1; v2; v3 is deﬁned by eigenvectors. The value of vd indicates the relative position from the ring center along the d-th eigenvector. Atomic coordinates in the system are computed by Eqs. (3) and (4). Next, for each ring r, spatial histograms for Si or O atom hSi;r v1; v2; v3 , hO;r v1; v2; v3 are computed using atomic coordinates in the new system. d Spatial correlation functions of Si or O atoms around rings hSi v1; v2; v3 , hO v1; v2; v3 are computed by averaging the spatial histograms, as expressed in Eq. (5). Fig. 2 Computational procedure of spatial correlation function around rings. a Illustration of a network topology of amorphous SiO2. Blue and red spheres represent Si and O atoms, respectively. Only 500 enumerated rings chosen from Nring ¼ 45; 423 ð Þ rings at random are visualized by highlighting their edges in green. b Eigenvectors of the variance-covariance matrix from atomic coordinates in each ring are computed. c For each ring, the new coordination system v1; v2; v3 is deﬁned by eigenvectors. The value of vd indicates the relative position from the ring center along the d-th eigenvector. Atomic coordinates in the system are computed by Eqs. (3) and (4). 91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat 3 COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w roughness, are included to remove the effect of the ring size. These indicators can be generalized by weighting atoms in computing ring centers and variance-covariance matrices. Variances of atomic positions weighted by atomic masses, called the radius of gyration, was proposed as a measure to evaluate ring compactions29. correlation function was computed based on a structural unit consisting of three atoms (i.e., a centered Si atom and two nearest O atoms in silica) to determine the coordination system of the correlation function. In contrast, our proposed approach determines the coordination system by using a ring as a larger unit consisting of over four atoms. This offers a more straightforward approach for visualizing and revealing intermediate-range structural orders in a network-forming amorphous material, which is demonstrated by identifying similarities in structural orders between amorphous and crystal- line materials of silica. Examples in Fig. 1e demonstrate that roundness and roughness appropriately evaluate these ring shapes. In general, there is no direct relationship between roundness and roughness. For example, roundness and roughness are not correlated for rings with sizes 9 and 11, as shown in Fig. 1e. However, they are negatively correlated for rings with sizes 6 and 8. Notably, these numerical values of small rings are restricted due to the small degrees of freedom in their shapes. Analysis of amorphous and crystalline silica structural models. In this study, we focused on amorphous and crystalline materials with corner-sharing tetrahedral motifs, which are represented by a structural model of amorphous silica (a-SiO2), along with those of nine crystalline polymorphs of silica: α-tridymite31, β- tridymite32, α-cristobalite33, β-cristobalite34, α-quartz35, β- quartz36, coesite I37, coesite II38, and stishovite39 (see Supple- mentary Tables 1–3 for data IDs, space groups, and lattice information, and Supplementary Discussion and Supplementary Figure 1 for structural statistics). Note that the coordination number of Si is six only in stishovite, while it is four in other crystals analyzed in this work. Spatial correlation function around rings. The second proposed procedure (Fig. 2) is used to visualize how the symmetry and/or anisotropy of a ring signiﬁcantly contribute to the local structural orderings in the intermediate-range scale. For the computational procedure of the spatial correlation function, we ﬁrst enumerate all the rings (Fig. 2a). For each ring, we compute the ring center by averaging atomic coordinates and the eigenvectors of the variance-covariance matrix from the atomic coordinates of the ring (Fig. 2b). COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w Then, we obtain the center cr ¼ crx; cry; crz , and eigenvectors er1, er2, er3 for the r-th ring, r ¼ 1; :::; Nring. Here, Nring is the number of enumerated rings, while Natom is the number of an element in the structural model, i.e., the number of Si or O atoms in silica. Next, for each element, i.e., Si or O atom in silica, we compute the spatial three-dimensional (3D) histo- gram around each ring by three steps (Fig. 2c): (1) Atomic coordinates of the element in the structural models xn ¼ xn; yn; zn T; n ¼ 1; :::; Natom are translated so that the ring cen- ter is the origin, which is computed by We generated a large-scale structural model of a-SiO2 by classical molecular dynamics (MD) simulation of a melt- quenching procedure, followed by reﬁnement using the reverse Monte Carlo (RMC)40 technique to reproduce XRD and ND data. Here, the side length of the simulation box for the model was assumed approximately 100 Å to ensure accurate statistics and suppress artifacts caused by the periodic boundary condition (see Methods and Supplementary Table 4 for the detailed procedure and structural statistics). RMC was implemented with some constraints to preserve the physically meaningful structure generated by MD (Methods and Supplementary Discussion). Hereafter, the generated structure is referred to as the MD–RMC model. The X-ray and neutron total structure factors S q of the model showed good agreement with the experimental data (Supplementary Figures 2 and 3). The coordination numbers around Si and O atoms, of over 99% in the ﬁrst coordination distance of the generated model, are four and two, respectively. It was established that a well-known network structure, consisting of an SiO4 tetrahedron sharing O atoms at the corner for SiO2 (NSiO ¼ 4, NOSi ¼ 2), is formed in the MD–RMC model (Supplementary Figures 4 and 5). xrn ¼ xn crx; yn cry; zn crz T ; n ¼ 1; :::; Natom ð3Þ ð3Þ where T is a transpose operator of a vector or a matrix. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w (2) When the coordinate axes of the histogram around r-th ring are deﬁned as er1, er2, er3, the atomic coordinates in the new coordination system are computed by vrn ¼ vrn1; vrn2; vrn3 T ¼ xT rner1; xT rner2; xT rner3 T; n ¼ 1; :::; Natom: ð4Þ (3) The 3D histogram of the element around the r-th ring in the new coordination system, hatom;r v1; v2; v3 is computed using atomic coordinates vrn; n ¼ 1; :::; Natom. After computing histo- grams for all rings, the spatial correlation function of each element, hatom v1; v2; v3 is computed by averaging histograms (Fig. 2d): Ring shape characterizations. Guttman ring15, King ring17, and primitive ring18,19 are well-known ring deﬁnitions. We selected the primitive ring for our investigations. A primitive ring is deﬁned as a ring that cannot be decomposed into two smaller rings18,19. This is equivalent to the condition that for any node pairs in the ring, there is no shortcut, which is a shorter path composed of edges outside the ring. Therefore, primitive rings are essential components in network-forming amorphous materials. Although the primitive criterion enumerates larger rings com- pared to Guttman’s rings, it can avoid enumerating redundant larger rings unlike those in King’s criteria. Please see Yuan’s paper19 for some illustrative examples of primitive rings. As a ﬁrst step, our analysis exhaustively enumerated rings in each network of the structural models based on the shortest-path algorithms (See Methods for the detail). Figure 3a shows the distributions of ring sizes, i.e., the numbers of Si atoms in a ring in amorphous and crystalline materials. Among the crystalline materials, cris- tobalite and tridymite phases have only six-fold rings, although the coordination number of each Si and O atom is the same in all hatom v1; v2; v3 ¼ 1 Catom ∑ Nring r¼1 hatom;r v1; v2; v3 : ð5Þ ð5Þ The normalization coefﬁcient is Catom ¼ d3 wNringNatomV1 box, where dw is the bin width of the histogram, and Vbox is the volume of the simulation box. Normalization is necessary to compare structural models with different atomic densities. In crystals, regions with large spatial correlation are point-like and are extremely small for visualization. To improve the visibility of the large correlation regions of crystalline materials, we used a Gaussian ﬁlter for their computed correlation functions to spatially expand them. ARTICLE ARTICLE COMMUNICATIONS MATERIALS \| (2023) 4:91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w Next, for each ring r, spatial histograms for Si or O atom hSi;r v1; v2; v3 , hO;r v1; v2; v3 are computed using atomic coordinates in the new system. d Spatial correlation functions of Si or O atoms around rings hSi v1; v2; v3 , hO v1; v2; v3 are computed by averaging the spatial histograms, as expressed in Eq. (5). “roughness” is deﬁned as “roughness” is deﬁned as deﬁned as rt ¼ s3ﬃﬃﬃﬃﬃﬃﬃ s1s2 p : ð2Þ ð1Þ rt ¼ s3ﬃﬃﬃﬃﬃﬃﬃ s1s2 p : ð2Þ ð2Þ ð1Þ It evaluates how close the ring is to a perfect circle. The roundness value for a perfect circle is 1. The second indicator It evaluates the ﬂatness of the ring. These normalizations, i.e., s1 for roundness and the geometric mean of s1 and s2 for the COMMUNICATIONS MATERIALS \| (2023) 4:91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat 3 3 COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w Fig. 3 Ring characterizations of SiO2 materials. Proportion of the number of rings in crystalline and amorphous materials as a function of ring size (a), roundness (b), and roughness (c). Ring size is deﬁned by the number of Si atoms in a ring. Roundness and roughness of a ring are deﬁned as Eqs. (1) and (2), respectively. Fig. 3 Ring characterizations of SiO2 materials. Proportion of the number of rings in crystalline and amorphous materials as a function of ring size (a), roundness (b), and roughness (c). Ring size is deﬁned by the number of Si atoms in a ring. Roundness and roughness of a ring are deﬁned as Eqs. (1) and (2), respectively. crystalline materials. Therefore, these structures are topologically ordered even while considering the network connectivity patterns over the ﬁrst coordination spheres. In contrast, other crystal phases have rings of different sizes. Notably, the ring sizes of coesite I and II are widely distributed, as in the amorphous material shown in the bottom panel, thus indicating that the structures of coesite I and II are topologically disordered, owing to its high density. The ring size of a-SiO2 is also widely dis- tributed, with the peak of the ring size distribution located at approximately sizes 6 or 7, similar to the cases of phases of cristobalite and tridymite. useful for enumerating isomorphic rings in crystalline materials because these values are identical when the ring shapes are identical. The isomorphic rings identiﬁed in crystalline SiO2 and their shape characterizations are summarized in Fig. 4 and Supplementary Table 5. Both β- and α-cristobalite have only one isomorphic ring, whereas β- and α-tridymite, β- and α-quartz, coesite I, coesite II, and stishovite have two, three, four, ﬁve, eleven, thirty-four, and four isomorphic rings, respectively. This indicates that the number of isomorphic rings in a crystalline material is a measure of its symmetry. Next, we analyzed linkages between ring symmetries and space groups of crystals. As a factor of the ring symmetry, the point group of each isomorphic ring is listed in Supplementary Table 5. The result concludes that only β-cristobalite and β-tridymite have a direct linkage between the point group of a ring and the space group of a crystal. For example, β-cristobalite includes only an isomorphic ring (A6-1) with the highest symmetry evaluated by both point group and our shape measures, as shown in Supplementary Table 5. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w Our proposed correlation function is a generalized formulation of the results from a recent study30. In the study, the spatial 4 ARTICLE COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w COMMUNICATIONS MATERIALS \| (2023) 4:91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w We found that the three-fold rotational symmetry of the ring in β-cristobalite directly links to the three- fold symmetry of the cubic space group, as illustrated in Fig. 5a, b. Because β-cristobalite includes only one isomorphic ring, this situation leads to the formation of a cubic crystal system that has the highest symmetry among the crystalline materials of SiO2. We also found a direct linkage in β-tridymite, as illustrated in Fig. 5c–e. The ring with three-fold symmetry (C6-1), whose shape For these structural models, we applied the proposed ring- shape characterizations, as summarized in Fig. 1. Figure 3b, c show our proposed indicators of ring shape, i.e., roundness and roughness, in crystalline and amorphous silica materials. It was found that β-cristobalite has only six-fold rings with the largest roundness and smallest roughness compared to other crystals. Thus, compared to other crystalline materials, β-cristobalite has the highest ring symmetry, which contributes to the cubic nature of the crystal system, as discussed later. In contrast, the rings of α- cristobalite are less symmetric than those of β-cristobalite but with higher symmetry than those of coesite I and II. Among these structures, the order of the ring symmetries is identical to the order of the ring symmetries in crystal systems. We demonstrated that a set of three eigenvalues of a ring can be used as an indicator of the ring’s shape. Notably, the set is also 5 COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w ARTICLE Fig. 4 Isomorphic primitive rings in crystalline SiO2. a The top view (the normal vector) of rings. b The side view (the second eigenvector). Numeric values of ring shape characteristics and the point group of each ring are listed in Supplementary Table 5. Fig. 4 Isomorphic primitive rings in crystalline SiO2. a The top view (the normal vector) of rings. b The side view (the second eigenvector). Numerical values of ring shape characteristics and the point group of each ring are listed in Supplementary Table 5. Fig. 4 Isomorphic primitive rings in crystalline SiO2. a The top view (the normal vector) of rings. b The side view (the second eigenvector). Numerical values of ring shape characteristics and the point group of each ring are listed in Supplementary Table 5. We further investigated the linkage between ring symmetries and crystal polymorphs based on the pressure–temperature phase diagram, as shown in Fig. 4 in a previous paper41. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w Two noteworthy ﬁndings regarding the phase diagrams are as follows: ﬁrst, that high- temperature phases are more symmetric than the low-temperature ones. The symmetry is evaluated by the number of isomorphic rings and ring symmetries (such as roundness, roughness, and point group). For example, β-phases (such as β-cristobalite) are more symmetric than α-phases (such as α-cristobalite). In addition, cristobalites are more symmetric compared with tridymites. Second, except for stishovite, which has different atomic coordination numbers from other crystals, low-pressure phases are more symmetric than the high-pressure ones. For example, phases of cristobalite and tridymite are more symmetric than those of quartz and coesite. These two ﬁndings are consistent with the fact that β- cristobalite is the most symmetric among SiO2 crystal polymorphs since the phase is located at the lowest pressure and highest temperature in the phase diagram. is identical to that of the ring (A6-1) in β-cristobalite, can yield six-fold screw symmetry combined with the partial translational operation along the screw axis. Lastly, rings in α-cristobalite, α- tridymite, β-quartz, α-quartz, coesite I, and coesite II are unrelated to the space groups. Although α-cristobalite includes only an isomorphic ring similar to β-cristobalite, the point group indicates no symmetry, small roundness, and large roughness, resulting in a less symmetric space group than that of β- cristobalite, as illustrated in Supplementary Fig. 6. In this case, the conﬁguration of rings with the lowest symmetry constructs four- fold screw symmetry. A similar situation is also found in α- tridymite, β-quartz, and α-quartz, as illustrated in Supplementary Figs. 7–9. The cases of coesite I and coesite II become extremely complicated, and it is hard to ﬁnd any linkages between ring symmetries and space groups in such cases. Overall, this analysis concludes that there is a strong linkage between the ring symmetry and the crystal symmetry, i.e., highly symmetric rings build a highly symmetric crystal system. is identical to that of the ring (A6-1) in β-cristobalite, can yield six-fold screw symmetry combined with the partial translational operation along the screw axis. Lastly, rings in α-cristobalite, α- tridymite, β-quartz, α-quartz, coesite I, and coesite II are unrelated to the space groups. Although α-cristobalite includes only an isomorphic ring similar to β-cristobalite, the point group indicates no symmetry, small roundness, and large roughness, resulting in a less symmetric space group than that of β- cristobalite, as illustrated in Supplementary Fig. 6. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w All rings of C6-2 are orthogonal to ring C6-1. Si atoms are colored blue or cyan, which indicate site equivalence whether the z coordinate is lower or higher from the ring plane. e Screw symmetry 63 in β-tridymite, which is along the direction [001] with C6-1 rings. Numbers marked in prime and double prime indicate trajectories of the screw operation, which demonstrates the direct linkage between the point group of the ring and the space group of β-tridymite. The bottom panels in Fig. 3b, c show the computed results for a- SiO2. Similar to the ring size distribution, roundness and roughness are broadly distributed. This indicates that various rings are included in a-SiO2. The ring-shape evaluation with two-dimensional distributions of both measures is shown in Fig. 6. The probability density in a-SiO2 was computed by a kernel density estimation with Gaussian kernel, whose band width was determined using the Scott’s rule42. Characterization results for crystalline materials are repre- sented by symbols due to the limited number of isomorphic rings. Figure 6 shows that these values of α-cristobalite, β-cristobalite, α- tridymite, and β-tridymite are negatively correlated because of the small ring sizes. However, a correlation is not generally observed, instead, it is not adaptable for most cases. The ﬁgure shows that the peak position of the roundness–roughness distributions in a-SiO2 is close to those of the ring B6-1 in α-cristobalite and D6-3 in α- tridymite among the six-fold rings. This shows a resemblance between the major rings in a-SiO2 and those in α-cristobalite and α- tridymite. The broad distribution of the shape indicators in a-SiO2, covering those of all isomorphic rings in crystalline materials, suggests that the distribution in a-SiO2 is a mixture of isomorphic rings in crystalline materials and their variants. Fig. 6 Probability distribution of ring characteristic indicators (roundness and roughness) of amorphous and crystalline SiO2. The probability density of a-SiO2 was computed by a kernel density estimation with Gaussian kernel. The band width of the kernel was determined using the Scott’s rule42 ( = n−1/(d+4)), where n is the number of rings, and d is dimension. In the data, n = 45,423 and d = 2. COMMUNICATIONS MATERIALS \| (2023) 4:91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat 7 The bottom panels in Fig. 3b, c show the computed results for a- SiO2. Similar to the ring size distribution, roundness and roughness are broadly distributed. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w In this case, the conﬁguration of rings with the lowest symmetry constructs four- fold screw symmetry. A similar situation is also found in α- tridymite, β-quartz, and α-quartz, as illustrated in Supplementary Figs. 7–9. The cases of coesite I and coesite II become extremely complicated, and it is hard to ﬁnd any linkages between ring symmetries and space groups in such cases. Overall, this analysis concludes that there is a strong linkage between the ring symmetry and the crystal symmetry, i.e., highly symmetric rings build a highly symmetric crystal system. COMMUNICATIONS MATERIALS \| (2023) 4:91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat 6 ARTICLE COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w Fig. 5 Symmetry with rings in β-cristobalite and β-tridymite. a The view from the direction [111]. b The side view of that in (a). In these panels, all rings are identical to the ring A6-1 in Supplementary Table 5, some of which are colored magenta or green to visualize layers. This visualization shows the direct linkage between point group of the ring (3 m) to the space group of β-cristobalite (F d 3 m). c The view from the direction [001] of β-tridymite. The magenta ring is C6-1, whose point group is 3 m. d The view from the direction [010]. The orange ring is C6-2. All rings of C6-2 are orthogonal to ring C6-1. Si atoms are colored blue or cyan, which indicate site equivalence whether the z coordinate is lower or higher from the ring plane. e Screw symmetry 63 in β-tridymite, which is along the direction [001] with C6-1 rings. Numbers marked in prime and double prime indicate trajectories of the screw operation, which demonstrates the direct linkage between the point group of the ring and the space group of β-tridymite. Fig. 5 Symmetry with rings in β-cristobalite and β-tridymite. a The view from the direction [111]. b The side view of that in (a). In these panels, all rings are identical to the ring A6-1 in Supplementary Table 5, some of which are colored magenta or green to visualize layers. This visualization shows the direct linkage between point group of the ring (3 m) to the space group of β-cristobalite (F d 3 m). c The view from the direction [001] of β-tridymite. The magenta ring is C6-1, whose point group is 3 m. d The view from the direction [010]. The orange ring is C6-2. COMMUNICATIONS MATERIALS \| (2023) 4:91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w This indicates that various rings are included in a-SiO2. The ring-shape evaluation with two-dimensional distributions of both measures is shown in Fig. 6. The probability density in a-SiO2 was computed by a kernel density estimation with Gaussian kernel, whose band width was determined using the Scott’s rule42. Characterization results for crystalline materials are repre- sented by symbols due to the limited number of isomorphic rings. Figure 6 shows that these values of α-cristobalite, β-cristobalite, α- tridymite, and β-tridymite are negatively correlated because of the small ring sizes. However, a correlation is not generally observed, instead, it is not adaptable for most cases. The ﬁgure shows that the peak position of the roundness–roughness distributions in a-SiO2 is close to those of the ring B6-1 in α-cristobalite and D6-3 in α- tridymite among the six-fold rings. This shows a resemblance between the major rings in a-SiO2 and those in α-cristobalite and α- tridymite. The broad distribution of the shape indicators in a-SiO2, covering those of all isomorphic rings in crystalline materials, suggests that the distribution in a-SiO2 is a mixture of isomorphic rings in crystalline materials and their variants. Fig. 6 Probability distribution of ring characteristic indicators (roundness and roughness) of amorphous and crystalline SiO2. The probability density of a-SiO2 was computed by a kernel density estimation with Gaussian kernel. The band width of the kernel was determined using the Scott’s rule42 ( = n−1/(d+4)), where n is the number of rings, and d is dimension. In the data, n = 45,423 and d = 2. COMMUNICATIONS MATERIALS \| (2023)4:91 \| https://doi org/10 1038/s43246 023 0 Fig. 6 Probability distribution of ring characteristic indicators (roundness and roughness) of amorphous and crystalline SiO2. The probability density of a-SiO2 was computed by a kernel density estimation with Gaussian kernel. The band width of the kernel was determined using the Scott’s rule42 ( = n−1/(d+4)), where n is the number of rings, and d is dimension. In the data, n = 45,423 and d = 2. 7 MMUNICATIONS MATERIALS \| (2023) 4:91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w ARTICLE Fig. 7 Cross-sectional mappings of spatial correlation functions from v1, v2, and v3-axis directions for crystalline and amorphous SiO2. a–f Cross- sectional mappings of the spatial correlation functions of β-cristobalite (a), α-cristobalite (b), β-tridymite (c), α-tridymite (d), β-quartz (e), α-quartz (f), coesite I (g), coesite II (h), stishovite (i), and a-SiO2 (MD-RMC) (j). COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w These ﬁgures demonstrate that β-cristobalite and the functions of symmetric rings similar to a perfect circle (the spatial correlation function for region I in Fig. 8b) and non-symmetric rings that are dissimilar to a perfect circle (the function for region IV in Fig. 8b) in a-SiO2. This demonstrates that atomic conﬁgurations are locally symmetric around symmetric rings, even in a-SiO2 and that the lesser the symmetric rings, the lesser symmetric the local atomic conﬁgurations, as shown in spatial correlation functions for regions II–IV in Fig. 8b and Supplementary Fig. 13b. In a- SiO2, highly symmetric rings are scarce, while slightly less symmetric rings are predominant in region II, which includes the probability density mode. Therefore, the amorphous material is mainly composed of slightly less symmetric local structures in the present case. g g Figure 7j shows the spatial correlation function for the a-SiO2 model. These diagrams exhibit a ring around the center, which is the average of all primitive rings. The v1-v2 plane also shows that the averaged ring shape in a-SiO2 is not a perfect circle, but an ellipse, which is consistent with the results of the roundness analysis shown in Fig. 3b. As shown in Fig. 7j and Supplementary Figure 10j, the center ring is surrounded by multiple shells in the amorphous model. The shells of the O and Si atoms are alternately located from the center owing to the O–Si–O–Si linkage. In addition, these ﬁgures show planes parallel to the center ring in the upper and lower parts of the ring along the v3-axis. They also show partially parallel planes along the v2-axis, which is parallel to the v1-axis (the major axis of the rings), as shown in the middle panel of Fig. 7j. However, they do not exhibit such planes along the v1-axis. Owing to the anisotropic shapes of the rings, it can be concluded that a- SiO2 has anisotropic structural order around the rings. In particular, the planes consisting of rings are parallel to each other but orthogonal to the normal vector of a ring. Since this situation is similar to the arrangement of lattice planes in crystals such as β- cristobalite and β-tridymite, as shown in Fig. 5, the pseudo-Bragg condition for diffraction could also be considered locally even in amorphous materials. In fact, the normal vector of the ring becomes parallel to the scattering vector. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w k Color indicator: a region with blue/red color indicates a large density of Si/O atoms. The cross-sectional thickness t was set to 2 Å for all models. Fig. 7 Cross-sectional mappings of spatial correlation functions from v1, v2, and v3-axis directions for crystalline and amorphous SiO2. a–f Cross- sectional mappings of the spatial correlation functions of β-cristobalite (a), α-cristobalite (b), β-tridymite (c), α-tridymite (d), β-quartz (e), α-quartz (f), coesite I (g), coesite II (h), stishovite (i), and a-SiO2 (MD-RMC) (j). k Color indicator: a region with blue/red color indicates a large density of Si/O atoms. The cross-sectional thickness t was set to 2 Å for all models. Fig. 7 Cross-sectional mappings of spatial correlation functions from v1, v2, and v3-axis directions for crystalline and amorphous SiO2. a–f Cross- sectional mappings of the spatial correlation functions of β-cristobalite (a), α-cristobalite (b), β-tridymite (c), α-tridymite (d), β-quartz (e), α-quartz (f), coesite I (g), coesite II (h), stishovite (i), and a-SiO2 (MD-RMC) (j). k Color indicator: a region with blue/red color indicates a large density of Si/O atoms. The cross-sectional thickness t was set to 2 Å for all models. Spatial correlation functions around rings. The inﬂuence of ring symmetries on the local structural ordering was evaluated using our proposed spatial correlation function. To visualize the functions of SiO2 materials, corresponding cross-sectional map- pings at approximately v1 ¼ 0, v2 ¼ 0, or v3 ¼ 0 were computed by integrating the correlation function over the cross-section thickness t, as shown in Fig. 7a–j. The cross-sectional thicknesses t was set to 2 Å for all crystalline and amorphous models. In these ﬁgures, the blue and red regions exhibit high densities of Si and O atoms, respectively, as depicted by the color indicator (Fig. 7k). 8 ARTICLE COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w values. The upper and lower planes made by the large correlation regions for a-SiO2, which are pseudo-Bragg planes, are also consistent with those for the phases of cristobalite and tridymite. The correlation functions for the phases of quartz and coesite (Supplementary Figs. 11e–h and 12e–h) also contain regions partly similar to those for a-SiO2, although they appear different globally. The anisotropic nature of the a-SiO2 amorphous model is consistent with the analysis results for crystalline SiO2, especially with phases of cristobalite and tridymite. These mappings also show that β-, α-cristobalite, β-tridymite, and α-tridymite (Fig. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w 7a–d) have parallel planes along the v3-axis and concentric circles on the v1-v2 plane, whereas other crystalline materials (Fig. 7e–i) appear to be disordered. This is attributed to the fact that phases of cristobalite and tridymite have a smaller number of isomorphic rings with relatively larger roundness and smaller roughness than other crystalline materials. Among all crystalline materials, the function of β-cristobalite (Fig. 7a) is highly oriented and are surprisingly identical due to only one isomorphic ring (six-fold ring) with large roundness and small roughness. In contrast, the functions for α-cristobalite, β-tridy- mite, and α-tridymite (Fig. 7b–d) appear noisy compared to that of β-cristobalite. This is because the three crystalline materials have a larger number of isomorphic rings or smaller roundness and larger roughness of rings than those of β-cristobalite. Coesite II (Fig. 7h) is the most disordered due to the variety of non- symmetric isomorphic rings. From another visualization of 3D surfaces of large spatial correlations shown in Supplementary Figure 10, we could have similar ﬁndings and discussions. For the amorphous SiO2, we performed an additional analysis to ﬁnd a linkage between the roundness and roughness of rings and the symmetry of the intermediate-range atomic conﬁguration around rings. To conﬁrm the linkage, the β-cristobalite, which has the highest crystal symmetry and contains one type of symmetric ring with large roundness and small roughness, is used as a standard. In the analysis, we ﬁrst selected four subsets of rings using their roundness and roughness values, as shown in Fig. 8a. The subsets (or regions in the ﬁgure) are determined by the quantiles of roundness rc ¼ 0:73; 0:80; 0:87, and those of roughness rt ¼ 0:24; 0:30; 0:36. For example, the range of region I is 0:87<rc ≤1 and 0 ≤rt ≤0:24. Rings in region I are more symmetric than those in other three regions because of larger roundness and smaller roughness of rings in the region. On the contrary, rings in region IV are less symmetric than others. For each region, we computed the spatial correlation function using rings included in the region. Figure 8b shows the computed spatial correlation functions, along with the correlation function of β-cristobalite to discuss a symmetry of the intermediate-range atomic conﬁgurations around the rings. Direct comparisons of these functions of amorphous SiO2 with that of β-cristobalite in same diagrams are shown in Supplementary Fig. 13b. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w The ﬁrst model (Rand- Coord) was generated from a random conﬁguration, followed by a hard-sphere Monte Carlo (HSMC) simulation under two 9 9 MMUNICATIONS MATERIALS \| (2023) 4:91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w ARTICLE Fig. 8 Spatial correlations cross-sectional mappings of Si/O atoms using speciﬁc shapes of rings in a-SiO2. a Regions of roundness and roughness for computing spatial correlation functions around speciﬁc rings. The probability distribution of ring characteristic indicators of amorphous SiO2 is the same as that in Fig. 6. The regions are determined by the quantiles of roundness rc ¼ 0:73; 0:80; 0:87, and those of roughness rt ¼ 0:24; 0:30; 0:36. For example, the range of region I is in 0:87<rc 1 and 0 rt 0:24. Rings in region I, whose roundness is larger, and roughness is smaller than those in other regions, are more symmetric than those in other three regions. On the contrary, rings in region IV are less symmetric than others. Region II includes rings of major shapes because the region includes the mode, i.e., the point with the largest probability density. b The correlation function computed using only rings whose shapes are in a speciﬁc region (I–IV), together with that of β-cristobalite, which is same as that in Fig. 7a. The color indicator at the bottom shows values of spatial correlations of Si/O atom. The direct comparisons of the function of Si/O atom in each region with that of β-cristobalite are shown in Supplementary Fig. 13. Fig. 8 Spatial correlations cross-sectional mappings of Si/O atoms using speciﬁc shapes of rings in a-SiO2. a Regions of roundness and roughness for computing spatial correlation functions around speciﬁc rings. The probability distribution of ring characteristic indicators of amorphous SiO2 is the same as that in Fig. 6. The regions are determined by the quantiles of roundness rc ¼ 0:73; 0:80; 0:87, and those of roughness rt ¼ 0:24; 0:30; 0:36. For example, the range of region I is in 0:87<rc 1 and 0 rt 0:24. Rings in region I, whose roundness is larger, and roughness is smaller than those in other regions, are more symmetric than those in other three regions. On the contrary, rings in region IV are less symmetric than others. Region II includes rings of major shapes because the region includes the mode, i.e., the point with the largest probability density. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w b The correlation function computed using only rings whose shapes are in a speciﬁc region (I–IV), together with that of β-cristobalite, which is same as that in Fig. 7a. The color indicator at the bottom shows values of spatial correlations of Si/O atom. The direct comparisons of the function of Si/O atom in each region with that of β-cristobalite are shown in Supplementary Fig. 13. restrictions: the closest atom–atom distance and coordination number. The closest-distance restriction avoids unreasonable spikes in the partial pair distribution functions. The coordination number restriction forces Si atoms to coordinate to four O atoms, whereas O atoms coordinate to two Si atoms within a Si–O cutoff distance of 1.90 Å. The second structural model (Rand-Tetra) used the Rand-Coord model as the initial atomic conﬁguration and was then generated by implementing HSMC with restrictions on the coordination numbers and O–Si–O bond angle distribu- tion to create a network structure, consisting of regular SiO4 tetrahedra sharing O atoms at the corner. The third model (Rand- RMC) used the Rand-Tetra model as the initial atomic conﬁguration, subsequently generated by the RMC simulation to reproduce the X-ray and neutron S q data. Structural statistics, such as structure factors, coordination numbers, bond angles, and ring characters are summarized in Supplementary Discussion and Supplementary Figs. 14–17. This structured summary demonstrates that the randomly initialized models (i.e., Rand-Coord, Rand-Tetra, and Rand-RMC) are more disordered compared to the MD-RMC model. Among the structure factors, only Rand-RMC and MD-RMC exhibit FSDP because RMC ﬁts the structure factors. Although all models form restrictions: the closest atom–atom distance and coordination number. The closest-distance restriction avoids unreasonable spikes in the partial pair distribution functions. The coordination number restriction forces Si atoms to coordinate to four O atoms, whereas O atoms coordinate to two Si atoms within a Si–O cutoff distance of 1.90 Å. The second structural model (Rand-Tetra) used the Rand-Coord model as the initial atomic conﬁguration and was then generated by implementing HSMC with restrictions on the coordination numbers and O–Si–O bond angle distribu- tion to create a network structure, consisting of regular SiO4 tetrahedra sharing O atoms at the corner. The third model (Rand- RMC) used the Rand-Tetra model as the initial atomic conﬁguration, subsequently generated by the RMC simulation to reproduce the X-ray and neutron S q data. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w Since the spacing between the planes (~ 4 Å) corresponds to the position of FSDP in reciprocal space, the origin of FSDP is likely the formation of intermediate- range structural orders containing these parallel rings. We further investigated the consistency between the spatial correlation functions of the amorphous and crystalline models. Supplementary Figure 11 shows the cross-sectional mappings of the Si correlation functions in the amorphous model together with those in crystalline models: β-cristobalite (a), α-cristobalite (b), β-tridymite (c), α-tridymite (d), β-quartz (e), α-quartz (f), coesite I (g), coesite II (h), and stishovite (i). In these diagrams, the function of the amorphous model is shown by the blue image, whereas that of the crystal model is shown by a green image. Supplementary Figure 12 shows correlation functions of the O atom, wherein spatial correlations of the amorphous model and those of crystalline models are colored red and green, respectively. The rightmost diagrams (v1-v2 plane) show that the averaged ring of the amorphous model is similar to that of α-cristobalite (Supplementary Figs. 11b and 12b), which is consistent with the roundness of rings in Fig. 3b. The averaged ring shape of α- tridymite (Supplementary Figs. 11d and 12d) is also similar to that of a-SiO2, whereas those of β-cristobalite (Supplementary Figs. 11a and 12a) and β-tridymite (Supplementary Figs. 11c and 12c) are different, but both are consistent with these roundness Comparing amorphous models with various structural order- ings. A series of analyses showed an anisotropic local structural order in the amorphous silica (a-SiO2) model, whereas isotropic halo rings were observed in the reciprocal space by macroscopic diffraction data. A transition from anisotropic to isotropic order prevails while transitioning from microscopic to macroscopic viewpoints. Notably, the anisotropic local structure originates from the rings, which is an essential topological motif in network- forming materials. Here, we discuss the inﬂuence of the aniso- tropic local structural order on the degree of structural order in amorphous structures through the comparison of some additional amorphous models. p To validate our analyses further, we generated three additional amorphous structural models from a random conﬁguration. The same number of atoms and the same size of the simulation box were used for these additional models. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w Structural statistics, such as structure factors, coordination numbers, bond angles, and ring characters are summarized in Supplementary Discussion and Supplementary Figs. 14–17. This structured summary demonstrates that the randomly initialized models (i.e., Rand-Coord, Rand-Tetra, and Rand-RMC) are more disordered compared to the MD-RMC model. Among the structure factors, only Rand-RMC and MD-RMC exhibit FSDP because RMC ﬁts the structure factors. Although all models form a network of chemical bonds, the structural orders of these amorphous models are highly different. Structures of three models without MD (Rand-Coord, Rand-Tetra and Rand-RMC) are considerably more disordered than MD-RMC models, as demonstrated in Supplementary Figure 17, wherein all variances of ring size, roundness, and roughness of these three models are larger than those of the MD-RMC models. These statistics indicate that RMC does not construct structural order, instead, it generates disordered structures43. g Figure 9 shows the spatial correlation functions of Rand-Coord (a), Rand-Tetra (b), Rand-RMC (c), and MD-RMC (d), wherein all models exhibit anisotropic structural orders around the rings. This indicates that anisotropy is related only to the network formation of amorphous materials. A clearer shell structure was observed in the MD-RMC model compared to other models. This is due to the ring size distribution in the MD-RMC model, concentrated at size 6 (see Supplementary Fig. 18), whereas other three models showed broad distributions. Hence, the correlation functions computed using only six-fold rings, as shown in Supplementary Fig. 18, present clearer shell structures in all models. The correlation functions for Rand-RMC and MD-RMC in Fig. 9c, d and Supplementary Figs. 18c, d exhibit parallel planes COMMUNICATIONS MATERIALS \| (2023) 4:91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat 10 ARTICLE COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416- Cross-sectional mappings of spatial correlations of a-SiO2 models. a–d Cross-sectional mappings of spatial correlations of Rand-Coord (a), Rand- b), Rand-RMC (c), and MD-RMC (d) models. e Color indicator: a region with blue/red color indicates a large density of Si/O atoms. The cross- al thickness t is 2 Å. Green arrows indicate parallel planes above and below the rings along the v1 and v2 axes. Fig. 9 Cross-sectional mappings of spatial correlations of a-SiO2 models. a–d Cross-sectional mappings of spatial correlations of Rand-Coord (a), Rand- Tetra (b), Rand-RMC (c), and MD-RMC (d) models. e Color indicator: a region with blue/red color indicates a large density of Si/O atoms. The cross- sectional thickness t is 2 Å. Discussion h d The coefﬁcients used in the simu- lation were Bij ¼ 21:39 ´ 1016 J and ρij ¼ 0:174 Å for an atomic pair of Si and O, and Bij ¼ 0:6246 ´ 1016 J and ρij ¼ 0:362 Å for a pair of O atoms. In the potential function, the interactions between the Si atoms were ignored, similar to our previous work20. Although numerous studies have investigated the empirical potential functions for SiO2 materials, potentials that assume Si–Si interaction are limited. One example is the reference study48, wherein the potential function with Si–Si interactions was determined using ab Initio quantum-chemical methods. However, the effect of Si–Si interactions is smaller than those of other interaction terms. A comparison of these potentials reported in another study49 showed that the potential function without Si–Si interactions is more accurate than that with Si–Si interaction. Because of the discussion in previous works, we considered an empirical potential function without Si–Si interaction. The atomic conﬁguration was initialized at random, and the system was equilibrated at 4000 K for 100,000 steps. Subse- quently, it was cooled to 300 K for 5,000,000 steps and annealed at 300 K for 100,000 steps. The generated model was reﬁned via RMC modeling using XRD and ND measurements. RMC was implemented with the constraints of the coordination numbers, bond angles of O–Si–O, and the partial pair-distribution functions within the ﬁrst coordination shell. The constraint of the bond angle was performed by preserving the distribution of the structures by the MD simulation. These constraints were added to preserve the physically meaningful structure generated by MD, which is discussed in detail along with some structural statistics. This model was named MD-RMC. p To link structural orders and material properties and realize data-driven material design, quantitative evaluations for various structural models are necessary. In crystalline materials, the macroscopic properties are linked to the lattice structure and their symmetry because they include all structural information. Conversely, amorphous materials do not have such linkages because they do not have the lattice and symmetric structures. Therefore, we should explore the linkages through another approach using structural motifs such as rings. An example of this approach is using different ﬁctive temperatures45, which revealed the linkage between the properties and ring size dis- tributions in SiO2 glass. Discussion h d This study proposed two analysis methods for the structural orders of rings and those around rings based on the ring shapes in network-forming materials. The proposed analysis approach for crystalline and amorphous SiO2 ﬁrst demonstrated that the dis- tribution of ring shape characteristics is a strong tool for ana- lyzing the amorphous models of SiO2. The roundness–roughness distribution showed that the major rings in amorphous SiO2 are similar to those in two crystals, α-cristobalite and α-tridymite, whose mass densities are similar to those of amorphous SiO2. In addition, it was found that the rings in quartz and coesite phases are included as minor rings in amorphous SiO2. This indicates that various rings found in crystalline polymorphs are necessary to form bulk amorphous materials44. Furthermore, the spatial correlation analysis showed that the anisotropic nature of struc- tural orders was found in both crystalline SiO2, with different polymorphisms, and amorphous SiO2. The analysis further showed that pseudo-Bragg planes, parallel to the rings in amor- phous SiO2, are consistent with those of four crystals: α-cristo- balite, α-tridymite, β-cristobalite, and β-tridymite. In addition, this study found that rings with low roughness tend to construct planes parallel to the central rings, as shown by the ring shape characterization of isomorphic rings on crystalline materials. To validate the applicability of our approaches, three amorphous SiO2 structural models with different ordering degrees were prepared. The corresponding anisotropic local environments, as described above, were conﬁrmed in all models. Although the actual degree of order for amorphous SiO2 is still arbitrary, observations in this study showed that even if amorphous SiO2 satisﬁes the experimental structure factors, it has anisotropic local structures. Furthermore, our analysis assumed that the observed pseudo-Bragg planes, presumably yielding the ﬁrst sharp dif- fraction peak, are correlated with the intermediate-range struc- tural orders in amorphous SiO2. ϕij ¼ Bij exp r ρij ! þ e2 4πϵ0 ZiZj r ; ð6Þ ð6Þ where r is the interatomic distance between the atoms i and j, Bij and ρij represent the magnitude (1016 J) and softness (Å) of the Born-Mayer term, respectively, Zi is the effective charge on atom i (ZSi ¼ 2:4 and ZO ¼ 1:2), e is the elementary charge, and ϵ0 is the permittivity of a vacuum. Methods above and below rings along the v3-axes indicated by green arrows. This relates to the intermediate-range structural orders exhibited by FSDP in the diffraction experiments. Conclusively, the anisotropy of structural orders around rings is essential for building intermediate-range structural orders. Generation of a structural model of amorphous silica. Struc- tural models of amorphous silica were generated by a melt- quenching procedure in classical MD simulation, followed by RMC techniques to ﬁt the diffraction measurements of the syn- thesized materials40. MD simulations and RMC were imple- mented using the LAMMPS code46 and the RMC++ code47, respectively. In the MD simulation of amorphous silica, the simulation box was a cube with a length of 100 Å. The density of this system was 2:2 g cm3. The system had 66,156 atoms (22,052 Si atoms and 44,104 O atoms) within the NVT ensemble. A time step of 1 fs was used in the Verlet algorithm. Furthermore, the interactions were described by pair potentials with short-range Born-Mayer repulsive and long-range Coulomb terms: COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w Green arrows indicate parallel planes above and below the rings along the v1 and v2 axes. Fig. 9 Cross-sectional mappings of spatial correlations of a-SiO2 models. a–d Cross-sectional mappings of spatial correlations of Rand-Coord (a), Rand- Tetra (b), Rand-RMC (c), and MD-RMC (d) models. e Color indicator: a region with blue/red color indicates a large density of Si/O atoms. The cross- sectional thickness t is 2 Å. Green arrows indicate parallel planes above and below the rings along the v1 and v2 axes. Fig. 9 Cross-sectional mappings of spatial correlations of a-SiO2 models. a–d Cross-sectional mappings of spatial correlations of Rand-Coord (a), Rand- Tetra (b), Rand-RMC (c), and MD-RMC (d) models. e Color indicator: a region with blue/red color indicates a large density of Si/O atoms. The cross- sectional thickness t is 2 Å. Green arrows indicate parallel planes above and below the rings along the v1 and v2 axes. 11 COMMUNICATIONS MATERIALS \| (2023) 4:91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat 11 ARTICLE Acknowledgements 17. King, S. V. Ring conﬁgurations in a random network model of vitreous silica. Nature 213, 1112–1113 (1967). This research was supported by JSPS KAKENHI Grant Numbers JP20H05878 (to M.S.), JP20H05884 (to M.S.), JP23K17837 (to A.H.), JP20H05881 (to A.H. and Y.O.), JP20H05882 (to H.M.), JP20H04241 (to M.S., A.H., and Y.O.), and JP19K05648 (to Y.O.) and JST PRESTO Grant Number JPMJPR16N6 (to M.S.). We would also like to thank Dr. Shinji Kohara for the helpful discussions. 18. Goetzke, K. & Klein, H. J. Properties and efﬁcient algorithmic determination of different classes of rings in ﬁnite and inﬁnite polyhedral networks. J. Non- Cryst. Solids 127, 215–220 (1991). y 19. Yuan, X. & Cormack, A. N. Efﬁcient algorithm for primitive ring statistics in topological networks. Comput. Mater. Sci. 24, 343–360 (2002). p g p 20. Onodera, Y. et al. Structure and properties of densiﬁed silica glass: h i i h d i hi di d NPG A i M 12 85 (2 20. Onodera, Y. et al. Structure and properties of densiﬁed silica glass: characterizing the order within disorder. NPG Asia Mater. 12, 85 (202 p p g characterizing the order within disorder. NPG Asia Mater. 12, 85 (2020). aracterizing the order within disorder. NPG Asia Mater. 12, 85 ( Data availability g g Chem. Rev. 121, 10037–10072 (2021). All data needed to evaluate the conclusions in the paper are present in the paper and/or Supplementary information. 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P., van Beest, B. W. H. & van Santen, R. A. Interatomic force ﬁelds for silicas, aluminophosphates, and zeolites: b d b l l h ( ) 49. Kramer, G. J., Farragher, N. P., van Beest, B. W. H. & van Santen, R. A. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w generated by connecting two different shortest paths that do not share internal nodes. Upon inspecting the primitive criterion for the generated candidates and removing candidates that do not satisfy the criterion, the ring enumeration algorithm is termi- nated. The ring enumerations in this study were implemented using our in-house python package. 23. Deringer, V. et al. Origins of structural and electronic transitions in disordered silicon. Nature 589, 59–64 (2021). 24. Erhard, L. C., Rohrer, J., Albe, K. & Deringer, V. L. A machine-learned interatomic potential for silica and its relation to empirical models. npj Comput. 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Crystal structure of low cristobalite at 10, 293, and 473 k: Variation of framework geometry with temperature. J. Appl. Phys. 57, 1045–1049 (1985). 34. Wyckoff, R. W. G. Ix. die kristallstruktur von 2-cristobalit SiO2 (bei hohen temperaturen stabile form). Z. Kristallogr. Cryst. Mater. 62, 189–200 (1925). y , 2 ( temperaturen stabile form). Z. Kristallogr. Cryst. Mater. 62, 189–200 (1925). 35. Kihara, K. An X-ray study of the temperature dependence of the quartz structure. Eur. J. Mineral. 2, 63–78 (1990). p ) g y ( ) 35. Kihara, K. An X-ray study of the temperature dependence of the quartz structure. Eur. J. Mineral. 2, 63–78 (1990). Discussion h d Various additional numerical descriptors such as geometric information of rings like roundness and roughness, and those of structural ordering from diverse per- spectives are needed for ﬁnding new linkages between macro- scopic properties and structural orders. For network-forming materials, we developed new descriptors and demonstrated useful applications for analyzing the structural orders of SiO2 materials, although the applicability of the approach is not limited to SiO2. Furthermore, the analysis of other network-forming materials using our proposed methods and a comprehensive comparison of the results should provide further information to help understand intermediate-range structural orders in network-forming materials. Ring enumeration. Under the assumption that the ﬁrst coordi- nation distance is less than 2.0 Å for Si–O, a network was gen- erated for each structural model of SiO2. Primitive rings can be efﬁciently enumerated based on the shortest-path algorithms19. The enumeration algorithm ﬁrst computes the distances of the node pairs in the network using the shortest path algorithm. Next, node pairs whose distances in the network are less than the threshold, which should be set to half of the maximum number of atoms counting both Si and O atoms in the primitive rings, are enumerated. Subsequently, all the shortest paths between the enumerated node pairs are enumerated. Ring candidates are 12 COMMUNICATIONS MATERIALS \| (2023) 4:91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat ARTICLE ARTICLE ARTICLE COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-023-00416-w manuscript. M.S., A.H., Y.O. and H.M. contributed to the writing and editing of this manuscript. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. p g The authors declare no competing interests. p g The authors declare no competing interests. Competing interests 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/. Author contributions 21. Shi, Y. et al. Revealing the effect of medium-range structure on silicate glass hardness. Phys. Rev. Mater. 7, 013602 (2023). M.S., A.H. and Y.O. designed the study. M.S. developed the analysis methods and computational codes. M.S. and Y.O. built the structural models and analyzed the con- M.S., A.H. and Y.O. designed the study. M.S. developed the analysis methods and computational codes. M.S. and Y.O. built the structural models and analyzed the con- ﬁgurations. A.H. designed the analysis on the linkage between ring symmetries and structural symmetries, and A.H. and M.S. performed them. M.S. prepared a draft of the 22. Minamitani, E., Shiga, T., Kashiwagi, M. & Obayashi, I. Topological descriptor of thermal conductivity in amorphous Si. J. Chem. Phys. 156, 244502 (2022). ﬁgurations. A.H. designed the analysis on the linkage between ring symmetries and structural symmetries, and A.H. and M.S. performed them. M.S. prepared a draft of the MMUNICATIONS MATERIALS \| (2023) 4:91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat 13 © The Author(s) 2023 Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s43246-023-00416-w. Correspondence and requests for materials should be addressed to Motoki Shiga. Peer review information Communications Materials thanks Sébastien Le Roux, Jin Bai, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Jet-Sing Lee. A peer review ﬁle is available. Reprints and permission information is available at http://www.nature.com/reprints © The Author(s) 2023 14 COMMUNICATIONS MATERIALS \| (2023) 4:91 \| https://doi.org/10.1038/s43246-023-00416-w \| www.nature.com/commsmat
https://openalex.org/W3113444203	https://biblio.ugent.be/publication/8685580/file/8685581.pdf	English	null	A Novel Feature Set for Low-Voltage Consumers, Based on the Temporal Dependence of Consumption and Peak Demands	Energies	2,020	cc-by	13,349	Keywords: load proﬁling; consumer categorization; clustering; load duration curve; peak demand; feature construction Citation: Claeys, R.; Azaioud, H.; Cleenwerck, R.; Knockaert, J.; Desmet, J. A Novel Feature Set for Low-Voltage Consumers, Based on the Temporal Dependence of Consumption and Peak Demands. Energies 2021, 14, 139. https://doi.org/10.3390/en14010139 energies energies Article A Novel Feature Set for Low-Voltage Consumers, Based on the Temporal Dependence of Consumption and Peak Demands bbert Claeys * , Hakim Azaioud , Rémy Cleenwerck , Jos Knockaert and Jan Desmet Robbert Claeys * EELab/Lemcko, Department of Electromechanical, Systems and Metal Engineering, Ghent University, 8500 Kortrijk, Belgium; Hakim.Azaioud@UGent.be (H.A.); Remy.Cleenwerck@UGent.be (R.C.); Jos.Knockaert@UGent.be (J.K.); JanJ.Desmet@UGent.be (J.D.) * Correspondence: Robbert.Claeys@UGent.be Abstract: This paper proposes a novel feature construction methodology aiming at both clustering yearly load proﬁles of low-voltage consumers, as well as investigating the stochastic nature of their peak demands. These load proﬁles describe the electricity consumption over a one-year period, allowing the study of seasonal dependence. The clustering of load curves has been extensively studied in literature, where clustering of daily or weekly load curves based on temporal features has received the most research attention. The proposed feature construction aims at generating a new set of variables that can be used in machine learning applications, stepping away from traditional, high dimensional, chronological feature sets. This paper presents a novel feature set based on two types of features: respectively the consumption time window on a daily and weekly basis, and the time of occurrence of peak demands. An analytic expression for the load duration curve is validated and leveraged in order to deﬁne the the region that has to be considered as peak demand region. The clustering results using the proposed set of features on a dataset of measured Flemish consumers at 15-min resolution are evaluated and interpreted, where special attention is given to the stochastic nature of the peak demands. Citation: Claeys, R.; Azaioud, H.; Cleenwerck, R.; Knockaert, J.; Desmet, J. A Novel Feature Set for Low-Voltage Consumers, Based on the Temporal Dependence of Consumption and Peak Demands. Energies 2021, 14, 139. https://doi.org/10.3390/en14010139 1. Introduction First, consumption proﬁles obtained from metering data with similar behavior have to be grouped together via a clustering algorithm. Unsupervised learning techniques are often used to detect underlying structures in large datasets, with K-means and hierarchical clustering among the most prevalent methods [5]. These algorithms can be performed either on the chrono- logical data itself, or on a feature set obtained by transforming this chronological data. Common examples for chronological data include clustering based on daily or weekly proﬁles [6,7]. The seasonal inﬂuence can be taken into account by either performing a clustering process for each individual season [8], or by determining the recurring daily load proﬁles on an annual basis [9]. Typical load proﬁles can now be found by using statistical measures on the grouped chronological data, such as the mean or the median value at each time step [10]. This illustrates the main drawback during the construction of synthetic load proﬁles for purposes related to peak demands, such as emerging capacity-based tariffs. The averaging process results in a loss of important time-sensitive information unique to the individual household, and less volatile proﬁles are obtained [11]. As mentioned, an alternative method to clustering via the chronological measurements is grouping consumers based on similar properties, also called features. This work follows the feature construction and evaluation approach. The advantages of using a limited set of features during a clustering process are multifold. First, artiﬁcial overﬁtting due to high dimensional data can be avoided [12]. Furthermore, computational time is saved and allows easier interpretation if the features are chosen to be application-dependent [13]. Features can be constructed by performing operations on the default chronological features, e.g., combining all daytime consumption in one single feature. However, more advanced features can also be constructed, ranging from features generated in the frequency do- main [14,15], to features related to the shape of the distribution of the load, such as the load factor [16]. Features constructed in literature are often application-dependent, i.e., depending on the goal of the work. One goal of this work is to investigate the temporal connection between consumption and peak demand behavior, to gain insight in the stochasticity of residential peak demands. Therefore, the features in this work are linked to either the consumption or the occurrence of peak demands. 1. Introduction In different regions and countries in the European Union, including Flanders, the reg- ulator for the electricity market has proposed an update to the traditional tariff structure for consumers connected to the low-voltage distribution grid [1,2]. The goal of this update is to obtain a tariff structure that better reﬂects the real costs associated with operating the distribution grid, as well as to incentivize consumers to change their consumption behavior. The emergence of digital meter technology and its rollout in European countries allows policymakers to implement these changes. Simultaneously, due to the higher time resolution, consumers are given a tool to gain additional insight in their consumption and related electricity invoice. Received: 26 November 2020 Accepted: 18 December 2020 Published: 29 December 2020 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institutional afﬁliations. y Residential and low-voltage consumers form a particularly challenging group from the viewpoint of grid operators and parties responsable for local grid balancing. Individual household consumption proﬁles are very behavior-dependent and often described as being peak-intensive and stochastic [3]. They often exhibit short peak demands, while simulta- neously being characterized by large periods during the day and at night with very low energy demand. Therefore, regulators are proposing tariff structures that include cost elements related to both energy and capacity. 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/). Consequently, the research attention has shifted to the mitigation of the peak demands by applying e.g., peak shaving programs or demand respons initiatives, as well as anal- yses on the predictability and stochasticity of these peaks. The research ﬁeld related to customer categorization and load proﬁling aims to support policymakers and stakeholders https://www.mdpi.com/journal/energies Energies 2021, 14, 139. https://dx.doi.org/10.3390/en14010139 Energies 2021, 14, 139 2 of 24 by providing insights into (i) the types of consumers, and (ii) the behavior and differences between their load proﬁles. The traditional example of this is the construction of synthetic or representative load proﬁles. For residential consumers, the Flemish regulator currently offers two synthetic load proﬁles on an annual basis, i.e., households with and without electric heating. g In standard modeling techniques, the following steps are taken [4]. 1. Introduction Previous studies incorporating temporal properties of these peaks in the clustering process either take the timing and the amplitude of the daily peak demand into account [17,18], or use statistical measures of the distribution of the measurement data [19]. In this work, the load duration curve (LDC), also called the demand frequency distribution graph, of each individual consumer is used to deﬁne which of its measurements constitute a peak demand on annual basis. The LDC is obtained by ordering the measurements in descending order. At the macrogrid level, the LDC has traditionally been used by electric utility engineers for network planning purposes, to analyse the utilisation of power plants, as well as characterizing the cyclic behavior of electricity demand [20–22]. While the LDC has not traditionally been used to model individual consumers, it was successfully used by Poulin et al. [23] to investigate the value of peak shaving for industrial and commercial consumers. Encouraged by these ﬁndings, the analytical form of the load duration curve is used in this work to construct the peak-based features. Based on the shape of the LDC, a threshold unique to each consumer is proposed, and every demand higher than this threshold can be considered as a peak. By determining the time of occurrence of these peak demands, the temporal properties of the peaks are taken into account. The main contribution of this work thus is the introduction of features related to the peak demands. The introduced methodology combines both frequency- and time-based Energies 2021, 14, 139 3 of 24 information to determine these features. The remainder of this work is organized as follows. In Section 2.1, the dataset and the preprocessing steps are described. Section 2.2 then proposes and validates an analytic expression for the load duration curves of low-voltage consumers. This expression is subsequently used in Section 2.3 to deﬁne the region that can be considered as peak demands. The features related to the timing of the consumption and peak demands are constructed in Section 2.4. This feature set is used for two purposes, and depending on the methodology, a different feature transformation has to be performed. The clustering algorithm, as well as the methodology used to analyse the stochastic nature of the peak demands, are described respectively in Sections 2.5.1 and 2.5.2. 2.1. Consumption Proﬁles The used dataset used in this work comprises 1422 consumers on the low-voltage distribution grid in two small Flemish towns in a suburban area, measured at a 15-min resolution during one year, leading to 35,040 time points per consumer. The data were provided by Fluvius cvba, the Flemish distribution network operator. The metering infrastructure was installed during a proof-of-concept study on digital meters in Flanders during the period 2010–2014. As more than 3000 households spanning different generations and compositions participated in this study, the dataset can be considered sufﬁciently diverse for consumers on the low-voltage distribution grid. Several preprocessing steps were undertaken to obtain the ﬁnal dataset, leading to a reduction from over 3.000 load proﬁles to 1422 data entries. These preprocessing steps are as follows: – A ﬁrst preprocessing step involving possible missing data was performed by the distribution network operator before providing the dataset for this research; – A ﬁrst preprocessing step involving possible missing data was performed by the distribution network operator before providing the dataset for this research; – Only meters that had measurements for the full year 2013 were included, given the purpose of this work; – Households equipped with a PV installation were excluded from the analysis, as it is known that the presence of a PV installation can induce behavioral changes to increase PV self-consumption [24]. Furthermore, the metering data for households with PV installations merely included information on the net consumption and injec- tion, not the gross consumption which is necessary for the proposed methodology; g p y p p gy – Following the Eurostat classiﬁcation [25], meters indicating an annual consumption lower than 1000 kWh or higher than 15,000 kWh were excluded, as these were assumed to not be representative for typical household behavior, or could include small and medium-sized enterprises (SMEs), meaning commercial meters, on the low-voltage distribution grid. After the preprocessing, the 1422 individual timestamped proﬁles are subsequently categorised based on the thermal images obtained via heatmaps of their demand proﬁle. The introduced categories will not be used as input for the clustering algorithm, merely used for a post-hoc validation and interpretation of the obtained clusters in Section 3.1. This heatmap is the visualisation of the matrix obtained by reshaping the 35,040 ×1 vector of the chronological data to a 96 × 365 matrix. 1. Introduction Section 3.1 reports on the ﬁndings and performance of the clustering algorithm, while Section 3.2 considers the relations between the consumption and peak demands in certain time periods to shed light on the stochasticity of these peak demands. Finally, Section 4 concludes this paper. 2.1. Consumption Proﬁles The entries belonging to the days of the start and end of daylight saving time are removed before reshaping the matrix, resulting in a 96 × 363 matrix. These days contain 92 and 100 data points, and would therefore distort the heatmap. Based on the obtained heatmaps, ﬁve categories are introduced that are able to describe the typical low-voltage consumers in Flanders: four behavior-speciﬁc categories and one so-called regular residential consumer for all consumers that do not ﬁt Energies 2021, 14, 139 4 of 24 one of the four special categories, inspired by the common Synthetic Load Proﬁles (SLPs) for Flanders. one of the four special categories, inspired by the common Synthetic Load Proﬁles (SLPs) for Flanders. The ﬁve categories are as follows: The ﬁve categories are as follows: – SME proﬁle: consistent load proﬁle with a 9–18 h behavior on weekdays and absent on weekends, as shown on Figure 1a; – Electric heating: consumption late in the evening and at night, superimposed upon a regular consumption proﬁle. Two substructures are observed: – Ripple control heating: These proﬁles exhibit the same moment during week- days when the heating is turned on, and a different behavior is observed for weekdays and weekends, as shown on Figure 1b; – Continuous heating: Unlike the ripple control heating, the moment of switch- ing on the heating is stochastic and no difference in heating behavior between weekdays and weekends can be observed, as shown on Figure 1c; – Air conditioning: proﬁles with a signiﬁcant electric load during summer months, superimposed upon a regular consumption proﬁle. This heatmap is not shown for brevity. y Regular residential consumer: the remaining load proﬁles not belonging to one of the above categories. There are typically (but not necessarily) characterized by a morning and evening peak, with demands concentrated during the evening as shown on Figure 1; y – Regular residential consumer: the remaining load proﬁles not belonging to one of the above categories. There are typically (but not necessarily) characterized by a morning and evening peak, with demands concentrated during the evening as shown on Figure 1; Figure 1. 2.2. Load Duration Curves 2.2. Load Duration Curves The load duration curve of an individual consumer is obtained by ordering its meter- ing data in a descending order rather than the traditional chronological order. The analytic expression introduced by Poulin et al. [23] for commercial, institutional and industrial consumers is taken as the starting point for the analysis on the low-voltage consumers considered in this work. Let Pi(t) denote the chronological demand data of a speciﬁc consumer i, its corresponding LDC Pi(τ) can subsequently be written as: Pi(τ) = 1 −aτ −bτc + d 1 + e f (τ−g) − d 1 + e f g (1) (1) The variables τ and Pi in the expression of the LDC respectively denote the normalized time and normalized demand, i.e., both scaled such that their range spans the interval [0, 1]. This allows for a scale-independent comparison between consumers, merely comparing the behavior of the demand curves. The six parameters included in Equation (1) show a clear connection to customer operations, and thus are relevant for consumer clustering purposes. The peak height and duration are correlated with b and c respectively, while parameters d, f and g are linked to respectively the height, slope and location of the step. Finally, a yields information about the general slope of the curve. These six parameters and their relation to the general shape of the LDC given by Equation (1) are given in Figure 2a. g p g y q g g While the six-parameter expression was previously validated to accurately model the LDC of individual and aggregated residential consumers [26], this work aims to both simplify this six-parameter expression, as well as to link the parameters in its simpliﬁed expression to properties of the consumer, such as the annual consumption. Therefore, Figure 2b displays the shape of the ﬁrst proposed improvement for low-voltage consumers, a 5-parameter model. Intuitively one could indeed expect households to spend the majority of their year on a certain baseload, i.e., the aggregated standby demand of the appliances in the household. As such, this would correspond to a saturation effect towards this standby demand being present in the household LDC for limτ→1 Pi(τ), in contrast to the decreasing slope that is present in Equation (1). Consequently, a ﬁve-parameter LDC model for low-voltage distribution grid con- sumers is proposed in Equation (2). The linear term included in Equation (1) is omitted. 2.1. Consumption Proﬁles Examples of heatmaps for four different types of consumers on the low-voltage grid: (a) an SME proﬁle, (b) a consumer with ripple control electric heating with a ﬁxed start time of the heating, as well as differences between weekdays and weekends, (c) a consumer with continuous electric heating, and (d) a regular residential consumer. Figure 1. Examples of heatmaps for four different types of consumers on the low-voltage grid: (a) an SME proﬁle, (b) a consumer with ripple control electric heating with a ﬁxed start time of the heating, as well as differences between weekdays and weekends, (c) a consumer with continuous electric heating, and (d) a regular residential consumer. As can be seen in Table 1, the majority of the considered consumers on the low-voltage distribution grid does not fall within a category with speciﬁc features such as the SME or the electric heating proﬁles, but can be considered a regular household. Table 1 gives an overview of the number of proﬁles in each category, split for different consumption ranges. The density of the regular demand proﬁles is the highest in the range of 2–3 MWh per year, which is consistent with the most common household consumption in Flanders. Simi- larly, the other categories are mostly concentrated at higher average yearly consumption. Energies 2021, 14, 139 5 of 24 Table 1. Dataset composition by the deﬁned proﬁle categories, by consumption ranges. Consumption Range Regular Ripple Control e-Heating Continuous e-Heating SME Airco 1–2 MWh 143 3 1 0 0 2–3 MWh 260 19 6 2 0 3–4 MWh 254 25 4 2 0 4–5 MWh 223 17 15 0 0 5–6 MWh 126 14 13 5 0 6–7 MWh 86 10 14 1 3 >7 MWh 114 19 18 10 15 Total 1.206 107 71 20 18 Table 1. Dataset composition by the deﬁned proﬁle categories, by consumption ranges. 2.2. Load Duration Curves Pi(τ) = 1 −bτc + d 1 + e f (τ−g) − d 1 + e f g (2) (2) The LDC based on the measurement data is constructed for each individual metering point and the values of the parameters included in Equations (1) and (2) are subsequently determined via a curve ﬁtting algorithm. This procedure is performed via the lmfit package in Python, where a Least-Squares minimization with a Trust Region Reﬂective method is used [27]. 6 of 24 Energies 2021, 14, 139 (a) (b) Figure 2. (a) Shape of the 6-parameter LDC for commercial, institutional and industrial consumers [23], and (b) shape of the proposed 5-parameter LDC for low-voltage consumers. (b) (a) (a) (b) Figure 2. (a) Shape of the 6-parameter LDC for commercial, institutional and industrial consumers [23], and (b) shape of the proposed 5-parameter LDC for low-voltage consumers. The parameters are constrained according to following boundaries: The parameters are constrained according to following boundaries:            0 ≤a, b, c ≤1 0.02 ≤d ≤1 25 ≤f ≤+∞ 0.025 ≤g ≤1            0 ≤a, b, c ≤1 0.02 ≤d ≤1 25 ≤f ≤+∞ 0.025 ≤g ≤1 The boundaries were chosen nearly identical to those used for the LDC ﬁtting pro- cedure in [23]. The only deviation from the boundaries in [23] is the lower bound of the g parameter. Therein, a lower bound of 0.1 for g was assumed. However, as mentioned in Section 1, residential consumers are more peak-intensive and their peaks are more stochastic. It is expected that this behavior is reﬂected in the shape of the LDC with a shorter duration of the peak and step. Therefore, the lower bound for g, the parameter linked to the location of the step, can be taken smaller than the aforementioned 0.1. A value of 0.025 was chosen for this lower bound. On average, the correlation coefﬁcient R2 is 0.977 for both expressions. However, for 91% of the consumers in the dataset, both the Akaike and the Bayesian information criterion point toward Equation (2) as the most suitable expression to describe the LDC. This is further supported by the very small value of a for the 6-parameter expression: on average, a has a value of 0.0026, corresponding to a near negligible slope in Equation (1). 2.2. Load Duration Curves g p g g g p q However, describing consumers via their load duration curve has several disadvan- tages. One inherent disadvantage of the load duration curve is the loss of all temporal information, which forms the subject of Section 2.4. A second disadvantage is related to the use of the expression for the demand-normalized LDC. While this allows for a more straightforward and scale-independent comparison of the parameters describing the behavior of individual consumers, all information related to the original peak demand is lost, and no information on the traditional properties such as the annual consumption is retained. Therefore, the second step of this analysis entails incorporating possible correlation between the values of the parameters in Equation (2) and the annual consumption. The re- lation between the individual parameters of the LDC and the yearly consumption of the consumer is given in Figure 3. Despite a large spread being present in the scatterplot, the parameter c describing the power law in Equation (2) is noticeably correlated with the yearly consumption. Consequently, Equation (3) ﬁxes the parameter c at the value c0 + c1Y. The values of c0 and c1 are determined by an ordinary least-squares ﬁtting procedure on the relation between Y, the yearly consumption in kWh, and c, as shown in Figure 3. Pi(τ) = 1 −bτc0+c1Y + d 1 + e f (τ−g) − d 1 + e f g (3) (3) 7 of 24 Energies 2021, 14, 139 Figure 3. Scatterplots of the parameters included in Equation (2), plotted versus the yearly consumption. Figure 3. Scatterplots of the parameters included in Equation (2), plotted versus the yearly consumption. With an obtained value of 0.0345 and 0.456 × 10−6 for c0 and c1 respectively, an analo- gous ﬁtting procedure of the LDC to Equation (3) is performed. The result and a comparison with the previous two models is listed in Table 2. While a decrease in median R2 value can be observed, this value is still acceptable. However, the observed mean value is signif- icantly lower and exhibits an increasing difference with the median value, highlighting that the 4-parameter model of Equation (3) leads to a worse ﬁt for a non-negligible amount of consumers. Table 2. Comparison of the ﬁtting result of the three considered LDC models. 2.2. Load Duration Curves Model Median R2 Value Mean R2 Value 6-parameter model, Equation (1) 0.987 0.977 5-parameter model, Equation (2) 0.987 0.977 4-parameter model, Equation (3) 0.968 0.937 Table 2. Comparison of the ﬁtting result of the three considered LDC models. As expected, given the high spread in the linear relation between c and the yearly consumption, the reduction in accuracy of modeling the LDC is a trade-off that has to be made in order to incorporate the dependency on the consumer’s yearly consumption. Given the importance of the ﬁtted parameters of the LDC in the remainder of this work, the further analyses are performed on Equation (2), the model that exhibited superior performance in the ﬁtting procedure. 2.3. Deﬁnition of Peak Demands The curvature function κ(τ) of Equation (4) is given by: κ(τ) = (Pi)′′(τ) h 1 + (Pi)′(τ) 2i 3 2 (5) (5) Maximizing κ(τ) with respect to τ yields following value for the point of maximum curvature: Maximizing κ(τ) with respect to τ yields following value for the point of maximum curvature: τ∗= c −2 b2c2(2c −1) 1 2(c−1) (6) (6) The histogram of the calculated values of τ∗and the corresponding value Pi(τ∗) for the considered dataset is given in Figure 4. A beta probability density function is successfully ﬁtted and shown to be able to describe the density functions, as shown overlaid in Figure 4. The distribution of τ∗has a 10–90 percentile range of [0.017, 0.041], with a mean value of 0.028. Translating this mean value of the normalised time τ to a yearly basis means that, on average across the distribution, 2.8% of the values on a yearly basis can be labeled as peaks, corresponding with 981 values of the 35.040 data points. Furthermore, the distribution of Pi(τ∗) shows the large potential of peak shaving initiatives: the mean value of Pi(τ∗) is 0.35, i.e., 35% of the original maximum demand. Figure 4. Density histogram of the calculated values of τ∗and Pi(τ∗), as deﬁned by Equation (6). Figure 4. Density histogram of the calculated values of τ∗and Pi(τ∗), as deﬁned by Equation (6). 2.3. Deﬁnition of Peak Demands The validated analytic expression of the load duration curve can now be used to introduce a binary classiﬁcation for peak demands, i.e., all values Pi(τ) for τ smaller than a certain threshold τ∗can be considered peaks for the individual consumer while all other values cannot. The challenge now lies in determining τ∗, the value of this threshold. The only condition a proposed expression or value for τ∗has to fulﬁll for the purposes intended in this work is that it has to be sufﬁciently small in order to yield usable results. Although the term “usable” implies a certain level of arbitrariness, it should be clear that a threshold value that labels 50% of all demands on yearly basis as peaks is not practical for e.g., peak shaving algorithms. Therefore, given the continuous nature of the load duration curve, it is inevitable that any proposed threshold value will have its own advantages and disadvantages. g This work proposes using the point of maximum curvature as this threshold, for the function for τ sufﬁciently small. Intuitively, the curvature of a function is the amount by Energies 2021, 14, 139 8 of 24 which this function deviates from a straight line in a certain point. Therefore, the maximum of this curvature function denotes the point where the curve has the sharpest bend. which this function deviates from a straight line in a certain point. Therefore, the maximum of this curvature function denotes the point where the curve has the sharpest bend. p p For τ sufﬁciently small, the LDC as deﬁned in Equation (2) can be approximated by Equation (4), which is dominated by the power law responsible for the peak demand features and the steep decay of the LDC: Pi(τ) ≈1 −bτc. (4) (4) Using the point of maximum curvature of Equation (4) as the threshold value to deﬁne the area of peak demands has two major advantages. First, this threshold is different for each individual as it depends on the shape of the individual load duration curve, allowing for a differentiation among low-voltage consumers. Second, the point of maximum curva- ture for an analytic function can be unambiguously described analytically. 2.4. Feature Construction The aim of this work is to construct a feature set that can be used for dual purposes. First, to group similar consumers together in so-called clusters based on properties that are relevant for (i) distribution network operators with respect to their operating and tarifﬁng purposes, as well as for (ii) individual consumers, i.e., for the local applicability of distributed energy resources and how evolving tariff structures impact them. Second, to investigate the stochastic nature of peak demands, given the current trend of countries to introduce capacity-based tariff schemes for low-voltage consumers combined with the uptake of small-scale storage systems that can be used for peak shaving purposes Energies 2021, 14, 139 9 of 24 9 of 24 in residential areas. Given the intended purposes and the large temporal dependence of renewable yields, we choose to explicitly incorporate the temporal inﬂuence on the demand proﬁle in the construction of the feature set. Two different temporal levels relevant for low-voltage distribution grids are con- sidered for these time-dependent features: the daily and the weekly level. At the daily level, the time periods are deﬁned based on the time of day. Similarly, at the weekly level the distinction between weekday and weekend is maintained. In order to distinguish between intervals I deﬁned on either the daily level and the weekly level, two notations are introduced: Id and Iw. The superscript d stands for daily, w for weekly. p p y, y While the deﬁnition of the intervals Iw is unambiguous, i.e., weekdays versus week- ends, distinct intervals at the daily level Id for residential consumers are not universally agreed upon. In [17], Haben et al. identiﬁed four key time periods for residential con- sumers: overnight, breakfast, daytime and evening period. Inspired by their ﬁndings, the distinction as listed in Table 3 is introduced for the daily level. In this work, the daytime period is further subdivided in a morning and afternoon range. Furthermore, while the daytime period in Ref. [17] ended at 15:30, it is extended to 18:00 for this work. Table 3. Definition of the considered time periods Id at the daily level, based on the hour of the day, h. Id Deﬁnition Early morning h ∈[ 06:00–08:30 ] Morning h ∈[ 08:30–12:00 ] Afternoon h ∈[ 12:00–18:00 ] Evening h ∈[ 18:00–22:30 ] Night h ∈[ 22:30–06:00 ] Table 3. 2.4. Feature Construction Definition of the considered time periods Id at the daily level, based on the hour of the day, h. Other temporal levels can easily be incorporated in the feature set, e.g., the seasonal inﬂuence by including four time periods at the annual level corresponding with the seasons. However, this seasonal variation is omitted in this work, as these features were found to not signiﬁcantly impact the clustering result in Section 3.1 and rather obfuscated the results, limiting the ease of interpretation. Based on these time periods at two different temporal levels, a two-pronged approach is introduced in the subsequent subsections. The ﬁrst class of features, which will be discussed in Section 2.4.1, considers the relation between the temporal property and the consumption: which fraction of the demand occurs during a certain predeﬁned time interval? In contrast, the second class of features, discussed in Section 2.4.2, considers the temporal properties of the peak demands: when do these peak demands occur? In order to unambiguously deﬁne which values constitute a peak, the analysis performed on the analytic form of the load duration curve in Section 2.3 is used. The individual features are suitable to characterize consumers, e.g., for assessing household compatibility with renewable energy sources (households with high daytime consumption are more favorable for rooftop-integrated PV installations without a battery), or for the timing of the individual peak demands, which is beneﬁcial information for distribution network operators. However, it is the knowledge on the fraction of the demand combined with the simultaneous occurence or absence of peak demands in that time period that can clarify the stochastic nature of these peak demands. Consumers that consistently exhibit a disproportionate amount of peak demands in a certain time period can be targeted for peak shaving initiatives, either via demand response programs or by utilising an energy storage system. 2.4.1. Temporal Dependence of Consumption 2.4.2. Temporal Dependence of Peak Demands 2.4.2. Temporal Dependence of Peak Demands The features related to the peak demands are treated in a different way than those linked to the consumption. While the amplitude of the demand Pi(t) at a certain point in time is important to determine the fraction of consumption that happens in a time interval, only the presence of peak demands is of importance for the second set of features, not the size of the peaks. Let Di be the subset of all measured demand values Pi(t) of consumer i that can be considered as a peak demand, as deﬁned in Section 2.3. As the LDC is normalized with respect to the annual peak demand Pi max, the value Pi(τ∗) has to be rescaled: Di = Pi(t)\| Pi(t) ≥Pi(τ∗) · Pi max (9) (9) Analogous to the previous section, let Di Ix now be the subset of Di that occurs in time period Ix: Analogous to the previous section, let Di Ix now be the subset of Di that occurs in time period Ix: Di Ix = Pi(t) \| Pi(t) ≥Pi(τ∗) · Pi max ∧t ∈Ix , x ∈{d, w} (10) (10) The number of peak demands per time interval can be found via the cardinality of the set Di Ix, i.e., Di Ix . The fraction of peak demands for consumer i in a certain time period, f i,p Ix , can therefore be found via Equation (11). Analogous to the features related to the temporal aspect of the consumption behavior, this leads to another ﬁve linearly independent features. Consequently, this brings the number of considered linearly independent features for the clustering algorithm up to ten parameters. f i,p Ix = Di Ix Di (11) (11) These 14 features can now describe the temporal behavior and distribution of the consumption and peak demands, as illustrated for one randomly chosen regular household, household 802, in Figure 5. Both the fraction of the consumption and the fraction of peaks are shown for each time period at the daily and the weekly level. Major differences between the distribution describing the consumption and peaks can be observed. At the weekly level, 65% of the household’s peaks are observed in the weekend, while only 35% of the consumption occurs during weekends. 2.4.1. Temporal Dependence of Consumption Let Bi Ix be the subset of all measured values Pi(t) of consumer i that occur in one of the previously deﬁned time periods Ix, with the superscript x denoting the considered temporal level. This yields following deﬁnition of this subset: Energies 2021, 14, 139 10 of 24 Bi Ix = Pi(t) \| t ∈Ix , x ∈{d, w} (7) Bi Ix = Pi(t) \| t ∈Ix , x ∈{d, w} (7) The fraction f i,c Ix of the demand of consumer i in time period Ixi is given by: f i,c Ix = ∑y∈Bi Ix y ∑t Pi(t) (8) (8) This deﬁnition yields a total of seven features: ﬁve features for the daily level, two for the weekly level. However, as the subsets Bi Ix for a given temporal level x are disjoint by construction, the sum of f i,c Ix over all Ix for a ﬁxed x is equal to 1. Therefore, this reduces down to ﬁve linearly independent features: four for the daily level, one for the weekly level. 2.5.1. Clustering Algorithm No additional information or metadata is included in the dataset of load proﬁles. As such, the true underlying structure or the optimal amount of clusters to segmentate the dataset into is unknown. Therefore, unsupervised machine learning is used to cluster those proﬁles that exhibit similar behavior. The majority of the rich body of literature available on the unsupervised clustering of load proﬁles, whether chronologically ordered proﬁles or based on a constructed feature set, is based on one of two techniques: either a variant of the K-means clustering algorithm or via agglomerative clustering. In the structured literature review on the classiﬁcation of consumption proﬁles performed by Tureczek and Nielsen, 65% of the considered papers included a K-means-based method, while another 29% performed analyses based on agglomerative clustering [4]. There are several differences between K-means and agglomerative clustering, both from a conceptual viewpoint, as well as the computational aspect. Agglomerative clustering offers a visualisation in a so-called dendrogram of the clustering results, intuitively showing how substructures in the dataset emerge when dividing or merging clusters. Furthermore, when a feature set is used as input for the agglomerative clustering, further analysis on the merging of clusters offers the possibility of tracking which features are the driving force that distinguish clusters. However, agglomerative clustering is a so-called greedy algorithm: at each step, the two closest clusters as deﬁned by a linkage method are merged together. Therefore, agglomerative clustering techniques are prone to yield a sub-optimal solution instead of a global optimum. In contrast, given an input k, the number of desired clusters, a K-means algorithm partitions the dataset into k clusters. However, K-means tends to get stuck in a local minimum instead of the global minimum. The main challenge for a K-means approach lies in ﬁnding the optimal amount of clusters. From a computational point of view, K-means is preferable for larger datasets as the time complexity for K-means algorithms typically is linear in the input data size, O(n), while the time complexity for agglomerative clustering is quadratic, O(n2). In this work, an agglomerative clustering algorithm with Ward’s linkage method is used, as implemented in Python’s scipy package [28,29]. The main contribution of this work is introducing and validating a novel feature set. Therefore, the visualisation and emergence of substructures in the dataset in the clustering process is of major importance, justifying the choice for an agglomerative clustering algorithm. 2.5. Transformation & Analyses on the Feature Set One additional step is performed on the proposed set of features before proceeding to either the clustering algorithm or the distributional analysis: the feature transformation. De- pending on the proposed methodology for each analysis, a different feature transformation is more appropriate. Therefore, this section discusses both the proposed methodology for each performed analysis, as well as the corresponding most suitable feature transformation. 2.4.2. Temporal Dependence of Peak Demands Similarly, more than 25% of consumption for this consumer happens at night, as deﬁned by Table 3, while 10% of the peak demands lie in this time period. It is this difference between distributions of consumption and peak behavior at the same temporal level that forms the subject of the following sections, as the presence or absence of differences can clarify whether or not peak demands tend to be more stochastic or more deterministic. 11 of 24 11 of 24 Energies 2021, 14, 139 Figure 5. Example of the 14 features describing the temporal behavior of the consumption and peak demands at the daily and weekly level of household 802 in the dataset. Figure 5. Example of the 14 features describing the temporal behavior of the consumption and peak demands at the daily and weekly level of household 802 in the dataset. 2.5.1. Clustering Algorithm The proposed linkage method minimizes the total within-cluster variance for each merging step, which tends to yield approximately equally sized clusters. Following the arguments presented by Kang and Lee in [30], it is a necessary condition for clusters to have a roughly equal size, in order to be useful in real life applications according to expert opinions. Therefore, Ward’s linkage Energies 2021, 14, 139 12 of 24 12 of 24 method can be deemed appropriate, as the tendency of clustering algorithms to propose singular clusters that contain outliers is avoided. method can be deemed appropriate, as the tendency of clustering algorithms to propose singular clusters that contain outliers is avoided. For the proposed feature set, Ward’s linkage method for agglomerative clustering relies on the Euclidean distance between the 10 linearly independent features in the 10- dimensional feature space. Therefore, obtained results will depend on the scale of the input features. However, when looking at both Table 3 and Figure 5 it is clear that the proposed features are not yet at the same scale. By construction, the proposed time periods are not of the same scale, e.g., the weekend period is not the same length as the weekday period, nor is the early morning of similar length as the night interval. Therefore, even a uniform distribution would not lead to similarly scaled features, leading to a distortion of importance of several features. Therefore, an initial transformation is performed that rescales the features based on the length of their time period such that in the case of a uniform distribution, the value of all features f i Ix would be equal to 1. Any deviation of a uniform distribution will then lead to a deviation of this unity value for each parameter, while avoiding an artiﬁcial inﬂation of the importance of an individual feature or one set of features. However, of the ten proposed linearly independent features, eight are deﬁned on daily basis, while only two are deﬁned on weekly basis. While this initially proposed transformation aims to give each individual feature the same weight, the two sets of features deﬁned on different temporal levels are not a priori equally represented in the feature set. 2.5.1. Clustering Algorithm Consequently, instead of transforming the features on weekly basis to be equal to 1 in the case of a uniform distribution, they are assigned an additional weighting factor equal to 2 to partially offset the numerical advantage of daily features. In summary, the two sets of features proposed in Section 2.4 are transformed in a two-step transformation before being used as input for the hierarchical clustering, using a Ward’s linkage method. First, the features are rescaled based on the length of the time interval in which they are deﬁned, which leads to individual features of the same scale. In the second step, an additional weighting factor is assigned based on the amount of features for each temporal level. A weighting factor of 2 is proposed for the weekly-level features, which partially offsets the numerical advantage daily-level features have in the proposed feature set. Further increasing this weighting factor would put a higher emphasis on the difference between weekdays and weekends in the clustering algorithm. 2.5.2. Distribution Analysis Therefore, the features are rescaled based on the length of the interval for which they are deﬁned such that a uniform consumption distribution leads to identical consumption-related features. Furthermore, Equation (12) is deﬁned for probabilities p(x). As such, the features deﬁned on the ﬁve periods at the daily level are rescaled to 0.2, while those deﬁned at the two periods at the weekly level are rescaled to 0.5. The second part of the distributional analysis entails a comparison between the distributions of the consumption behavior and the occurrence of peak demands at the daily and weekly level. As such, a measure for the distance between these two distributions has to be introduced. In this work, the Wasserstein-1 distance is used to characterize the distance metric between two probability distributions [32]. Here, the formal deﬁnition of the Wasserstein-1 distance as integrated in Python’s scipy package is used [29]: l1(p, q) = inf π∈Γ(p,q) Z R×R\|x −y\|dπ(x, y) (13) (13) Here, p and q are two distribution functions, and Γ(p, q) is the set of probability func- tions on R × R whose marginals are p and q on the ﬁrst and second marginals respectively. This Wasserstein distance is also commonly called the earth-mover’s distance, as it origi- nated in the ﬁeld of optimal transport issues. Intuitively, it can be seen as the minimum amount of “work” that has to be done to transform one distribution into the other, if each distribution could be considered as a pile of earth. The “work” takes into account both the distance it has to move, as well as the amount of earth it has to move. As such, distributions P and Q that are different over “long” (horizontal) regions will be far away from each other in the Wasserstein distance sense [33]. It is this property of the Wasserstein distance that is appropriate for this work. As the time periods at the daily level were introduced in an ad hoc way in Table 3, a distance metric that takes the horizontal difference into account instead of performing a pointwise comparison partly compensates the arbitrary nature of the deﬁnition. For a given con- sumption behavior, this allows us to identify distributions of the peak demands that are closer in time. This property is illustrated in Figure 6, where an artiﬁcal distribution of the consumption at the daily level is compared with two peak demand distributions. 2.5.2. Distribution Analysis The distribution of features f i Ix at the daily or weekly level x can yield interesting information. As mentioned before, households with high daytime consumption are ideal candidates for PV installations, whereas households that exhibit a large amount of peak demands in a certain time interval, could be targeted via demand response initiatives. However, it is the difference between the distributions describing the consumption and peak behavior at the daily or weekly level that yields information about the disproportion- ate presence of peak demands at a certain time interval, and thus about how stochastic the presence of peak demands are for an individual household. Therefore, two measures are proposed to investigate these distributions. At the level of the individual distributions, we propose using the concept of entropy at the daily or weekly level to characterize the variability of household behavior. Similar to the goal of this work, Ref. [9] introduced entropy to study the variability of households, not with respect to features based on consumption of peak behavior, but based on the variability of consumption behavior described by the frequency of different representative daily load shapes during the year. Shannon entropy as introduced in information theory is deﬁned in Equation (12), with xi being a possible outcome and p(xi) the probability of this outcome [31]. H(x) = − k ∑ i=1 p(xi) ln p(xi) (12) (12) Energies 2021, 14, 139 13 of 24 13 of 24 This deﬁnition of entropy has several interesting properties for this research. First, in the case of a uniform distribution, the entropy reaches its maximum and thus yields maxi- mum uncertainty. Second, any deviation from a uniform distribution results in a decreasing entropy, and thus less uncertainty. If there is no uncertainty, then the entropy becomes 0. In order for these properties to hold in the analysis of the introduced features, it is important that these features are consistent with the assumptions in the Shannon entropy. First, in the case of a uniform distribution, the entropy becomes maximal. A uniform distribution for the consumption behavior would entail having the same consumption at each time period. p However, as mentioned in Section 2.5.1, the periods as deﬁned in Section 2.4 are not of equal length, which leads to inequal features in the case of a uniform consumption distribution. 2.5.2. Distribution Analysis As the ﬁrst probability distribution of the peak demands has a maximum chronologically closer to that of the normalised consumption, the Wasserstein distance is lower. The chronological ordering of the time periods as given in Figure 6 is chosen for the remainder of this analysis. Energies 2021, 14, 139 14 of 24 Figure 6. Illustration of how the Wasserstein distance is able to capture chronological differences between the probability distributions of consumption and peak demands. Figure 6. Illustration of how the Wasserstein distance is able to capture chronological differences between the probability distributions of consumption and peak demands. 3. Results First, the results for the agglomerative clustering algorithm are illustrated based on the calculated dendrogram. The results for a low number of features are benchmarked to the available synthetic load proﬁles in Flanders, subsequently highlight how differences in feature behavior lead to the emergence of distinct and compact clusters, and argue how this knowledge can be leveraged from the viewpoint of demand response programs or peak shaving initiatives. Second, the distributions of the features at the same time levels are analysed. On the one hand, the Shannon entropy is used to characterize the variability of each type of feature. On the other hand, the Wasserstein-1 distance is used for an in-depth analysis of the stochastic nature of the peak demands, by comparing the distributions describing the household consumption and peak demand behavior. 3.1. Clustering Result The dendrogram visualizing the hierarchical clustering process using Ward’s linkage method on the proposed feature set is shown in Figure 7. Two horizontal cuts are included in the ﬁgure. The black line at y = 30 denotes the height where three clusters are obtained. This can serve as an initial benchmark, as there are three synthetic load proﬁles available for low-voltage consumers in Flanders: residential with and without electric heating, and non-residential. The red line was chosen such that 10 disjoint clusters emerge, leading to the color threshold of the highlighted clusters in the dendrogram. This threshold of 10 clusters was chosen based on two independent studies stating that for practical considerations, the total number of clusters should not exceed 10 [13,30]. This argument is based on the opinions of industrial experts, as these clusters are often used for tarifﬁng or marketing purposes. p p First, it is necessary to benchmark the clustering result to the available residential SLPs in Flanders. As the color threshold and further discussion in this section is based on 10 clusters, the analysis is performed based on the highlighted 10 clusters. By tracking the merging clusters into the three branches of the dendrogram at the cut y = 30, a bench- mark can be performed. Figures 8 and 9, displaying respectively the distributions of the 14 untransformed features for the individual clusters, and the distributions of the yearly consumption of the consumers assigned to each clusters, allow for an interpretation of the obtained clusters based on consumer properties. The ﬁrst branch separates into clusters 1–3, the second into clusters 4–5, while the ﬁnal branch leads to clusters 6–10. The clusters originating from these three branches are partitioned by dashed lines in Figure 8 for an easier comparison. The following discussion on the benchmarking of the results is based on the observed feature distributions in Figure 8. The ﬁrst branch groups consumers with a high fraction of consumption and peaks in the evening, which is typical for regular households. The second branch, containing clusters 4–5, groups consumers with a high fraction of the consumption Energies 2021, 14, 139 15 of 24 15 of 24 and peaks at night. This is encouraging, as this could indicate the presence of electric heating, one of two major categories of residential consumers. and peaks at night. 3.1. Clustering Result This is encouraging, as this could indicate the presence of electric heating, one of two major categories of residential consumers. Figure 7. Dendrogram obtained via hierarchical clustering, with a color threshold highlighting 10 clusters. Individual proﬁles are given on the x-axis, while the y-axis denotes the distance. Figure 7. Dendrogram obtained via hierarchical clustering, with a color threshold highlighting 10 clusters. Individual proﬁles are given on the x-axis, while the y-axis denotes the distance. The interpretation of the third branch is less straightforward, as the properties of the clusters composing this branch are more diffuse: (i) clusters 6–7 group consumers with a disproportionate amount of peaks during the weekend, (ii) cluster 8 collects the consumers with a signiﬁcant amount of peaks during the early morning, whereas (iii) clusters 9–10 exhibit a large number of peaks during the morning and afternoon. Figure 8. Boxplots visualizing the distribution of the 14 untransformed features for 10 clusters, with the 10 features at the daily level displayed on the left and the 4 features at the weekly level on the right. The whiskers of the boxplots describe the [10, 90] percentiles. Figure 8. Boxplots visualizing the distribution of the 14 untransformed features for 10 clusters, with the 10 features at the daily level displayed on the left and the 4 features at the weekly level on the right. The whiskers of the boxplots describe the [10, 90] percentiles. 16 of 24 16 of 24 Energies 2021, 14, 139 Figure 9. Density histograms with shared y-axes, displaying the distribution of the yearly consumption of the individual consumers assigned to each of the 10 considered clusters. The black plot denotes the density for the individual cluster, while the plot in yellow indicates the density of the full dataset. Figure 9. Density histograms with shared y-axes, displaying the distribution of the yearly consumption of the individual consumers assigned to each of the 10 considered clusters. The black plot denotes the density for the individual cluster, while the plot in yellow indicates the density of the full dataset. It is clear that for each time period and in the same branch of the dendrogram, the differences between the fractions of total consumption for that period are limited. Rather, the temporal behavior of the peak demands is the driving force to further separate clusters in each of the three major branches of the dendrogram. 3.1. Clustering Result Furthermore, the clustering process yields compact clusters with comprehensive results. This illustrates the usefulness of a feature set that includes the temporal properties of peak demands, especially with the advent of capacity-based tariff schemes for low-voltage consumers. With the introduction of capacity-based tariffs, it is no longer sufﬁcient to know when consumption occurs. Additional knowledge about when peak demands tend to happen is vital to offer consumers the most suitable techno-economic solution. As a post-hoc validation of the performance of the proposed feature set in determining customer categories, the clusters of the different consumer types in the dataset as introduced in Section 2.1 are determined and given in Table 4. Clusters 4 and 5 are predominantely populated by households with electric heating, while cluster 10 groups households with high daytime consumption as well as the majority of the SMEs. However, not all proﬁles with electric heating are categorised inside clusters 4–5. This is further investigated in Figure 9, which displays the density plots of the yearly consumption for each individual cluster compared to the density plot of the full dataset. Matched against the density plot of the complete dataset, clusters 4, 6, 8 and 10 are skewed towards households with lower to average yearly consumption in the Eurostat classiﬁcation. This distribution for cluster 4 is expected and can clarify the diffusion of households with electric heating over different clusters. As the demand proﬁles of these households can be considered an aggregation of the proﬁle of a regular household with a load proﬁle of an electric heating appliance, the features connected to the peak demands are intrinsically linked to the behavior of that load proﬁle and the timing of the peak demands without the electric heating. The heating load proﬁle for households with otherwise relatively low yearly consumption dominates the aggregated load proﬁle, and consequently encounter the majority of their consumption peaks during the night, consistent with the behavior of cluster 4. For households with electric heating in e.g., cluster 3, the consumption and peak demands during the evening outweigh those during the night. Energies 2021, 14, 139 17 of 24 Table 4. Relative frequency of consumer categories over the 10 different clusters. 3.1. Clustering Result # of Proﬁles 1 2 3 4 5 6 7 8 9 10 SME 20 0% 5% 0% 0% 0% 0% 0% 10% 10% 75% Ripple control heating 107 2% 6% 6% 18% 36% 9% 20% 2% 2% 0% Continuous heating 77 14% 8% 15% 11% 31% 1% 10% 4% 4% 0% Air conditioning 18 17% 17% 6% 0% 6% 0% 33% 6% 17% 0% Regular consumer 1206 17% 25% 5% 0.1% 1% 7% 17% 8% 16% 5% Table 4. Relative frequency of consumer categories over the 10 different clusters. It can be concluded that the proposed feature set is able to capture the known con- sumer categories from existing SLPs, and thus passes our self-imposed benchmark test. Three clusters can be attributed to known differences in behavior for low-voltage con- sumers: the presence of electric heating is captured in clusters 4–5, while the high daytime consumption of SME proﬁles is present in cluster 10. Deviations from these two clusters for electric heating can be traced back to differing contributions of the electric heating load to the total yearly consumption of the households. 3.2. Stochastic Nature of Peak Demands The variability of the daily and weekly consumption and peak patterns are described by the entropy of their probability distribution, where the individual fractions are nor- malized with respect to the length of the considered time period. A uniform distribution with maximum uncertainty leads to a maximal value of the entropy, while the absence of uncertainty leads to an entropy value of 0. For example, a situation where all peak demands occur during the night due to an electric heating would lead to 0 entropy at the daily level for the consumption probability distribution. The obtained distributions for the entropy at the daily and weekly level for the consumption and peak probability distributions of the full dataset are given in Figure 10. At the daily level, the peak demands exhibit a much larger variability than the consumption. This is unsurprising, given the continuous nature of the consumption. At the weekly level, this difference is less pronounced. Figure 10. Density histograms of the entropy of the consumption and peak demands at the daily and weekly level, with a ﬁtted beta probability density function overlaid in yellow. Figure 10. Density histograms of the entropy of the consumption and peak demands at the daily and weekly level, with a ﬁtted beta probability density function overlaid in yellow. A beta distribution was successfully ﬁtted to each individual density histogram. The 2-parameter beta probability distribution, deﬁned on the interval [0,1], is deﬁned as follows, with a > 0 and b > 0: f (x, a, b) = Γ(a, b)xa−1(1 −x)b−1 Γ(a)Γ(b) (14) (14) Energies 2021, 14, 139 18 of 24 18 of 24 The beta function offers several properties that make it suitable to describe the ob- tained distributions. First, it has a ﬁnite support: the regular 2-parameter beta function in Equation (14) has a [0,1] support. As the entropy can vary from 0 to a maximum of −ln(0.2) for the daily level and −ln(0.5) for the weekly level, the ﬁnite support of a rescaled and shifted beta function is appropriate. Second, as can be observed in Figure 10, the shapes of the daily and weekly behaviors differ signiﬁcantly. The two shape parameters a and b in the deﬁnition of the beta probability function allow us to describe the four distributions with the same formula. 3.2. Stochastic Nature of Peak Demands For the distributions shown in Figure 10, it merely means that b > 1 for the distributions at the daily level, while b < 1 for those at the weekly level. y y The relation between the entropy and the clusters obtained in Section 3.1 is inves- tigated in Figure 11, which displays the mean values of the entropy for each individual cluster. The signiﬁcantly lower entropy of the probability distribution describing the peak demands can be traced back to the clustering results. The overwhelming presence of peak demands during the night period results in low entropy for cluster 4, while cluster 10 exhibited a majority of its peaks during daytime. Similarly, half of the peak demands for cluster 1 occurred during the evening. On a weekly basis, clusters 6–7 showed a signiﬁcant amount of peak demands during the weekend, leading to a lower entropy for this period. A low entropy of the probability function describing the peak demands can be taken as an indicator for the presence of a large amount of peaks in a certain time period, which can be leveraged to target demand response programs or peak shaving via an energy storage system. Furthermore, a clear relation can be observed between the obtained clusters on the introduced feature set and the entropy values of the peak demands. The lower entropy values in certain clusters can be traced back to differing intercluster consumer operations at the daily or weekly level. Figure 11. Mean values of the calculated entropy of the distributions of the normalized consumption and peak demands at the daily and weekly level of the individual consumers populating each cluster. Figure 11. Mean values of the calculated entropy of the distributions of the normalized consumption and peak demands at the daily and weekly level of the individual consumers populating each cluster. However, the stochastic nature of these peak demands remains an open question. The probability distributions of the peak demands tend to be signiﬁcantly more variable than those of the consumption behavior, according to the entropy. Even so, this entropy as a single variable does not reveal anything about whether or not the amount of peaks in a certain time period is disproportional relative to the consumption in that time period. 3.2. Stochastic Nature of Peak Demands Therefore, the Wasserstein-1 distance is used to quantify the difference between the probability distributions of the consumption and peak demands at the daily and weekly level for each individual consumer. A larger distance corresponds to a stronger deviation of the peak distribution from the distribution of the consumption, and thus peaks are more deterministic. Figures 12 and 13 yield the distributions for the Wasserstein-1 distances at the daily and weekly level, separated by individual cluster. Analogous to Figure 9, the distribution of the Wasserstein-1 distance calculated for each proﬁle in the full dataset is included for comparison to cluster-speciﬁc behavior. 19 of 24 19 of 24 Energies 2021, 14, 139 Figure 12. Histograms of the Wasserstein-1 distance between the distributions of the consumption and peak demands probability functions at the daily level. The black plot denotes the density for the individual cluster, while the plot in yellow indicates the density of the full dataset. Figure 12. Histograms of the Wasserstein-1 distance between the distributions of the consumption and peak demands probability functions at the daily level. The black plot denotes the density for the individual cluster, while the plot in yellow indicates the density of the full dataset. Figure 13. Histograms of the Wasserstein-1 distance between the distributions of the consumption and peak demands probability functions at the weekly level. The black plot denotes the density for the individual cluster, while the plot in yellow indicates the density of the full dataset. Figure 13. Histograms of the Wasserstein-1 distance between the distributions of the consumption and peak demands probability functions at the weekly level. The black plot denotes the density for the individual cluster, while the plot in yellow indicates the density of the full dataset. The distributions of the Wasserstein-1 distances further conﬁrm the ﬁndings concern- ing the behavior of consumers constituting each cluster. At the weekly level, clusters 6 and 7 show a major deviation from the dataset behavior, due to the presence of a dispropor- tionate amount of peak demands in the weekend. Similarly for the daily level, cluster 4 displays a large Wasserstein-1 distance, pointing to the electric heating which pushes nearly all peak demands to nighttime. Clusters 1 and 2 exhibited similar behavior for their consumption at the daily level in Figure 8. 3.2. Stochastic Nature of Peak Demands However, households in cluster 1 are characterized by an even higher amount of peak demands in the evening than those in cluster 2, translating to a higher than average Wasserstein-1 distance for cluster 1 at the daily level. This variability and disproportionate amount of peaks in a certain time interval offers insight in possibilities for targeted demand Energies 2021, 14, 139 20 of 24 20 of 24 response initiatives or peak shaving via a residential energy storage system. While cluster 6– 7 and 8–9 have a similar consumption pattern, the time of occurrence of peak demands is signiﬁcantly different, which leads to distinct solutions. response initiatives or peak shaving via a residential energy storage system. While cluster 6– 7 and 8–9 have a similar consumption pattern, the time of occurrence of peak demands is signiﬁcantly different, which leads to distinct solutions. As peak demands are typically generated by the simultaneous use of individual appli- ances, targeted demand response initiatives can be effective for cluster 6 and 7, where the majority of peaks occurs in the weekend. Scattering the use of individual appliances over different days or being mindful of the simultaneous use in the weekend by inducing behavioral changes can reduce the number of peak demands. However, this requires a trigger for the behavioral changes and for these appliances to be available in different time periods. If this is not an option, investing in an energy storage system applying a peak shaving algorithm during weekends, while e.g., maximising the PV self-consumption during weekdays could offer an alternative, although the economic viability depends on the local tariff structure and the investment cost. In contrast, cluster 8 is characterized by peak demands in the early morning and during the daytime, while households in cluster 9 exhibit peaks during the whole day. Consequently, for these households, a PV installation combined with a storage system can already offer a solution to reduce the demand from the grid, while maintaining a high self-consumption. As a ﬁnal check on the stochastic nature of peak demands, the relationship between the consumption in a time period and the presence of peak demands is investigated. Figure 14 displays the relations between the (untransformed) fractions of the consumption and peak demands at the daily level, with an ordinary least-squares (OLS) regression ﬁt overlaid given the observed linear relation. 3.2. Stochastic Nature of Peak Demands The coefﬁcients obtained in the OLS regres- sion for f p = a × f c + b, with f p and f c the fraction of respectively the peak demands and consumption in that time period, are given in Table 5. As the presence of electric heating heavily skewed previous results for the consumption and peak demands at night, consumers with and without electric heating are treated separately for this analysis. A correlation between the fraction of the consumption and that of the peak demands is present in Figure 14 and 15. As the presence of consumption in a certain time period is a prerequisite for a peak demand, some relation between the two types of parameters was expected. At ﬁrst sight, the linear relation could be interpreted as an indication of predictability of peak demands in a certain time period. However, it is the spread on this relation that is the indicator of the stochasticity of the peak demands. For example, if 30% of a household’s total consumption is observed occurring during the evenings, the results shown in Figure 14 suggests that 30–60% of the peak demands can occur in this same time period. This large uncertainty, which is present for each of the considered time periods, severely limits the usability of this linear relation, observed for the full dataset. Figure 14. Relation between the fraction of consumption and peak demands in the time periods at the daily level, with an OLS regression estimate overlaid. Figure 14. Relation between the fraction of consumption and peak demands in the time periods at the daily level, with an OLS regression estimate overlaid. 21 of 24 Energies 2021, 14, 139 Figure 15. Relation between the fraction of consumption and peak demands in the time periods at the weekly level, with an OLS regression estimate overlaid. Figure 15. Relation between the fraction of consumption and peak demands in the time periods at the weekly level, with an OLS regression estimate overlaid. However, the knowledge of the introduced clusters can partly alleviate this uncertainty. This is illustrated in Figure 16 for clusters 1–3, which group households with a large fraction of their consumption during the evening, with a high number of peak demands simultaneously occurring in this time period. 4. Conclusions The introduction of capacity-based tariffs poses a major challenge for residential load modeling. The construction of representative load proﬁles traditionally involves an averaging process. However, this involuntarily leads to less volatile proﬁles and time-sensitive information about the peak demands is smoothened out. Consequently, we set out to construct a new feature set that would be able to capture the stochastic behavior of the peak demands. An expression for the load duration curve of individual low-voltage consumers was initially validated. Using the point of maximum curvature of the exponential decay as threshold, it was possible to deﬁne the individual’s region of peak demands. p Two types of features were subsequently constructed. First, the fraction of consump- tion that occurs in a certain time period at the daily or weekly level. Second, the fractions of peak demands that occur in these same periods. The proposed feature set was used in a hierarchical clustering process to build 10 clusters from a dataset of 1.422 proﬁles of low-voltage consumers from a suburban region in Flanders. The clustering algorithm yielded compact clusters that showed a clear connection to real-life applications concerning the peak demands such as demand response initiatives, or the applicability of e.g., battery storage systems for peak shaving purposes. Furthermore, differences in the behavior of the peak demands were found to be the main drivers of the clustering procedure. The presence of electric heating could be identiﬁed for several clusters, while others exhibited high daytime consumption during weekdays, which is typical for SMEs. In the ﬁnal analysis of this work, the stochastic nature of the peak demands was investigated by considering the relation between the consumption and the presence of peak demands in the same time period. The disproportionate presence of peak demands in a certain time period was quantiﬁed, and a linear relation was observed between the fraction of the consumption and peak demands in each time period. The spread on the results quantiﬁed the stochasticity of the peak demands, which limited the general applicability of the found relations. The obtained clusters showed a clear relation to the predictability and variability of the consumption and peak behavior, reducing the stochasticity of these peak demands and when they tend to occur. The constructed feature set and performed clustering algorithm have several impli- cations for the integration and application of new technologies on the low-voltage grid, which can be further investigated. 3.2. Stochastic Nature of Peak Demands While we should be cautious drawing conclusions based on clusters that only include a limited amount of households, it appears that the spread on the fraction of peak demands for the individual clusters is smaller than those in Figure 14 for the full dataset, while the linear correlation that was observed before is nearly non-existent in some relations. Figure 16. Relation between the fraction of consumption and peak demands in three time periods at the daily level for clusters 1–3. Figure 16. Relation between the fraction of consumption and peak demands in three time periods at the daily level for clusters 1–3. Figure 16. Relation between the fraction of consumption and peak demands in three time periods at the daily level for clusters 1–3. Energies 2021, 14, 139 22 of 24 Table 5. Coefﬁcients of the OLS regressions shown in Figures 14 and 15 at the daily and weekly level. Time Period a b Early morning 1.88 −0.11 Morning 2.58 −0.22 Afternoon 2.34 −0.31 Evening 2.53 −0.33 Night (regular consumer) 1.41 −0.21 Night (electric heating) 1.99 −0.33 Weekday 3.06 −1.50 Weekend 3.06 −0.56 4. Conclusions Abbreviations The following abbreviations are used in this manuscript: SME Small and medium-sized enterprises 4. Conclusions First, knowledge about the time of occurrence of peak demands throughout the day and week allows for value stacking of residential energy stor- age systems combined with PV installations for capacity-based tariffs. This can be achieved by e.g., performing a control strategy that maximizes the PV self-consumption during the week, but applies a peak shaving algorithm during the weekend if peaks predominantly occur then. Second, the integration of electric vehicles for low-voltage consumers can be further investigated. Optimal charging schemes can be suggested based on periods where the household is typically occupied, but peak demands are absent. This could avoid additional costs related to higher peak demands for capacity-based tariffs. In both Energies 2021, 14, 139 23 of 24 23 of 24 cases, the clustering result can be used to propose generic strategies for consumers with similar intracluster behavior. However, future experimental research and knowledge of upcoming capacity-based tariffs are both necessary to validate the economic and technical feasibility of these applications. A ﬁnal possible research direction is related to the use of stochastic load models. The obtained relation between the fraction of consumption and peak demands in certain time periods can be used to validate and improve existing stochastic load models. This could allow for more accurate stochastic modeling of the temporal dependency of peak demands. Author Contributions: Conceptualization, R.C. (Robbert Claeys) and J.D.; methodology, R.C. (Robbert Claeys), R.C. (Rémy Cleenwerck) and J.D.; software, R.C. (Robbert Claeys); validation, R.C. (Robbert Claeys), H.A. and J.D.; formal analysis, R.C. (Robbert Claeys); investigation, R.C. (Robbert Claeys); data curation, J.D.; writing—original draft preparation, R.C. (Robbert Claeys); writing—review and editing, R.C. (Robbert Claeys), H.A., R.C. (Rémy Cleenwerck), J.K. and J.D.; visualization, R.C. (Robbert Claeys) and R.C. (Rémy Cleenwerck); supervision, J.K. and J.D. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: The authors would like to thank the Flemish distribution system operator Fluvius for providing the dataset of consumption proﬁles. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. VREG. 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W3007558289.txt	http://www.ajnr.org/content/ajnr/41/4/658.full.pdf	en		The FRED for Cerebral Aneurysms of the Posterior Circulation: A Subgroup Analysis of the EuFRED Registry	American journal of neuroradiology	2,020	cc-by	3,880	ORIGINAL RESEARCH INTERVENTIONAL The FRED for Cerebral Aneurysms of the Posterior Circulation: A Subgroup Analysis of the EuFRED Registry C.J. Griessenauer, M.A. Möhlenbruch, P. Hendrix, C. Ulfert, C. Islak, M. Sonnberger, T. Engelhorn, E. Müller-Thies-Broussalis, T. Finkenzeller, M. Holtmannspötter, J.-H. Buhk, W. Reith, A. Simgen, H. Janssen, N. Kocer, and M. Killer-Oberpfalzer ABSTRACT BACKGROUND AND PURPOSE: Flow diversion for the posterior circulation remains a promising treatment option for selected posterior circulation aneurysms. The Flow-Redirection Intraluminal Device (FRED) system has not been previously assessed in a large cohort of patients with posterior circulation aneurysms. The purpose of the present study was to assess safety and efﬁcacy of FRED in this location. MATERIALS AND METHODS: Consecutive patients with posterior circulation aneurysms treated at 8 centers participating in the European FRED study (EuFRED) between April 2012 and January 2019 were retrospectively reviewed. Complication and radiographic and functional outcomes were evaluated. RESULTS: Eighty-four patients (median age, 54 years) with 84 posterior circulation aneurysms were treated with the FRED. A total of 25 aneurysms (29.8%) had previously ruptured, even though most aneurysms were diagnosed incidentally (45.2%). The intradural vertebral artery was the most common location (50%), and saccular, the most common morphology (40.5%). The median size was 7 mm. There were 8 (9.5%) symptomatic thromboembolic and no hemorrhagic complications. Thromboembolic complications occurred mostly (90.9%) in nonsaccular aneurysms. On last follow-up at a median of 24 months, 78.2% of aneurysms were completely occluded. Functional outcome at a median of 27 months was favorable in 94% of patients. All mortalities occurred in patients with acute subarachnoid hemorrhage and its sequelae. CONCLUSIONS: The largest cohort of posterior circulation aneurysms treated with the FRED to date demonstrated favorable safety and efﬁcacy proﬁles of the device for this indication. Treatment in the setting of acute subarachnoid hemorrhage was strongly related to mortality, regardless of whether procedural complications occurred. F low diversion for aneurysms of the posterior circulation continues to be more controversial than other endovascular treatment modalities in that location. Whereas it is established in the anterior circulation, flow diversion in the posterior circulation remains “off-label” according to the FDA, which has not granted approval to any flow-diverting device for that indication. Still, flow-diverting stents are used at increasing frequency in those locations.1-3 The Received October 23, 2019; accepted after revision January 14, 2020. From the Research Institute of Neurointervention (C.J.G., E.M.-T.-B., M.K.-O.), Paracelsus Medical University, Salzburg, Austria; Department of Neurosurgery (C.J.G.), Geisinger, Danville, Pennsylvania; Department of Neuroradiology (M.A.M., C.U.), Heidelberg University Hospital, Heidelberg, Germany; Department of Neurosurgery (P.H.), Saarland University Medical Center and Saarland University Faculty of Medicine, Homburg/Saar, Germany; Department of Neuroradiology (C.I., N.K.), Cerrahpasa Medical School, Istanbul University, Istanbul, Turkey; Department of Neuroradiology (M.S.), Kepler Universitätsklinikum, Linz, Austria; Department of Neuroradiology (T.E.), University Hospital Erlangen, Erlangen, Germany; Institute of Radiology and Neuroradiology (T.F., M.H.), Klinikum Nuernberg Sued, Paracelsus Medical University, Nuernberg, Germany; Department of Neuroradiology (T.F.), Klinikum Weiden, Weiden, Bavaria, Germany; Department of Neuroradiology (J.-H.B.), University Hospital Hamburg Eppendorf, Hamburg, Germany; Clinic for Diagnostic and Interventional Neuroradiology (W.R., A.S.), Universitätsklinikum des Saarlandes, Homburg/Saar, Homburg, Saarland, Germany; and Institute for Neuroradiology (H.J.), Klinikum Ingolstadt, Ingolstadt, Germany. Due to equal contributions, the 2 last authors share senior authorship. 658 Griessenauer Apr 2020 www.ajnr.org largest studies on posterior circulation flow diversion are currently available for the Pipeline Embolization Device (PED; Covidien, Irvine, California).4,5 Aneurysms of the vertebral artery have been found to be particularly amenable to flow diversion.5,6 Larger cohorts of other flow diverters used in the posterior circulation are currently lacking. Here, we performed a subgroup analysis of posterior circulation cases treated with the Flow-Redirection Endoluminal Device (FRED; MicroVention, Tustin, California) at centers participating in the European FRED study (EuFRED).7 MATERIALS AND METHODS Study Design and Data Collection The EuFRED study was a retrospective, multicenter postmarket registry of consecutive patients with intracranial aneurysms treated with the FRED at European high-volume neurovascular Please address correspondence to Christoph J. Griessenauer, MD, Department of Neurosurgery, Geisinger, 100 N Academy Ave, Danville, PA 17822; e-mail: christoph.griessenauer@gmail.com; @cgriessenauer Indicates article with supplemental on-line table. http://dx.doi.org/10.3174/ajnr.A6447 FIG 1. A 66-year-old woman presenting with an unruptured left vertebral artery aneurysm measuring 5 mm in maximum diameter. A, Lateral view of a left vertebral artery digital subtraction angiogram. B, Rotational 3D reconstruction. C, Angiogram post-FRED placement (arrows show the proximal and distal ends of the ﬂow diverter). D, Stasis in the aneurysm after ﬂow-diverter placement. E, Conebeam CT with contrast 1 month after stent placement shows complete occlusion of the aneurysm. F, Conﬁrmation of aneurysm occlusion at 1 year. centers as previously described.7 The decision to use the FRED was at practitioner discretion to reflect real-world indications. An independent study nurse not involved in the clinical decisionmaking collected all raw data of the original EuFRED dataset. For this subgroup analysis of the posterior circulation aneurysms treated, 8 centers that had treated posterior circulation aneurysms provided additional consecutive cases up to January 2019. Aneurysms were classified according to the morphology as saccular, fusiform, dissecting, and blister/pseudoaneurysm with and without intraluminal thrombus, with the maximum diameter measured in millimiters. The categorization of aneurysm morphology was performed at the individual centers and in accordance with prior classification of posterior circulation aneurysms treated with flow diversion.5 Details on platelet function testing and antiplatelet regimen were obtained. Complications were classified as thromboembolic, hemorrhagic, and other and divided in symptomatic and asymptomatic. Clinical and imaging follow-up time points were at the discretion of the participating center as was the imaging technique. Angiographic outcome was evaluated locally at the individual center by a radiologist not involved in the procedure and graded as completely occluded, near-completely occluded with neck remnant, or incompletely occluded. Functional outcomes were assessed locally and by using the mRS. Internal review board approval was obtained at all participating institutions, and all patients signed approved consent forms. Statistical Analyses Continuous variables are presented as median and range, and categoric variables are presented as frequency and percentage. Analyses were performed using x 2 and Fisher exact tests. P values , .05 were considered statistically significant. RESULTS Baseline Patient and Aneurysm Characteristics A total of 84 aneurysms in 84 patients (median age, 54 years) were treated with the FRED. All patients with electively treated aneurysms had a good preprocedural functional status (mRS, 0–2). A history of aneurysm rupture was present in 25 (29.8%) aneurysms, with 12 aneurysms (14.3%) treated in the acute phase (within 48 hours) of aneurysm rupture. The intradural vertebral artery (50%) (Fig 1) and basilar artery (26.1%) were the most common locations of the aneurysm. The aneurysms were saccular (40.5%), fusiform (25.0%), dissecting (22.6%), or blister (11.9%) and had a median maximum diameter of 7 mm (range, 2–67 mm). Intraluminal thrombus was present in 8.3% of aneurysms (Table 1). Treatment Characteristics and Outcomes Most aneurysms were treated with a single FRED (95.2%). Adjunctive coiling was performed in 16.7%. Platelet function testing was available in 95.2% of cases and resulted in a regimen change in 2.4%. The most common postprocedural antiplatelet medication regimen was aspirin and clopidogrel (86.9%). Of the 12 aneurysms treated in the acute setting of subarachnoid hemorrhage, 9 patients (75%) were started on tirofiban and transitioned to aspirin and clopidogrel (8 patients) or aspirin and ticagrelor (1 patient), while 2 (16.7%) were loaded with aspirin and clopidogrel, and 1 (8.3%), with aspirin and ticagrelor. There were 11 (13.1%) thromboembolic infarctions, most frequently affecting the cerebellum and occurring mostly in nonsaccular aneurysms (90.9%). Symptomatic infarctions occurred in 8 (9.5%) patients. Imaging follow-up was available for 77 (91.7%) patients. Imaging modalities included MRA (50%), DSA (45.6%), conebeam CT with IV contrast (2.9%), and CTA (1.5%). At a median follow-up of 24 months, 78.2% of aneurysms were completely and 9% nearcompletely occluded. Retreatment with another flow diverter was performed in 7.1% of patients (Fig 2). Functional outcome at a median of 27 months was favorable in 94% of patients (mRS, 0–2) and available for 98.8% of patients. There were 5 (6%) mortalities, all occurring in patients treated in the acute setting of subarachnoid hemorrhage. In one of those patients, mortality was due to a thromboembolic infarction from the flow-diversion procedure (Table 2). AJNR Am J Neuroradiol 41:658–62 Apr 2020 www.ajnr.org 659 Table 1: Baseline characteristics Parameter No. of patients No. of aneurysms Females Median age (range) (yr) Pretreatment mRS of patients without SAH 0 1 2 3–5 Index aneurysm presentation Acute SAH (within 48 hours) ,2 weeks since SAH .2 weeks since SAH Stroke Brain stem compression Brain stem infarction Incidental Othera Aneurysm location Vertebral artery (intradural) Basilar artery Posterior cerebral artery Superior cerebellar artery Vertebral artery (extradural) Posterior inferior cerebellar artery Vertebrobasilar junction Aneurysm morphology Saccular Fusiform Dissecting Blister/pseudoaneurysm Index aneurysm pretreatedb Aneurysm size (median) (range) (mm) Intra-aneurysmal thrombus No. 84 84 42 (50.0%) 54 (15–81) 58 53 (91.4%) 4 (6.9%) 1 (1.7%) 0 (0.0%) 12 (14.3%) 6 (7.1%) 7 (8.3%) 2 (2.4%) 5 (6.0%) 2 (2.4%) 38 (45.2%) 12 (14.3%) 42 (50%) 22 (26.1%) 7 (8.3%) 4 (4.8%) 4 (4.8%) 3 (3.6%) 2 (2.4%) 34 (40.5%) 21 (25.0%) 19 (22.6%) 10 (11.9%) 7 (8.3%) 7 (2–67) 7 (8.3%) Predictors of Complication and Occlusion Status at Last Follow-Up The small number of cases with complications and incomplete occlusion status limited the ability to perform statistical analysis. All thromboembolic complications occurred in aneurysms not treated with adjunctive coiling, albeit this relationship did not reach statistical significance. All patients with thromboembolic complications had undergone platelet function testing, and no adjustments to the antiplatelet regimen were warranted. There were no associations of occlusion status or complications with any of the recorded variables (On-line Table). DISCUSSION The present cohort is the largest reported on the FRED for aneurysms of the posterior cerebral circulation. The FRED was successfully implanted in 84 patients with 84 aneurysms, most commonly located along the intradural vertebral artery. Symptomatic thromboembolic complications occurred in 9.5% of patients, whereas no hemorrhagic complications were reported. Complete or near-complete occlusion was achieved in 87.2% of aneurysms at a median follow-up of 24 months. At a median clinical follow-up of 27 months, all living patients had a favorable functional outcome. Mortalities occurred only in patients treated in the acute setting of subarachnoid hemorrhage. The Flow-Redirection Intraluminal Device The FRED is a dual-layer braided device with an inner low-porosity stent that acts as the flow diverter and an outer part that serves as a scaffold for the inner stent. The EuFRED study with 579 aneurysms treated with the FRED is the largest to date. In aneurysms followed for .1 year, complete occlusion was a Other Headache (n = 7), vertigo (n = 1), syncope (n = 1), TIA (1), dizziness (n = 1), achieved in 95.3%. Transient morbidity, permanent morbidity, Traumatic brain injury (n = 1). b Pretreatment endovascular (n = 6), endovascular, and open microsurgery and mortality rates were 3.2%, 0.8%, and 1.5%, respectively.7 (n = 1). The Italian core lab–adjudicated FRED registry included 165 aneurysms.8 Device-related morbidity and mortality occurred in 6.1% and 2.4% of patients. Complete or near-complete occlusion at 12 to 24 months was 77% and 19%, respectively. The study also included 29 aneurysms in the posterior circulation. Even though this subgroup was not discussed in detail, posterior circulation aneurysms contributed to mortality in particular. Among the 6 delayed deaths, 3 occurred in basilar artery aneurysms.8 The Safety and efficacy Analysis of FRED Embolic device in aneurysm treatment (SAFE) study is the third largest on FRED, enrolling 103 aneurFIG 2. A 68-year-old male patient who had an SAH with a Hunt and Hess score of 3 (left). ysms; posterior circulation aneurysms Diagnostic angiography reveals a 3-mm dissecting aneurysm of the basilar artery at the origin of were not included.9 the anterior inferior cerebellar artery (middle). Implantation of a FRED into the basilar artery with In the United States, the FRED is good wall apposition and complete coverage of the aneurysm neck and anterior inferior cerebellar artery origin was achieved. The patient was administered tiroﬁban for the ﬁrst 24 hours after not approved currently. Still, in a comimplantation (right). During recovery, the patient presented with cranial nerve IX to XII palsies parative study of EuFRED anterior and slight hemiparesis on the left. MR imaging showed only minimal DWI lesions in both cerebelcirculation aneurysms matched to lar hemispheres (MRI not shown). Diagnostic angiography 7 days after the primary intervention aneurysms treated with the PED at US revealed occlusion of the aneurysm and occlusion of the anterior inferior cerebellar artery. The centers, both safety and efficacy profiles patient recovered to a mRS 2 after 3 months. 660 Griessenauer Apr 2020 www.ajnr.org Table 2: Treatment characteristics and outcomes Parameter No. of FREDs per aneurysm 1 FRED 2 FREDs 4 FREDs Adjunctive coiling required Platelet function testing Antiplatelet regimen changed after testing Postprocedural antiplatelet regimen ASA þ clopidogrel Tiroﬁban loading followed by ASA þ clopidogrel ASA þ ticagrelor Prasugrel ASA þ prasugrel ASA þ tiroﬁban þ ticagrelor Thromboembolic infarction due to procedure Symptomatic thromboembolic complication Location of infarction Cerebellum Brain stem Posterior cerebral artery territory Thalamus Hemorrhagic complications Other complications Parent artery occlusion Time of radiographic follow-up (median) (range) (mo)a Occlusion status last imaging follow-upa Complete occlusion (100%) Near-complete occlusion (.90%) Incomplete occlusion (,90%) Imaging techniqueb MRA DSA Conebeam CT with IV contrast CTA Retreatmentc Time of functional outcome follow-up (median) (range) (mo)d Functional outcomee mRS 0–2 mRS 3–5 mRS 6 No. 80 (95.2%) 3 (3.6%) 1 (1.2%) 14 (16.7%) 80 (95.2%) 2 (2.4%) 62 (73.8%) 11 (13.1%) 6 (7.1%) 3 (3.6%) 1 (1.2%) 1 (1.2%) 11 (13.1%) 8 (9.5%) 7 (8.3%) 2 (2.4%) 1 (1.2%) 1 (1.2%) 0 (0.0%) 3 (3.6%) 2 (2.4%) 24 (0.03–72) 61 (78.2%) 7 (9%) 10 (12.8%) 34 (50%) 31 (45.6%) 2 (2.9%) 1 (1.5%) 6 (7.1%) 27 (0.03–72) 78 (94%) 0 5 (6%) Note:—ASA indicates acetylsalicylic acid. Data missing in 6 patients. b Data missing in 16 patients. c Data missing in 1 patient. d Data missing in 6 patients. e Data missing in 1 patient. a of both flow diverters were comparable.10 To overcome the limitation of short angiographic follow-up of 6.6 months in the initial EuFRED publication,7 we updated both angiographic and clinical follow-up for the current study. With median follow-ups of 24 and 27 months, respectively, this study also presents some of the longest follow-up data on the FRED.8,9 Occlusion of Posterior Circulation Aneurysms with Flow Diverters Per FDA regulations, flow diversion in the posterior circulation continues to be considered off-label. Nevertheless, flow diversion is increasingly used for posterior circulation aneurysms with most data available for the PED.4-6,11 The largest single-center series on posterior circulation aneurysms treated with either the PED or the p64 (phenox, Bochum, Germany) included 58 nonsaccular aneurysms.11 The cohort represents the most challenging aneurysm morphologies in the posterior circulation.6 Angiographic occlusion or minor remnants were reported in 100% (5/5) of dolichoectatic, 95.5% (21/22) of fusiform, and 63.2% (12/19) of transitional morphology at a mean follow-up of 25.2 months. Up to 8.3 flow diverters were used, on average, in fusiform basilar artery aneurysms. A metaanalysis of nonsaccular posterior circulation aneurysms treated with flow diverters reported the average device number at 4.33 per aneurysm.6 In contrast, the present study included 50 nonsaccular aneurysms classified as fusiform, dissecting, and blister, with a median diameter of 7 mm. The median number of FREDs used was 1. Among patients with angiographic follow-up and nonsaccular aneurysms, the rates of complete and near-complete occlusion were 81.8% and 4.6%, respectively, at 18.5 months. Given the complexity of these lesions, however, any comparison must be interpreted with caution. Still, data are promising in terms of occlusion of some of the most challenging cerebral aneurysms. The second largest single-center series was published in 2019, including 55 aneurysms treated with the PED.4 The classification of aneurysms was similar to that in the current study as saccular, fusiform, and dissecting/pseudoaneurysm. One PED was used in 84% of procedures. Angiographic follow-up was short, with only 8 aneurysms imaged at 24 months. On last follow-up, however, complete occlusion or trace filling was achieved in 72% and 9%, respectively, which is comparable with the present study, especially given the shorter follow-up. The largest series on posterior circulation flow diversion was a multicenter study, including 131 aneurysms, all treated with the PED.5 At a median of 11 months, complete or near-complete occlusion was achieved in 78.1% of aneurysms, with dissecting and fusiform aneurysms occluding most and least frequently, respectively. A predictor of occlusion in dissecting aneurysms was age, with older aneurysms occluding less often, a relationship that has also been demonstrated for anterior circulation aneurysms after flow diversion.12 In fusiform aneurysms, no predictors of occlusion were identified. Assessed variables were aneurysm size and location, intraluminal thrombus, adjunctive coiling, and the number of devices used. Saccular aneurysms of the basilar artery occluded less often than saccular aneurysms in other posterior circulation locations, though this relationship was not significant (P ¼ .08).5 Complications Associated with Posterior Circulation Flow Diversion The posterior circulation is characterized by unique neurovascular anatomy with end arterial perforating vessels to the brain stem arising from the distal vertebral artery, basilar trunk, and proximal posterior cerebral arteries. Placement of flow diverters inevitably results in coverage of smaller or larger side branches of the vertebrobasilar system.13 Reported morbidity and mortality are important. An aforementioned meta-analysis of 131 posterior circulation aneurysms treated with flow diversion reported overall morbidity and mortality rates of 26% and 21%, respectively. Favorable clinical outcome (mRS, #2) was most frequently achieved in the vertebral AJNR Am J Neuroradiol 41:658–62 Apr 2020 www.ajnr.org 661 artery as opposed to other locations, likely owing to the relative paucity of perforators in that location.6 In the present study, 11 thromboembolic complications occurred. Even though there was not a predilection for a specific location, 10 of 11 (90.9%) thromboembolic complications occurred in nonsaccular aneurysms, and 5 of 11 (45.5%), in aneurysms that had previously ruptured. The multicenter study of 131 posterior circulation aneurysms treated with the PED also identified fusiform aneurysm morphology as a risk factor for major complications.5 Nevertheless, data from the present study show that even nonsaccular aneurysms of the posterior circulation can be treated safely with flow diverters as long as treatment is performed in unruptured aneurysms. Several key aspects, as summarized by Bhogal et al,11 are critical to minimize complications. These include rigorous platelet function testing and regimen adjustments, performed in 95.2% of patients in the current study; treatment of not acutely symptomatic patients, either due to rupture or brain stem compression or ischemia; the use of fewer flow diverters per aneurysm (median of 1 in the current series); and adjunctive coiling. Applying those principles in the current study may have explained the very favorable safety profile. Strengths and Limitations The most significant limitation is the heterogeneity of posterior circulation aneurysms. Given their variability in size and involvement of various segments of the posterior circulation tree, comparative analyses are challenging. Thus, any related study is exposed to significant selection bias across different neurovascular centers and individual neurovascular practitioners. Data collection was performed at the individual institution and was not core lab–adjudicated, introducing potential bias. Nevertheless, the dataset was nearly complete. The inclusion of multiple institutions, on the other hand, improved the generalizability of the findings. Another limitation was variability in the type of platelet function test used at the individual institution, albeit most patients underwent platelet function testing. CONCLUSIONS The largest cohort of posterior circulation aneurysms treated with the FRED to date demonstrated favorable safety and efficacy profiles of the device for this indication. Regardless of location and morphology, flow diversion using the FRED represents a viable treatment option for those challenging lesions. Thromboembolic complications occurred mostly in nonsaccular aneurysms. Treatment in the setting of acute subarachnoid hemorrhage, however, was strongly related to mortality, regardless of whether procedural complications occurred. Disclosures: Markus A. Möhlenbruch—UNRELATED: Board Membership: Codman Neurovascular; Consultancy: Medtronic, MicroVention, Stryker; Grants/Grants Pending: Balt, MicroVention; Payment for Lectures Including Service on Speakers Bureaus: Medtronic, MicroVention, phenox, Stryker.* Christian Ulfert— UNRELATED: Payment for Lectures Including Service on Speakers Bureaus: Cerenovus; Travel/Accommodations/Meeting Expenses Unrelated to Activities Listed: Stryker, MicroVention, Balt. Markus Holtmannspötter—UNRELATED: Consultancy: Medtronic, MicroVention, Sequent Medical, Stryker, Mentice, Route 92, Comments: consultancy and proctoring agreement; Payment for Lectures Including Service on Speakers Bureaus: Medtronic, MicroVention, Sequent Medical. Jan-Hendrik 662 Griessenauer Apr 2020 www.ajnr.org Buhk—UNRELATED: Consultancy: Acandis, Cerenovus, Codman Neurovascular, Medtronic, MicroVention, Stryker, Comments: consultancy and proctoring. Naci Kocer —UNRELATED: Consultancy: MicroVention, Medtronic, Comments: consultant and proctorship agreement; Payment for Lectures Including Service on Speakers Bureaus: MicroVention, Comments: lectures on FRED. Hendrik Janssen—UNRELATED: Consultancy: MicroVention/Sequent Medical, Comments: proctor; Travel/ Accommodations/Meeting Expenses Unrelated to Activities Listed: MicroVention, Comments: travel expenses for conferences. Monika KillerOberpfalzer—RELATED: Grant: MicroVention/Terumo; Consulting Fee or Honorarium: MicroVention/Terumo; UNRELATED: Consultancy: Medtronic, Stryker, Cerus; Grant: MicroVention/Terumo, Comments: research grant; Support for Travel to Meetings for the Study or Other Purposes: MicroVention/Terumo; Travel/Accommodations/Meeting Expenses Unrelated to Activities Listed: MicroVention, Stryker, Medtronic. Civan Islak— UNRELATED: Consultancy: MicroVention; Patents (Planned, Pending or Issued): US 2015/0327868A1.*Money paid to institution. REFERENCES 1. Petr O, Brinjikji W, Cloft H, et al. Current trends and results of endovascular treatment of unruptured intracranial aneurysms at a single institution in the flow-diverter era. AJNR Am J Neuroradiol 2016;37:1106–13 CrossRef Medline 2. Ogilvy CS, Jordan NJ, Ascanio LC, et al. Surgical and endovascular comprehensive treatment outcomes of unruptured intracranial aneurysms: reduction of treatment bias. World Neurosurg 2019;126: e878–87 CrossRef Medline 3. Wang CB, Shi WW, Zhang GX, et al. Flow diverter treatment of posterior circulation aneurysms: a meta-analysis. Neuroradiology 2016;58:391–400 CrossRef Medline 4. Bender MT, Colby GP, Jiang B, et al. Flow diversion of posterior circulation cerebral aneurysms: a single-institution series of 59 cases. Neurosurgery 2019;84:206–16 CrossRef Medline 5. Griessenauer CJ, Ogilvy CS, Adeeb N, et al. Pipeline embolization of posterior circulation aneurysms: a multicenter study of 131 aneurysms. J Neurosurg 2018;130:923–35 CrossRef Medline 6. Kiyofuji S, Graffeo CS, Perry A, et al. Meta-analysis of treatment outcomes of posterior circulation non-saccular aneurysms by flow diverters. J Neurointerv Surg 2018;10:493–99 CrossRef Medline 7. Killer-Oberpfalzer M, Kocer N, Griessenauer CJ, et al. European multicenter study for the evaluation of a dual-layer flow-diverting stent for treatment of wide-neck intracranial aneurysms: the European Flow-Redirection Intraluminal Device Study. AJNR Am J Neuroradiol 2018;39:841–47 CrossRef Medline 8. Piano M, Valvassori L, Lozupone E, et al. FRED Italian Registry: a multicenter experience with the Flow Re-direction Endoluminal Device for intracranial aneurysms. J Neurosurg 2019;1–8 CrossRef Medline 9. Pierot L, Spelle L, Berge J, et al. SAFE study (Safety and efficacy Analysis of FRED Embolic device in aneurysm treatment): 1-year clinical and anatomical results. J Neurointerv Surg 2019;11:184–89 CrossRef Medline 10. Griessenauer CJ, Thomas AJ, Enriquez-Marulanda A, et al. Comparison of Pipeline Embolization Device and Flow Re-Direction Endoluminal Device flow diverters for internal carotid artery aneurysms: a propensity score-matched cohort study. Neurosurgery 2019;85: E249–55 CrossRef Medline 11. Bhogal P, Pérez MA, Ganslandt O, et al. Treatment of posterior circulation non-saccular aneurysms with flow diverters: a single-center experience and review of 56 patients. J Neurointerv Surg 2017; 9:471–81 CrossRef Medline 12. Adeeb N, Moore JM, Wirtz M, et al. Predictors of incomplete occlusion following Pipeline embolization of intracranial aneurysms: is it less effective in older patients? AJNR Am J Neuroradiol 2017;38:2295–300 CrossRef Medline 13. Adeeb N, Griessenauer CJ, Dmytriw AA, et al. Risk of branch occlusion and ischemic complications with the Pipeline embolization device in the treatment of posterior circulation aneurysms. AJNR Am J Neuroradiol 2018;39:1303–09 CrossRef Medline
https://openalex.org/W2625355169	http://www.journalijar.com/uploads/451_IJAR-17667.pdf	English	null	QUALITY OF WORK LIFE AND INFLUENCING FACTORS.	International journal of advanced research	2,017	cc-by	3,876	Introduction:- Quality of work life plays a key role in any organization and has an effect on the people, their work, performance and self- development as well as organization’s development. It refers to relationship between the employees and the work environment in which they work. It focuses on creating a working environment where employees work co – operatively and achieve results collectively. Thus a better quality of work life improves the growth of the employee's along with the organization growth (Pugalendhiet al.2011). Objectives of the Study:- The main objective of this study is to develop a clear understanding of the recent studies published in the field of Human Resource Development. The fundamental problem motivating this review is the need to know and understand the different factors influencing the quality of work life.  To provide an archive of past research points related to studies of quality of work life.  To provide an archive of past research points related to studies of quality of work life.  To explore the different influencing factors contributing for positive quality of work life among the employees.  To have a clearer view for future research studies and methodologies this in turn will contribute to the related accumulated knowledge in the field of human resource development.  To have a clearer view for future research studies and methodologies this in turn will contribute to the related accumulated knowledge in the field of human resource development. QUALITY OF WORK LIFE AND INFLUENCING FACTORS. (An insights from the literature review) Copy Right, IJAR, 2017,. All rights reserved. Copy Right, IJAR, 2017,. All rights reserved. Copy Right, IJAR, 2017,. All rights reserved. …………………………………………………………………………………………………….... Introduction:- ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 5(5), 1328-1332 Journal Homepage: -www.journalijar.com Article DOI:10.21474/IJAR01/4252 DOI URL: http://dx.doi.org/10.21474/IJAR01/4252 Review of Literature:- Narehan Hassan et al. (2014), in the paper titled, ‘The Effect of Quality of Work Life (QWL) Programs on Quality of Life (QOL) among Employees at Multinational Companies in Malaysia’, investigated the relationship between quality of work life (QWL) programs and quality of life (QOL) among 179 employees presently working at multinational companies in Bintulu, Sarawak, Malaysia. The studies showed that there was a positive and significant relationship between quality of work life (QWL) programs and quality of life (QOL). The most influence factor on quality of life (QWL) were work environment followed by job facets. Others were emotional well-being, personal development, social inclusion and interpersonal relations. It can be concluded that organization should consider continuously introducing, improving and enforcing the quality of work life programs within the organization as quality of work life programs enriches employee job motivation and job performance, employee loyalty and commitment to the organization. Sarang S. Bhola and Jyoti J. Nigade (2012), in the paper titled, ‘A Study of Quality of Work Life (QWL) in Small Scale Unionized and Non -Unionized Organization’, conducted a comparative study of quality of work life between small scaled unionized and non-unionized organizations in Satara Industrial Estate with a total sample size of 100 employees which examined the significant role of union in developing and maintaining high degree of quality of work life in organization. The study revealed that quality of work life in unionized organization is better than non- unionized organization. It also concluded that union plays an important role in the improvement of quality of work life in the organization resulting into high degree of satisfaction, productivity and greater organizational effectiveness. KuldeepKaur and Dr. (Ms.) GurpreetRandhawa (2012), in the paper titled, ‘Quality Of Work Life Issues: A Comparative Study Of Multinational And Indian Food Companies Of Punjab’, investigated and compared employees’ perception about various Quality of work life issues in MNCs and INCs with 100 employees of food industry in Punjab. The study revealed that a significant difference exists between the MNCs and INCs employee’s perception over various quality of work life issues such as job characteristics, welfare facilities, personal growth and development and social relevance of work. It implies that the employees of MNCs are slightly more satisfied than INCs as their job being more creative, prestigious, higher paid with more promotional avenues. Review of Literature:- It can be concluded that the organizations should take effect steps to encourage professional and personal-life balance of employees. AnshulJaiswal (2014), in the paper titled, ‘Quality Of Work Life’, mentioned that Quality of work life (QWL) is an indicator of the overall quality of the human experience at the workplace. A commitment to quality of work life by the management of an organisation can be considered a keystone of organizational behaviour. Quality of work life creates a workplace that enhances employee well-being and satisfaction. The general goal of quality of work life program is to satisfy the full range of employee needs. The researcher concluded that the quality of work life program will certainly be improved to the desired levels if the organization properly adopts the techniques of job redesign, career development, flexible work schedules and job security. Dr.Yogesh Jain &Renil Thomas (2014), in the paper titled, ‘A study on quality of work life among the employees of a leading pharmaceuticals limited company of Vadodara district’, conducted a survey of 70 employees of a leading pharmaceutical company of Vadodara district to understand the relationship among various components of quality of work life mentioned by Walton. The results show that there exist significant relationship between the Job Satisfaction and components of the quality of work life variables namely adequate and fair compensation, appropriate career development, Organisational commitment, effective Constitutionalisation of the work environment and social relevance of the work life. Whereas greater correlation was shown towards career development that indicated company should have a proper career advancement graph to retain knowledgeable people. Further the results showed that there exist significant relationship between the Organisational Commitment and components of the quality of work life variables and Organisational Commitment was greatly correlated to work and the total life space, job satisfaction, Constitutionalisation of work environment and social relevance of work life. The researchers concluded that to have excellent quality of work life organizations must go extra miles in order to retain talented employees that can provide the best results to the organization. Hend Al Muftah and HananLafi (2011), in the paper titled, ‘Impact of QWL on employee satisfaction case of oil and gas industry in Qatar’, examines the factors that influence quality of work life and also investigated the relationship between quality of work life and satisfaction among employees working in the Oil and Gas companies in the State of Qatar. Methodology:- gy The nature of study conducted are from the relevant research materials related to human resource development and especially quality of work life published from various national and international journals. Corresponding Author:-SulbhaWaghmare. 13 Corresponding Author:-SulbhaWaghmare. Address:-Assistant Professor, Department of Management (MBA), Indira Institute of Management, Pune, India. 1328 Corresponding Author:-SulbhaWaghmare. Address:-Assistant Professor, Department of Management (MBA), Indira Institute of Management, Pune, India. 1328 Int. J. Adv. Res. 5(5), 1328-1332 ISSN: 2320-5407 Review of Literature:- The three essential factors identified by the researchers were physical, psychological and social 1329 Int. J. Adv. Res. 5(5), 1328-1332 Int. J. Adv. Res. 5(5), 1328-1332 Int. J. Adv. Res. 5(5), 1328-1332 ISSN: 2320-5407 ISSN: 2320-5407 factors as potential predictors of quality of work life in an organization. The result showed that all factors were positively associated with quality of work life and is positively and significantly related to employee satisfaction. The result of this study supports that the most important determinant of quality of work life is physical factors, followed by psychological factors and then social factors centrality have a significant relationship with quality of work life which indirectly impact employees’ satisfaction. It concluded from the results that individual’s family life correlates significantly with his/her level of quality of work life and that a successful family life carries over into one’s career and makes one more satisfied with personal achievements. Jana Cocuľová (2015), in the article titled, ‘Analytical view of the relation between selected factors of work life and work performance’, aim to determine the statistically significant difference in the evaluation of importance of quality of work life factors in terms of their effect on the work performance by employees. The factors identified for the study were reward system, work environment, nature and content of work, Work relations, corporate culture and opportunities for career development. The analysis showed that the highest importance was observed for the factor of the reward system by all groups of employees, irrespective of gender, position and duration of work experience and the lowest rate of the importance were assigned to the factor of opportunities for career development. The findings of the study also suggest that the required performance of employees can be achieved mainly through rewards and the organization should have proper system for evaluation of work performance that can lead to high motivation for work performance. The researcher therefore concluded that the effort to build a strong corporate culture can lead to higher performance. M.Vetrimani and Dr. K. KavithaMaheswari (2014), in the paper titled, ‘A Study on Factors Affecting Quality Of Work Life Among The Employees Of Cement Industry’, examine the perception of the existing quality of work life among the employees in the production unit of Dalmia cements, Ariyalur, Tamilnadu with a sample size of 120 employees. Review of Literature:- The study revealed that more than half of the employees perceived high level of quality of work life and its dimensions like proactive, human relations, learning organization and work life balance. Further it was revealed that there is no significant influence of monthly income, experience, designation and tenure of employment on overall quality of work life among the employees. The researchers concluded that the organization’s management, unions and employees should learn to work together in order to achieve and win goals of an improved quality of life at work for all members of the organization. NamrataSandhu and Rahul Prabhakar (2012), in the paper titled, ‘Factors Influencing the Quality of Work Life in the Indian Banking Industry – An Empirical Study’, identified the various factors that influence the quality of work life in the Indian banking sector. The sample size for the study consists of 235 professionals working in the banking industry. The study revealed the five factors such as appropriate remuneration, opportunities for personal growth, supportive leadership and structures, work environment and work life balance which most significantly influence the quality of work life of employees working in the Indian banking industry. The study therefore suggest that while framing retention policies the organization must be keep in mind these five factors. Barkha Gupta and Dr. Anukool M. Hyde (2013), in the paper titled, ‘Managerial Employees Prospective of Quality of Work Life in Banks’, examine the factors affecting Quality of Work Life of Managerial employees of Banks in Indore. The sample size consists of 150 employees. The study revealed that there is no significant impact of experience, age, gender, income and Managerial employee’s quality of work life. Managerial employees of Private and Nationalized Banks enjoy same level of quality of work life. It might be due to the factors like job security and status employee’s exhibits higher level quality of work life in Managerial employees of Nationalized and Private Banks. The researchers therefore concluded that the employees are satisfied with the job when money, social environment and physical environment are perfect at the needed level. And hence quality of work life increases the job involvement of officers in Banks. Dzeba, Ana. (2011), in the paper titled, ‘Quality of Work Life in Croatia: differences between the private and the public sector’, investigated the differences in the Quality of Work Life among Croatian employees in the private and public sector. Review of Literature:- The results showed that the quality of work life is higher in the private sector than in the public sector. The study showed the aspects of advancement prospects, good pay and participation in decision making regarding the choice of coworkers and when to take a vacation are higher in the private sector while aspects of job security and interesting job are higher in the public sector. The researcher finally concluded that the quality of work life is higher in the private sector and that there is still room for improvement of Quality of Work Life in the public sector. 1330 Int. J. Adv. Res. 5(5), 1328-1332 Int. J. Adv. Res. 5(5), 1328-1332 ISSN: 2320-5407 12. ShineyChib (2012), in the paper titled, ‘Quality Of Work Life And Organisational Performance Parameters At Workplace’, examine whether quality of work life has a significant relationship with organizational performance at work place of a private manufacturing unit, located at Nagpur, India through a structured questionnaire containing 31 items related to 6 variables, namely organizational performance, job satisfaction, quality of work life, wage policy, company policy and union policy. The researcher framed two models, one is organization performance depends on quality of work life, Job satisfaction, wage policy, company policy and union participation and the other one is quality of work life which depends on Organization performance, job satisfaction, wage policy, company policy and union participation. The collected data was analyzed using simple percentage, regression and correlation analysis. The study reveals that both the models stand true and quality of work life had significant relationship with organizational performance. It can be concluded that quality of work life is the degree of excellence brought about work and working conditions which contribute to the overall satisfaction and performance at the individual and at the organizational level. It tries to meet the higher order needs of employees as well as their more basic needs. P.Bhuvaneswari, N.S.Suganya, K.Vishnupriya (2010),in their article titled, ‘A Study on Quality of work life among employees in Neyveli Lignite Corporation Limited, Tamilnadu’, examined the perception of Quality of work life among employees working in Neyveli Lignite Corporation Limited, Tamilnadu. The study reveals that majority of the employees are satisfied with their job, nature of job, salary, co-operation with colleagues, training and development, freedom to work, rewards & recognitions, social & cultural Programs, health, safety & welfare measure and quality of work life. Review of Literature:- Further it is also revealed that all the employee benefits and other facilities on an average show above neutral on satisfaction and there is no significant relationship between gender and quality of work life. Thus the effort of NLC management for upgrading the living standard of the employees is encouraging and unique. It can be concluded that a good quality of work life leads a positive impact on the employee’s attitude towards their job and the working environment in the organization. RavneetRehan and R.S Arora (2014), in the paper titled, ‘Overall Quality of Work Life and Gap Analysis: A Study of Punjabi University Teachers’, attempts to examine the gap and overall satisfaction of Punjabi university teachers with regard to factors of quality of work life. They analyzed quality of work life considering 15 factors of teacher’s well- being. In these fifteen factors, job related policies provide more satisfaction to the employees and salary is the second best factor that gives more satisfaction. They found that there exists a significant gap between the perceived satisfaction and perceived importance of the employees with regard to the factors of quality of work life. Overall satisfaction score showed a moderate satisfaction of the employees and requires improvement in quality of work life of university teachers. It can be concluded that to achieve desired results organization should provide good Quality of Work Life. D. Vijayalakshmi and V. Loganayaki (2013) in the paper titled, ‘A Study on Quality of Work life at Telecom Industries with Special Reference to Coimbatore’, analyzed the perception of quality of work life among employees of Telecom Industry in Coimbatore. The study found that there was significant relationship between educational qualification and growth and development of employees as well as between self- motivation and working conditions of the employees and there was a significant difference between the educational qualification and the interpersonal relationship. Further, it was found that employee’s with less years of experience find it difficult to balance their work life and growth opportunities were available for qualified employees. The researchers therefore concluded that an appropriate working condition, interpersonal relationship, career growth and development, work life and relative facilities can lead to employee satisfaction which ensures the overall organization performance. References:- AnshulJaiswal, Quality of Work Life”, Blue Ocean Research Journals, BORJ, Vol 3, No 2 (2014). B kh G t d D A k l M H d (2013) M i l E l P ti f Q lit f , Q y , , , , ( ) 2. Barkha Gupta and Dr. Anukool M. Hyde (2013), Managerial Employees Prospective of Quality of Work Life in , Q y , , , , ( ) 2. Barkha Gupta and Dr. Anukool M. Hyde (2013), Managerial Employees Prospective of Quality of Work L 2. Barkha Gupta and Dr. Anukool M. Hyde (2013), Managerial Employees Prospective of Quality of W Banks International Journal of Research in Management Science and Technology Vol I Issue II 2. Barkha Gupta and Dr. Anukool M. Hyde (2013), Managerial Employees Prospective of Quality of Work Life in Banks, International Journal of Research in Management Science and Technology Vol. I Issue. II 2. Barkha Gupta and Dr. Anukool M. Hyde (2013), Managerial Employees Prospective of Quality Banks, International Journal of Research in Management Science and Technology Vol. I Issue. rkha Gupta and Dr. Anukool M. Hyde (2013), Managerial Employees Prospective of Quality of W nks, International Journal of Research in Management Science and Technology Vol. I Issue. II g gy 3. D. Vijayalakshmi and v. Loganayaki, ‘A Study On Quality Of Worklife At Telecom Industries With Special 3. D. Vijayalakshmi and v. Loganayaki, ‘A Study On Quality Of Worklife At Telecom Indust 3. D. Vijayalakshmi and v. Loganayaki, ‘A Study On Quality Of Worklife At Telecom Industries With Spec Reference To Coimbatore, International Journal of Management focus, apr-jun 2013, p5 3. D. Vijayalakshmi and v. Loganayaki, ‘A Study On Quality Of Worklife At Telecom Industries With Special R f T C i b I i l J l f M f j 2013 5 3. D. Vijayalakshmi and v. Loganayaki, A Study On Quality Of Worklife At Telecom In Reference To Coimbatore, International Journal of Management focus, apr-jun 2013, p5 j y g y y Q y Reference To Coimbatore, International Journal of Management focus, apr-jun 2013, p5 4. Dr.Yogesh Jain &Renil Thomas (2014), A study on quality of work life among the employees of a leading pharmaceuticals limited company of Vadodara district, International Journal of Applied Research 2016; 2(5): 926-934 4. Conclusion:- To ensure a positive outcome, attention to the factors identified in the suggested framework is important for improved quality of work life. Organizations must aim at promoting peaceful work environment and good organizational culture which is highlighted by the management and the employees. Since human resources is the backbone of every organization. QWL is an excellent tool for HR managers to frame policies and the scope is enormous to examine each aspect of QWL. References:- Dr.Yogesh Jain &Renil Thomas (2014), A study on quality of work life among the employees of a leading pharmaceuticals limited company of Vadodara district, International Journal of Applied Research 2016; 2(5): 926-934 5. Dzeba, Ana. (2011), Quality of Work Life in Croatia: differences between the private and the public sector. Online available at http;//darhiv.ffzg.hr/id/eprint/1542 on 10-12-12. 5. Dzeba, Ana. (2011), Quality of Work Life in Croatia: differences between the private and the public sector. Online available at http;//darhiv.ffzg.hr/id/eprint/1542 on 10-12-12. 6. Jana Cocuľová (2015), Analytical view of the relation between selected factors of work life and work performance, Journal of Management and Marketing, Vol.3, Issue 1, 6. Jana Cocuľová (2015), Analytical view of the relation between selected factors of work life and work performance, Journal of Management and Marketing, Vol.3, Issue 1, 7. KuldeepKaur and Dr. (Ms.) GurpreetRandhawa (2012), Quality Of Work Life Issues: GIAN JYOTI E- JOURNAL, Volume 2, Issue 3 (Jul – Sep 2012) 7. KuldeepKaur and Dr. (Ms.) GurpreetRandhawa (2012), Quality Of Work Life Issues: GIAN JYOTI E- JOURNAL, Volume 2, Issue 3 (Jul – Sep 2012) 8. M.Vetrimani and Dr. K. KavithaMaheswari (2014), A Study on Factors Affecting Quality Of Work Life Among The Employees Of Cement Industry, Indian Journal Of Applied Research, Vol. 4 Issue 12 8. M.Vetrimani and Dr. K. KavithaMaheswari (2014), A Study on Factors Affecting Quality Of Work Life Among The Employees Of Cement Industry, Indian Journal Of Applied Research, Vol. 4 Issue 12 9. Muftah, HendAl.Lafi, Hanan. (2011), Impact of QWL on employee satisfaction case of oil and gas industry in Qatar. International Scientific Press, 1 (2), 107-134. 10. NamrataSandhu and Rahul Prabhakar (2012), Factors Influencing the Quality of Work Life in the Indian Banking Industry – An Empirical Study, DOI: 10.7763/IPEDR. 2012. V55. 23 11. Narehan Hassan et al. (2014), The Effect of Quality of Work Life (QWL) Programs on Quality of Life (QOL) among Employees at Multinational Companies in Malaysia, Procedia - Social and Behavioral Sciences 112 ( 2014 ) 24 – 34 12. P.Bhuvaneswari, N.S.Suganya, K.Vishnupriya (2010), A Study on Quality of Work Life Among employees in Neyveli Lignite Corporation Limited, Tamilnadu., Research Journal of Commerce &Behavioural Science 13. RavneetRehan and R.S Arora (2014), Overall Quality of Work Life and Gap Analysis: A Study of Punjabi University Teachers, International Journal of Advance Research in Computer Science and Management Studies, Volume 2, Issue 9, 14. Sarang S. Key Observations:- y Reward system has a major impact on the work performance of employees that can lead to high motivation. The organization should have proper system for evaluation of work performance. Job related policies provide more satisfaction to the employees while salary is the second best factor that gives more satisfaction to the employees. There was significant relationship between educational qualification and growth and development of employees as well as between self- motivation and working conditions of the employees. Employees possessing less year of experience in the organization find it difficult to balance their work life as they require more time to learn their job. Growth opportunities were available for highly qualified employees. Factors like job security, human relations, learning organization and work life balance exhibits higher level Quality of Work Life among employees.There exist a positive and significant relationship between quality of work life 1331 ISSN: 2320-5407 Int. J. Adv. Res. 5(5), 1328-1332 Int. J. Adv. Res. 5(5), 1328-1332 Int. J. Adv. Res. 5(5), 1328-1332 (QWL) programs and quality of life (QOL) of employees. The most influence factor on quality of life (QWL) were work environment followed by job facets. There is also positive association of employee satisfaction with individual’s family life. While framing retention policies, the organization must be keep in mind the five factors such as appropriate remuneration, opportunities for personal growth, supportive leadership and structures, work environment and work life balance which most significantly influence the quality of work life of employees. References:- Bhola and Jyoti J. Nigade (2012), A Study of Quality of Work Life (QWL) in Small Scale Unionized and Non -Unionized Organization, Vishwakarma Business Review Volume II , Issue 1 (Jan 2012),16-24 15. ShineyChib( 2012 )," Quality of Work Life and Organisational Performance Parameters at Workplace", SEGi Review, Vol. 5, No.2 1332
https://openalex.org/W4280616663	https://www.frontiersin.org/articles/10.3389/fcvm.2022.874795/pdf	English	null	Is the Risk of Diabetes Lower in Patients With Atrial Fibrillation Treated With Direct Oral Anticoagulant Compared to Warfarin?	Frontiers in cardiovascular medicine	2,022	cc-by	5,658	SYSTEMATIC REVIEW published: 19 May 2022 doi: 10.3389/fcvm.2022.874795 SYSTEMATIC REVIEW published: 19 May 2022 doi: 10.3389/fcvm.2022.874795 SYSTEMATIC REVIEW published: 19 May 2022 doi: 10.3389/fcvm.2022.874795 Xuyang Liu1†, Shenghui Feng2†, Zhuohui Chen3†, Yue Zhou4, Kang Yin5, Zhengbiao Xue5 and Wengen Zhu3* 1 Department of Cardiology, Jinggangshan University, Ji’an, China, 2 Queen Mary School, Medical Department, Nanchang University, Nanchang, China, 3 Department of Cardiology, The First Afﬁliated Hospital of Sun Yat-sen University, Guangzhou, China, 4 State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China, 5 Department of Critical Care Medicine, The First Afﬁliated Hospital of Gannan Medical University, Ganzhou, China Edited by: Antonino Tuttolomondo, University of Palermo, Italy Background: The use of anticoagulants is an established strategy to prevent stroke, embolism, and cardiovascular mortality in patients with atrial ﬁbrillation (AF), but its role in the prevention of incident diabetes is unclear. We aimed to investigate this question by using participant data from cohort studies. Reviewed by: Francesco Poletto, University of Padua, Italy Dana Huskens, Synapse BV, Netherlands Correspondence: Kang Yin 474614909@qq.com Zhengbiao Xue 57438440@qq.com Wengen Zhu 602701970@qq.com; zhuwg6@mail.sysu.edu.cn †These authors have contributed equally to this work Methods: We conducted a meta-analysis of participants to investigate the impact of direct oral anticoagulants (DOACs) on the risk of new-onset diabetes in AF patients. The collection of related data was performed in the PubMed and EMBASE databases until December 2021, including studies associated with evaluating the correlation between DOACs and incident diabetes. The hazard ratios (HRs) and 95% conﬁdence intervals (CIs) were adjusted by the random-effects model with an inverse variance method. Results: Two cohort studies with a total of 24,434 patients were included in this study (warfarin: n = 6,906; DOACs: n = 17,528). Compared with warfarin, the use of DOACs could reduce the incident diabetic risk in AF patients (HR = 0.75, 95%CI: 0.68–0.82). Investigations about the effects of three major classes of DOACs showed that the individual use of dabigatran (HR = 0.76, 95%CI: 0.64–0.90), rivaroxaban (HR = 0.74, 95%CI: 0.64–0.87), apixaban (HR = 0.74, 95%CI: 0.60–0.92) and the combined use of rivaroxaban and apixaban (HR = 0.74, 95%CI: 0.66–0.84) could reduce the risk of new- onset diabetes compared with warfarin. This risk reduction effect could be observed in both male and female groups (HR = 0.73, 95%CI: 0.64–0.84, P < 0.00001; HR = 0.82, 95%CI: 0.82–0.99, P = 0.04). Specialty section: This article was submitted to Thrombosis, a section of the journal Frontiers in Cardiovascular Medicine Specialty section: This article was submitted to Thrombosis, Received: 13 February 2022 Accepted: 25 April 2022 Published: 19 May 2022 Keywords: atrial ﬁbrillation, non-vitamin K antagonist oral anticoagulants, diabetes mellitus, warfarin, meta- analysis Is the Risk of Diabetes Lower in Patients With Atrial Fibrillation Treated With Direct Oral Anticoagulant Compared to Warfarin? Xuyang Liu1†, Shenghui Feng2†, Zhuohui Chen3†, Yue Zhou4, Kang Yin5, Zhengbiao Xue5* and Wengen Zhu3* Citation: Liu X, Feng S, Chen Z, Zhou Y, Yin K, Xue Z and Zhu W (2022) Is the Risk of Diabetes Lower in Patients With Atrial Fibrillation Treated With Direct Oral Anticoagulant Compared to Warfarin? Conclusions: Treatment with DOACs compared with warfarin reduced the risk of new- onset diabetes in both male and female patients with AF. Front. Cardiovasc. Med. 9:874795. doi: 10.3389/fcvm.2022.874795 Front. Cardiovasc. Med. 9:874795. doi: 10.3389/fcvm.2022.874795 May 2022 \| Volume 9 \| Article 874795 Frontiers in Cardiovascular Medicine \| www.frontiersin.org DOACs vs. Warfarin in AF Liu et al. authors for data, techniques, and materials to recreate the results or the program. authors for data, techniques, and materials to recreate the results or the program. INTRODUCTION Atrial ﬁbrillation (AF) is the most common cardiac arrhythmia in the clinic, characterized by high rates of thromboembolic complications and related mortality. Study Quality Assessment Newcastle-Ottawa Scale (NOS) was used by authors to assess the quality of included studies. A total of nine points were divided into three domains, including the cohort selection (0–4 points), cohort comparability (0–2 points), and the outcomes evaluation (0–3 points) were assessed by the NOS tool. Studies with the NOS results < 6 points were considered as low quality. Literature Retrieval Two databases PubMed and Embase were used for systemic search in this study, retrieval keywords included (1) atrial ﬁbrillation OR AF AND (2) incident diabetes OR new-onset diabetes AND (3) Direct oral anticoagulants OR DOAC OR DOAC OR oral anticoagulants OR dabigatran OR rivaroxaban OR apixaban AND (4) vitamin K antagonists OR warfarin OR VKA. Two databases PubMed and Embase were used for systemic search in this study, retrieval keywords included (1) atrial ﬁbrillation OR AF AND (2) incident diabetes OR new-onset diabetes AND (3) Direct oral anticoagulants OR DOAC OR DOAC OR oral anticoagulants OR dabigatran OR rivaroxaban OR apixaban AND (4) vitamin K antagonists OR warfarin OR VKA. An integrated approach of AF care mainly contains rhythm and rate control therapy, anticoagulation therapy (OAC), and comprehensive upstream therapy (1). Among them, OAC is of vital importance in AF-related stroke prevention. As a traditional oral anticoagulant, vitamin K antagonists such as warfarin plays their role by antagonizing vitamin K epoxide reductase complex. Warfarin has a narrow therapeutic range, multiple drug and food interactions, and requires frequent blood monitoring of the international normalized ratio (INR) (2). Therefore, direct oral anticoagulants (DOACs, sometimes referred to as non-vitamin K antagonist anticoagulants) have been introduced in the clinic, reducing the risk of stroke or systemic embolism and bleeding compared with warfarin among patients with AF (3–5). Study Selection and Data Extraction Study Selection and Data Extraction Two independent researchers extracted data independently through screening the titles and abstracts to select potential studies for meta-analysis. Then full-text screening was carried out subsequently. Controversies were resolved by discussing with the third researcher. If multiple screened studies suitable for meta-analysis were from the same data source, the study that was more in line with predeﬁned criteria was included. Studies with later publication years and longer follow-up times were preferentially included. The relevant information of each available study included the ﬁrst author, publication year, study design, outcomes, types of DOACs, follow-up period, the sample size and the number of events in the warfarin or DOACs groups, hazard ratio (HR) and 95% conﬁdence intervals (CI) were collected independently by the fourth author. Inclusion and Exclusion Criteria The literature inclusion criteria of this study include (1) randomized controlled trials or observational cohort studies focusing on the risk of developing DM in AF patients treated by warfarin vs. DOACs (Apixaban, dabigatran, rivaroxaban or edoxaban), (2) The outcomes of studies include the appearance of new-onset diabetes, which meets the International Classiﬁcation of Diseases, the use of anti-diabetic medication, or death occurred during the investigation period. (3) All of the patients included in the cohort study were treated with at least one type of anticoagulant after AF diagnosis. The clinical follow-up time was unlimited. Speciﬁc literature forms including reviews, case reports, case series, editorials, meeting abstracts, and insuﬃcient clinical data were excluded. Patients with comorbid AF and DM have a higher risk of stroke, thromboembolism, and cardiovascular mortality (3, 6–9). Vitamin K has been suggested to regulate the activity of vitamin K-dependent proteins (VKDP) such as osteocalcin, which eﬀectively improve β cell proliferation and insulin secretion to reduce the risk of new-onset DM (9–14). Due to the diﬀerent eﬀects of warfarin and DOACs on vitamin K, the use of DOACs was considered with the potential of reducing DM risk in AF patients. Two cohort studies have been conducted to compare the risk of DM induction in AF patients treated by warfarin and DOACs (9, 15). The study of Cheung et al. included the data of 13,688 DOACs new users from the Clinical Data Analysis and Reporting System (CDARS) managed by the Hong Kong Hospital Authority (HA). Their results showed that dabigatran was signiﬁcantly related to incident diabetes risk reduction, and for Xa inhibitor anticoagulants, only the combination use of rivaroxaban and apixaban rather than individual drugs could decrease this risk. In this study, males were the only gender with a diabetic risk-reduction eﬀect. While in a cohort analysis performed by Huang et al., a total of 10,746 AF patients from Taiwan’s National Health Insurance Research Database (NHIRD) were ﬁtted into the study. All of the three diﬀerent DOACs (dabigatran, rivaroxaban, apixaban) were reported with the incident diabetic reduction eﬀect compared with warfarin. In this study, a similar trend of lowering new-onset DM risk was observed in both male and female groups treated with DOAC vs. warfarin. We used individual participants’ data from the two cohort studies to assess the impact of DOACs on new-onset diabetes risk in AF patients. Frontiers in Cardiovascular Medicine \| www.frontiersin.org Statistical Analysis The preferred reporting items for systematic review and meta- analysis (PRISMA) 2020 guidelines were used to conduct our present meta-analysis. Only published publications were included in our meta-analysis, so we did not need ethical permission. Readers can contact the corresponding We chose the Cochrane Q-test and I2 statistic to assess the consistency of the included studies. A P < 0.1 for the Q-test or I2 ≥50% result was considered as the existence of substantial heterogeneity. The Review Manager Version 5.3 (The May 2022 \| Volume 9 \| Article 874795 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 2 Liu et al. DOACs vs. Warfarin in AF Nordic Cochrane Center, The Cochrane Collaboration, 2014, Copenhagen, Denmark1) was used for all statistical analyses; P < 0.05 was considered statistically signiﬁcant. First, we collected the sample size and number of events in the warfarin or DOACs groups, then the crude events rates of DM induction risk were carried out and expressed by HRs and 95% CIs. Second, the HR of DM induction was calculated in both the warfarin group and DOACs group with respect to gender diﬀerences. The adjusted HRs were converted to the natural logarithms and standard errors. The inverse variance method was used to incorporate random eﬀect models. Nordic Cochrane Center, The Cochrane Collaboration, 2014, Copenhagen, Denmark1) was used for all statistical analyses; P < 0.05 was considered statistically signiﬁcant. First, we collected the sample size and number of events in the warfarin or DOACs groups, then the crude events rates of DM induction risk were carried out and expressed by HRs and 95% CIs. Second, the HR of DM induction was calculated in both the warfarin group and DOACs group with respect to gender diﬀerences. The adjusted HRs were converted to the natural logarithms and standard errors. The inverse variance method was used to incorporate random eﬀect models. may inﬂuence patients’ DM risk were recorded in the baseline characteristic table. Both of these two studies could meet our screening criteria. For the quality assessment, the NOS scores of both included studies were ≥6 points. The number of included studies was less than 10. Thus, there was no need for publication bias assessment. Crude Event Rates Between Direct Oral Anticoagulants vs Warfarin The crude rates of the occurrence of the incident DM in AF patients treated by warfarin or DOACs were reported in both cohort studies, shown in Table 2. Compared with warfarin, the incidence of new-onset diabetes was relatively lower in DOACs treated group (6.78% vs 7.68%). Both coagulation factor Xa inhibitors apixaban (5.38% vs 6.14%), rivaroxaban (8.03% vs 8.05%) and thrombin inhibitor dabigatran (6.73% vs 8.49%) show the eﬀectiveness in reducing the incidence of new-onset diabetes in AF patients, and this eﬀect in coagulation factor Xa inhibitors combination group also exist (6.82% vs 7.23%). Study Selection The retrieval ﬂow chart of this meta-analysis is shown in Figure 1. A total of 205 studies were acquired through online searching in the PubMed and Embase databases. After removing repeated investigations, 25 studies were chosen to develop title/abstract screening. Then 10 studies were evaluated in detail. On the basis of predeﬁned criteria, ﬁnally, two eligible cohort studies were included in our meta-analysis (9, 15). Exhibited in Table 1 was the baseline information of patients in the included studies. A total of 24,434 individual participants (warfarin: n = 6,906; DOACs: n = 17,528) from two cohort studies were included in our meta- analysis. The data of included cohort studies were from Taiwan’s National Health Insurance Research Database (NHIRD) and Clinical Data Analysis and Reporting System (CDARS) managed by the Hong Kong Hospital Authority (HA). Their study periods were not less than 5 years and the medical use conditions that Adjusted Data of Outcomes Between Direct Oral Anticoagulants vs Warfarin Both included studies have reported the adjusted data of new- onset DM in AF patients treated by DOACs vs warfarin (9, 15). The outcomes displayed in Figure 2 were able to conﬁrm that compared with warfarin, DOACs can reduce the risk of diabetes in AF patients (HR = 0.75, 95%CI: 0.68–0.82). Moreover, the outcomes of the gender subgroup according to the included studies are shown in Supplementary Figure 1. Compared with warfarin, the tendency of DOACs to reduce the incidence of new-onset diabetes can be observed in both male and female groups (male: HR = 0.73, 95%CI: 0.24–0.84; female: HR = 0.82, 95%CI: 0.68–0.99). 1https://community.cochrane.org/ Adjusted Data of Outcomes Between Direct Oral Anticoagulants vs Warfarin FIGURE 1 \| Retrieval ﬂow chart of this meta-analysis. FIGURE 1 \| Retrieval ﬂow chart of this meta-analysis. FIGURE 1 \| Retrieval ﬂow chart of this meta-analysis. Frontiers in Cardiovascular Medicine \| www.frontiersin.org May 2022 \| Volume 9 \| Article 874795 3 DOACs vs. Warfarin in AF Liu et al. TABLE 1 \| Main baseline characteristics of DOACs and warfarin treated patients in the included cohort studies. Huang et al. (9) Cheung et al. (15) Warfarin DOACs Warfarin DOACs Apixaban Dabigatran Rivaroxaban Basal characteristics Sample size, n 4,596 3,452 3,335 4,210 2,689 3,454 Age 70.9 (± 12.0) 70.6 (± 12.9) 78.1 (± 10.8) 74.4 (± 10.3) 74.9 (± 10.8) 72.9 (± 12.2) Female Sex,% 40.0 39.2 50.9 47.9 47.7 44.9 Median follow-up duration 2.4 year 2.3 years 363 (106–648) days 363 (84–700) days 392 (98–730) days 222 (36–704) days Data source From Taiwan’s National Health Insurance Research Database (NHIRD) Clinical Data Analysis and Reporting System (CDARS) managed by the Hong Kong Hospital Authority (HA) Country Taiwan, China Hong Kong, China Study period 5 years 6 years Outcomes new-onset DM requiring treatment with an anti-diabetic drug ICD-9-CM 250.xx including type 1 and type 2 diabetes or a prescription of anti-diabetic medication. Adjusted Data of Outcomes Between Direct Oral Anticoagulants vs Warfarin Comorbidities, n (%) Congestive heart failure 766 (31.1) 737 (29.9) 636 (19.1) 576 (13.7) 413 (15.4) 741 (21.5) Stroke 742 (30.1) 681 (27.6) 706 (21.2) 797 (18.9) 460 (17.1) 525 (15.2) COPD 357 (14.5) 350 (14.2) 304 (9.1) 338 (8.0) 215 (8.0) 313 (9.1) Fall – – 637 (19.1) 617 (14.7) 420 (15.6) 566 (16.4) Fracture – – 299 (9.0) 282 (6.7) 214 (8.0) 251 (7.3) Chronic liver disease/liver cirrhosis 55 (2.2) 56 (2.3) 16 (0.5) 14 (0.3) 4 (0.1) 23 (0.7) Osteoporosis – – 65 (1.9) 51 (1.2) 41 (1.5) 37 (1.1) Rheumatoid arthritis 20 (0.8) 22 (0.9) 39 (1.2) 36 (0.9) 22 (0.8) 29 (0.8) Chronic kidney disease 292 (11.9) 280 (11.4) 79 (2.4) 42 (1.0) 49 (1.8) 187 (5.4) Hypertension 1,616 (65.6) 1,619 (65.7) – – – – Coronary artery disease 821 (33.3) 801 (32.5) – – – – Hyperlipidemia 646 (26.2) 613 (24.9) – – – - Dementia 142 (5.8) 136 (5.5) – – – – Gout 265 (10.8) 251 (10.2) – – – – Malignancy 185 (7.5) 194 (7.9) – – – – Medication use condition ACE inhibitors – – 1,455 (43.6) 1,529 (36.3) 1,088 (40.5) 1,504 (43.5) Beta blockers 1,472 (59.7) 1,502 (60.9) 1,959 (58.7) 2,490 (59.1) 1,649 (61.3) 2,019 (58.5) Proton pump inhibitors – – 1,396 (41.9) 1,321 (31.4) 877 (32.6) 1,169 (33.8) Systemic corticosteroids – – 324 (9.7) 341 (8.1) 222 (8.3) 377 (10.9) Anti-depressants – – 202 (6.1) 199 (4.7) 128 (4.8) 162 (4.7) Statins 143 (5.8) 134 (5.4) – – – – Thiazides 199 (8.1) 191 (7.8) – – – – Antipsychotics 143 (5.8) 141 (5.7) – – – – Steroid 143 (5.8) 134 (5.4) – – – – Index year 2012 45 (1.8) 45 (1.8) – – – – 2013 476 (19.3) 476 (19.3) – – – – 2014 593 (24.1) 593 (24.1) 90 (2.7) 403 (9.6) 371 (13.8) 704 (20.4) 2015 694 (28.2) 694 (28.2) 256 (7.7) 578 (13.7) 525 (19.5) 727 (21.0) 2016 657 (26.7) 657 (26.7) 486 (14.6) 785 (18.6) 621 (23.1) 670 (19.4) 2017 – – 886 (26.6) 1,045 (24.8) 547 (20.3) 630 (18.2) 2018 – – 1,163 (34.9) 1,174 (27.9) 524 (19.5) 628 (18.2) 2019 – – 454 (13.6) 225 (5.3) 101 (3.8) 95 (2.8) COPD, Chronic Obstructive Pulmonary Disease; DOACs, direct oral anticoagulants. As shown in Table 2, the subgroup analysis of diﬀerent types of DOAC were also analyzed in our study. diabetes (Apixaban: HR = 0.74, 95% CI: 0.60–0.92; Dabigatran: HR = 0.76, 95% CI: 0.66–0.88; Rivaroxaban: HR = 0.74, 95% CI: 0.64–0.86). After pooling the data of rivaroxaban and apixaban, DISCUSSION The primary ﬁndings of this study included (1) Compared with warfarin, DOACs including thrombin inhibitor dabigatran, coagulation factor Xa inhibitor rivaroxaban and apixaban could reduce the risk of new-onset diabetes in AF patients. (2) The DM- reduced eﬀect of DOACs vs. warfarin can be observed in both male and female groups. In 2017, a novel drug betrixaban was approved by FDA as the ﬁfth DOAC that can be used in clinic. With low renal clearance and minimal hepatic metabolism, betrixaban was considered particularly beneﬁcial for patients with renal or hepatic dysfunction (28). However, the lack of an eﬀective reversal agent makes betrixaban has a longer terminal half-life compared with other approved DOACs (28, 29). The impact of betrixaban on the risk of new-onset diabetes has not been evaluated in current studies, and the relevant results are expected to be supplemented in the future. Compared with DOACs, warfarin has various limitations in the process of anticoagulant treatment. The changing international standardized ratio (INR) control and dose adjustment, various dietary or drug interactions (16, 17), narrow therapeutic window (17) result in the restrictions of warfarin in clinical use. Several meta-analyses and randomized controlled trials have reported the contrasts with the eﬀectiveness and safety between DOACs and warfarin (18–20). The role of reducing AF and diabetes-associated risk factors including major bleeding, renal decline and cardiac valve calciﬁcation can be observed in the use of DOACs rather than warfarin (21–25). Our study was based on two cohort studies with a total sample size of 24,434, which is the most comprehensive and latest study according to the risk of DOACs vs. warfarin in inducing new- onset diabetes in AF patients. The result in the cohort study from Cheung et al. (15) proposed that only dabigatran was signiﬁcantly associated with incident diabetes risk reduction, our results support that three of existing approved DOAC dabigatran, apixaban, and rivaroxaban with the function of reducing incident diabetic risk. At the same time, this eﬀect in factor Xa inhibitors rivaroxaban and apixaban were not obvious, and our ﬁndings conﬁrmed that all of these three drugs with the risk reduction ability. Adjusted Data of Outcomes Between Direct Oral Anticoagulants vs Warfarin All of the three evaluated DOAC i d i h h d d i k f i d i i id diabetes (Apixaban: HR = 0.74, 95% CI: 0.60–0.92; Dabigatra HR = 0.76, 95% CI: 0.66–0.88; Rivaroxaban: HR = 0.74, 95% C 0 64 0 86) Af li h d f i b d i b TABLE 1 \| Main baseline characteristics of DOACs and warfarin treated patients in the included cohort studies. As shown in Table 2, the subgroup analysis of diﬀerent types of DOAC were also analyzed in our study. All of the three evaluated DOACs associated with the decreased risk of inducing incident May 2022 \| Volume 9 \| Article 874795 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 4 DOACs vs. Warfarin in AF Liu et al. TABLE 2 \| Pooled HRs of diabetes between DOACs vs. warfarin in patients with AF. DOACs Dabigatran Rivaroxaban Apixaban Rivaroxaban + Apixaban Crude event rates 6.78% vs. 7.68% 6.73% vs. 8.49% 8.03% vs. 8.05% 5.38% vs. 6.14% 6.82% vs. 7.23% HRs and 95% CIs 0.75 (0.68–0.82) 0.76 (0.66–0.88) 0.74 (0.64–0.86) 0.74 (0.60–0.92) 0.74 (0.66–0.84) P value <0.00001 0.001 0.0001 0.007 <0.00001 I2 statistic 0% 30% 1% 0% 0% AF, atrial ﬁbrillation; HR, hazard ratio; CI, conﬁdence interval; DOACs, Direct oral anticoagulants. FIGURE 2 \| Comparing the primary outcomes of incident diabetes in DOACs vs. warfarin. DOACs, direct oral anticoagulants; HR, hazard ratio; CI, conﬁdence interval; SE, standard error; IV, inverse of the variance. TABLE 2 \| Pooled HRs of diabetes between DOACs vs. warfarin in patients with AF. FIGURE 2 \| Comparing the primary outcomes of incident diabetes in DOACs vs. warfarin. DOACs, direct oral anticoagulants; HR, hazard ratio; CI, conﬁdence interval; SE, standard error; IV, inverse of the variance. FIGURE 2 \| Comparing the primary outcomes of incident diabetes in DOACs vs. warfarin. DOACs, direct oral anticoagulants; HR, hazard ratio; CI, conﬁdence interval; SE, standard error; IV, inverse of the variance. a similar eﬀect could also be detected (HR = 0.74, 95% CI: 0.66– 0.84), which conﬁrms the reducing-eﬀect of coagulation factor Xa inhibitors on the risk of new onset diabetes. The insulin resistance ameliorating eﬀect of vitamin K was suggested through the inactivation of the NF-κβ signaling pathway to inhibit inﬂammatory responses and lipid-decreasing eﬀect (11, 12). Considering the vitamin K antagonizing function of warfarin, it can inﬂuence the incidence of diabetes. Adjusted Data of Outcomes Between Direct Oral Anticoagulants vs Warfarin However, the anticoagulation process of DOACs does not inﬂuence the vitamin K concentration in the circulatory system. Therefore, using DOACs compared with warfarin could reduce the risk of new-onset diabetes in AF patients. Frontiers in Cardiovascular Medicine \| www.frontiersin.org CONCLUSION Our ﬁndings of current analysis suggested that treatment with DOACs compared with warfarin reduced the risk of new-onset diabetes in both male and female patients with AF. AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcvm. 2022.874795/full#supplementary-material DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s. The results of our study suggested that DOACs could reduce the risk of incident diabetes, which is probably more suitable for AF patients with a higher risk of new-onset diabetes. More prospective clinical data about the risk of incident DM in AF patients treated by DOACs and warfarin could prove our point. FUNDING This study was funded by the National Natural Science Foundation of China (82100273). This study was funded by the National Natural Science Foundation of China (82100273). DISCUSSION Also, in the outcomes of Cheung et al., only a speciﬁc gender of AF patients with the advantage of incident diabetic risk reduction in DOACs treatment, whereas the result of our investigation suggested that this eﬀect could be observed in Warfarin plays its role by antagonizing vitamin K, which is an important inﬂuence factor of glucose homeostasis and insulin sensitivity. In animal tissues, vitamin K homolog menaquinone-4 (MK-4) might act as an incretin-like nutrient and a cofactor of microsomal γ-glutamyl carboxylase (14, 26, 27). It contributes to the post-translational carboxylation process of transferring glutamate to γ-carboxyglutamate (Gla) residues of VKDP. Insulin production could be promoted by VKDP–osteoblast-speciﬁc secreted osteocalcin in a bone- pancreas endocrine loop to regulate glucose metabolism (14). May 2022 \| Volume 9 \| Article 874795 5 Liu et al. DOACs vs. Warfarin in AF both male and female groups. In addition, the estimated crude events rates of new-onset diabetes were evaluated during our investigation process. Although available data is insuﬃcient to support the eﬀect of vitamin K in ameliorating prediabetes (the impaired glucose tolerance, fasting blood sugar, fasting serum insulin level would not be restored), the glucose and insulin levels of 2-h post-oral glucose tolerance test could be reduced by stable vitamin K support (10, 30, 31), which indicate that DOACs may not induce the rapid deterioration of prediabetes compared with warfarin. Also, the clinical trials have demonstrated that the new anti-diabetic drug sodium-glucose linked transporter inhibitors (SGLTi) with a beneﬁcial eﬀect on cardiovascular disease (32–34), regardless if diabetes exists or not (34). In the aspect of AF, SGLTi can counteract the production of cellular ROS in cardiomyocytes, which may change atrial remodeling and reduce the burden of AF (34). This suggests that the new anti-diabetic drug SGLTi and new oral anticoagulants DOAC may have similar eﬀects on the prevention of new-onset diabetes during AF treatment. with rheumatic heart disease, congenital heart disease, or valve replacement surgery are more likely to be treated with warfarin rather than DOAC (9). This group that may induce selection bias was not completely excluded in our study. Future research could carry out propensity score matching on the basis of incorporating more data to minimize the impact of confounding factors. LIMITATIONS This meta-analysis still had several limitations: (1) Only two cohort studies were included in our study, and the data were relatively limited, therefore the evaluation of the eﬀect of edoxaban and betrixaban on incident diabetes was not supported. (2) The included population in our study only contain AF patients from Hong Kong and Taiwan, thus the evaluation of new-onset diabetes risk just considered Asian AF patients. (3) The subtypes of DM were not be distinguished in this study, whether DOACs have the same eﬀect in reducing the risk of type 1 and type 2 diabetes in AF patients is still not clear. (4) The confounding factors cannot be completely excluded in observational studies. According to clinical guidelines, patients REFERENCES Rasekhi H, Karandish M, Jalali MT, Mohammad-Shahi M, Zarei M, Saki A, et al. 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https://openalex.org/W2471217505	https://revistas.ufpi.br/index.php/reufpi/article/download/4958/pdf	Portuguese	null	Kangoroo Care Method at Neonatal Intensive Care Unit / Método Canguru na Unidade de Cuidado Intermediário Neonatal Convencional / Unidad de Cuidados Canguro en el convencional Neonatal de Cuidados Intermedios	Revista de Enfermagem da UFPI	2,015	cc-by	6,221	ABSTRACT Obj ti Objective: To analyze the nursing care team in the first stage of the kangaroo method and discuss the precautions to be taken in order to avoid failures in achieving the objectives of the said method. Method: This is a descriptive qualitative study with 10 professionals of nursing, effective nurses and maternity in the city of Teresina, Piauí. Data were collected in December 2014, after approval by the Ethics Committee, through interviews, according to the criteria of Resolution of the National Health Council 466/12 and subjected to content analysis. Results: showed two categories: "Awareness of mothers to the Kangaroo Mother Care" through guidelines and the difficulties encountered by the team during this approach and the "Preventive measures for success in Kangaroo Care" guidelines that can be done gradually, with the partnership of other professionals, design and staff training for effective guidance and empathy for the mothers of these neonates in the Intermediate Care Unit. Conclusion: The lack of information about the method, the lack of recurrent training for professionals, proper sizing and a satisfactory physical structure are gaps that show that changes are needed in the implementation of the Kangaroo Method in motherhood. Key words: Kangaroo Mother Care. Neonatology. Nursing. RESUMO Objetivo: analisar o cuidado da equipe de enfermagem na primeira etapa do método canguru e discutir as precauções a serem tomadas com vistas a se evitar falhas no alcance dos objetivos do referido método. Método: Trata-se de um estudo qualitativo descritivo, realizado com 10 profissionais da área da enfermagem, enfermeiros e técnicos efetivos de uma maternidade na cidade de Teresina, Piauí. Os dados foram coletados no mês de dezembro de 2014, após aprovação do Comitê de Ética, por meio de entrevista, segundo os critérios da Resolução do Conselho Nacional de Saúde 466/12 e submetidos à análise de conteúdo. Resultados: evidenciaram duas categorias: “Conscientização das mães para o Método Canguru” através de orientações e as dificuldades encontradas pela equipe durante essa abordagem e as “Medidas preventivas para o sucesso no Método Canguru”, orientações que podem ser realizada de forma gradativa, com a parceria de outros profissionais, dimensionamento e capacitação pessoal para uma orientação efetiva e empatia com as mães desses neonatos internados na Unidade de Cuidados Intermediários. ISSN: 2238-7234 Mendes GVS, et al. ISSN: 2238-7234 Mendes GVS, et al. Kangaroo Care Method at Neonatal Intensive Care Unit Gabrielle Visgueira Soares Mendes1, Silvana Santiago da Rocha2, Jaqueline Carvalho e Silva Sales3, Olívia Dias de Araújo4, Liana de Oliveira Araújo5 ¹Enfermeira. Universidade Federal do Piauí. E-mail: bibivisgueira@hotmail.com ²Enfermeira. Doutora em Enfermagem. Docente da Universidade Federal do Piauí. E-mail: silvanasantiago27@gmail.com ³Enfermeira. Doutoranda em Enfermagem pela Universidade Federal do Piauí. E-mail: jaqueline- carvalho@uol.com.br 4Enfermeira. Doutoranda em Enfermagem pela Universidade Federal do Piauí. E-mail: oliviaenf@ufpi.edu.br 5Enfermeira. Faculdade Santo Agostinho. E-mail: lianaoaraujo@gmail.com INTRODUÇÃO O elevado número de neonatos de baixo peso ao nascimento (peso inferior a 2.500g, sem considerar a idade gestacional) constitui um importante problema de saúde e representa um alto percentual na morbimortalidade neonatal. Além disso, tem graves consequências médicas e sociais, muitos bebês são acometidos de distúrbios metabólicos, dificuldades em alimentar-se e para regular sua temperatura corporal, consequentemente as mães acabam sendo acometidas por humor depressivo, ansiedades, insônia, fadiga, dificuldade de concentração, sensação de incapacidade para cuidar do seu filho, sentimento de culpa, até ser diagnosticada com depressão pós parto(1). Com o objetivo de contribuir para a mudança de postura dos profissionais e visando à humanização da assistência ao recém-nascido, o Ministério da Saúde (2011) lançou, por meio da Portaria nº 693, de 5 de julho de 2000, a Norma de Atenção Humanizada ao Recém-nascido de Baixo Peso (Método Canguru)(8). A adoção do Método Canguru é estratégia essencial para mudança institucional na busca da atenção à saúde, centrada na humanização da assistência e no princípio da cidadania da família. Mas, a simples implantação do Método Canguru em uma instituição não alcança os objetivos almejados, pois é necessária a capacitação dos profissionais de saúde envolvidos no processo para transformação do modelo assistencial(9). À mortalidade neonatal é responsável por quase 70% das mortes no primeiro ano de vida e o cuidado adequado ao recém-nascido tem sido um dos desafios para reduzir os índices de mortalidade infantil em nosso País. A taxa de mortalidade infantil (crianças menores de 1 ano), de acordo com o Instituto Brasileiro de Geografia e Estatística (IBGE), 2010, teve expressiva queda nas última décadas no Brasil, graças às estratégias implementadas pelo governo federal, como ações para a diminuição da pobreza, ampliação da cobertura da Estratégia Saúde da Família, ampliação das taxas de aleitamento materno exclusivo, entre outras(2). O número de óbitos foi diminuído de 47,1 a cada mil nascidos vivos em 1990, para 15,6 em 2010(3). Este método foi desenvolvido para ser aplicado em três etapas, e a mudança de uma para outra vai depender da melhora do recém-nascido. Na primeira etapa, preconiza-se acesso precoce e livre dos pais à Unidade de Cuidados Intensivos Neonatal (UTIN) e Unidade de Cuidados Intermediários Neonatais Convencionais (UCINco), estímulo à amamentação e participação da mãe nos cuidados do bebê, bem como início do contato pele a pele logo que as condições clínicas do bebê permitam. Portuguese Rev Enferm UFPI. 2015 Oct-Dec;4(4):68-74. INTRODUÇÃO Na segunda etapa, mãe e bebê permanecem em enfermaria conjunta, e a posição canguru deve ser realizada pelo maior tempo possível(10). A terceira fase refere- se à alta hospitalar, porém permanece no método com acompanhamento ambulatorial até atingir o peso de 2.500 gramas. Em 2011 foram 44 mil óbitos, uma redução de 73%, bem maior do que a média global, que foi de pouco mais de 40%. No entanto, se comparados a índices de outros países, o Brasil ainda ocupa posição desconfortável (107ª) na tabela de mortes de crianças. Ainda, de acordo com o IBGE, à medida que o País tem avanços nas questões estruturais relacionadas às áreas de saneamento e acesso à saúde, a tendência é que os óbitos infantis se concentrem no componente neonatal precoce (óbitos de crianças menores de sete dias)(4). Esta pesquisa apresenta como objeto de estudo analisar o cuidado da equipe de enfermagem na primeira etapa do método canguru e discutir as precauções a serem tomadas com vistas a se evitar falhas no alcance dos objetivos do referido método em uma maternidade de referência do estado do Piauí. Entretanto, a meta de garantir o direito à vida e à saúde a toda criança brasileira ainda não foi alcançada, persistindo desigualdades regionais e sociais inaceitáveis(5). ABSTRACT Obj ti Estud mostrado que o método reduz ainda as com de saúde, tanto físicas quanto mentais, assoc baixo peso ao nascer, reduzindo em até de período de internação hospitalar dos prematu Com o objetivo de contribuir para a mud postura dos profissionais e visando à humani assistência ao recém-nascido, o Ministério d (2011) lançou, por meio da Portaria nº 693, julho de 2000, a Norma de Atenção Human Recém-nascido de Baixo Peso (Método Cangu A adoção do Método Canguru é e essencial para mudança institucional na b atenção à saúde, centrada na humaniz assistência e no princípio da cidadania da Mas, a simples implantação do Método Can uma instituição não alcança os objetivos al pois é necessária a capacitação dos profissi saúde envolvidos no processo para transform modelo assistencial(9). Este método foi desenvolvido para ser em três etapas, e a mudança de uma para o depender da melhora do recém-nascido. Na etapa, preconiza-se acesso precoce e livre d Unidade de Cuidados Intensivos Neonatal Unidade de Cuidados Intermediários N Convencionais (UCINco), estímulo à amame participação da mãe nos cuidados do be como início do contato pele a pele logo condições clínicas do bebê permitam. Na etapa, mãe e bebê permanecem em en conjunta, e a posição canguru deve ser pelo maior tempo possível(10). A terceira fas se à alta hospitalar, porém permanece no com acompanhamento ambulatorial até a peso de 2.500 gramas. Esta pesquisa apresenta como objeto d analisar o cuidado da equipe de enferma primeira etapa do método canguru e dis precauções a serem tomadas com vistas a falhas no alcance dos objetivos do referido em uma maternidade de referência do es Piauí. METODOLOGIA O presente estudo é uma pesquisa qualit natureza descritiva não se preocupando c ISSN: 2238-7234 Mendes GVS, et al. ISSN: 2238-7234 Mendes GVS, et al. Kangaroo Care Method at Neonatal Intensive Care Unit Kangaroo Care Method at Neonatal Intensive Care Unit Canguru” para “Método Canguru”. Estudos têm mostrado que o método reduz ainda as complicações de saúde, tanto físicas quanto mentais, associadas ao baixo peso ao nascer, reduzindo em até dez dias o período de internação hospitalar dos prematuros(7). ABSTRACT Obj ti Conclusão: a carência de informação a respeito do método, a falta de treinamentos recorrentes para profissionais, o dimensionamento adequado e uma estrutura física satisfatória são lacunas que evidenciam que são necessárias mudanças na implementação no Método Canguru na maternidade. p ç g Palavras-chave: Método Canguru. Neonatologia. Enfermagem. É Objetivo: Analizar el equipo de cuidados de enfermería en la primera etapa del método canguro y discutir las precauciones que deben tomarse con el fin de evitar fallos en la consecución de los objetivos de dicho método. Método: Se trata de un estudio cualitativo descriptivo con 10 profesionales de enfermería, enfermeras eficaces y maternidad en la ciudad de Teresina, Piauí. Los datos fueron recolectados en diciembre de 2014, después de la aprobacion del Comité de Ética, a través de entrevistas, de acuerdo con los criterios de la Resolución del Consejo Nacional de Salud 466/12 y sometidos a análisis de contenido. Resultados: mostraron dos categorías: "El conocimiento de las madres en el cuidado madre canguro" a través de directrices y las dificultades encontradas por el equipo durante este enfoque y las "medidas preventivas para el éxito en el cuidado canguro" directrices que se pueden hacer poco a poco, con la asociación de profesionales de otro tipo de formación, diseño y profesionales para la orientación y la empatía eficaz para las madres de estos recién nacidos en la Unidad de Cuidados intermedios. Conclusión: La falta de información sobre el método, la falta de entrenamiento recurrente para los profesionales, el tamaño adecuado y una estructura física satisfactoria son las lagunas que muestran que se necesitan cambios en la aplicación del método canguro en la maternidad. g Palabras clave: Cuidado de la Madre Canguro. Neonatología. Enfermería. g Palabras clave: Cuidado de la Madre Canguro. Neonatología. Enfermería. ¹Enfermeira. Universidade Federal do Piauí. E-mail: bibivisgueira@hotmail.com ²Enfermeira. Doutora em Enfermagem. Docente da Universidade Federal do Piauí. E-mail: silvanasantiago27@gmail.com ³Enfermeira. Doutoranda em Enfermagem pela Universidade Federal do Piauí. E-mail: jaqueline- carvalho@uol.com.br 4Enfermeira. Doutoranda em Enfermagem pela Universidade Federal do Piauí. E-mail: oliviaenf@ufpi.edu.br 5Enfermeira. Faculdade Santo Agostinho. E-mail: lianaoaraujo@gmail.com 68 Portuguese Rev Enferm UFPI. 2015 Oct-Dec;4(4):68-74. ISSN: 2238-7234 Mendes GVS, et al. Kangaroo Care Method at Neonatal Intensive Ca INTRODUÇÃO O elevado número de neonatos de baixo peso ao nascimento (peso inferior a 2.500g, sem considerar a idade gestacional) constitui um importante problema de saúde e representa um alto percentual na morbimortalidade neonatal. ABSTRACT Obj ti Além disso, tem graves consequências médicas e sociais, muitos bebês são acometidos de distúrbios metabólicos, dificuldades em alimentar-se e para regular sua temperatura corporal, consequentemente as mães acabam sendo acometidas por humor depressivo, ansiedades, insônia, fadiga, dificuldade de concentração, sensação de incapacidade para cuidar do seu filho, sentimento de culpa, até ser diagnosticada com depressão pós parto(1). À mortalidade neonatal é responsável por quase 70% das mortes no primeiro ano de vida e o cuidado adequado ao recém-nascido tem sido um dos desafios para reduzir os índices de mortalidade infantil em nosso País. A taxa de mortalidade infantil (crianças menores de 1 ano), de acordo com o Instituto Brasileiro de Geografia e Estatística (IBGE), 2010, teve expressiva queda nas última décadas no Brasil, graças às estratégias implementadas pelo governo federal, como ações para a diminuição da pobreza, ampliação da cobertura da Estratégia Saúde da Família, ampliação das taxas de aleitamento materno exclusivo, entre outras(2). O número de óbitos foi diminuído de 47,1 a cada mil nascidos vivos em 1990, para 15,6 em 2010(3). Em 2011 foram 44 mil óbitos, uma redução de 73%, bem maior do que a média global, que foi de pouco mais de 40%. No entanto, se comparados a índices de outros países, o Brasil ainda ocupa posição desconfortável (107ª) na tabela de mortes de crianças. Ainda, de acordo com o IBGE, à medida que o País tem avanços nas questões estruturais relacionadas às áreas de saneamento e acesso à saúde, a tendência é que os óbitos infantis se concentrem no componente neonatal precoce (óbitos de crianças menores de sete dias)(4). Entretanto, a meta de garantir o direito à vida e à saúde a toda criança brasileira ainda não foi alcançada, persistindo desigualdades regionais e sociais inaceitáveis(5). Em 1979, para o atendimento de RNs prematuros e os de baixo peso situação que envolvia uma alta Canguru” para “Método Canguru”. METODOLOGIA Diante das perguntas realizadas foi possível constatar que um dos principais cuidados prestados pelos profissionais de enfermagem que trabalham na UCINco, são as orientações realizadas para as mães como observado nos discursos abaixo: Na coleta de dados utilizou-se a entrevista semiestruturada, permitindo a organização flexível e ampliando questionamentos à medida que as informações foram fornecidas pelo entrevistado. O informante pôde discorrer sobre o tema proposto e o pesquisador em alguns momentos realizou perguntas adicionais provocando a recomposição do contexto da entrevista. “Quando a mãe chega a gente pergunta se ela deseja colocar o bebê no colo, colocar dentro da roupa dela, no método canguru, a gente pergunta se ela quer colocar. Se ela não quer naquele momento, a gente também não obriga a mãe colocar, fica a critério dela, mas a gente orienta que ali dentro o bebê vai ficar mais aquecido, vai se desenvolver melhor...” (E1). A análise de conteúdo consistiu na leitura detalhada da transcrição das falas e interpretação das respostas fornecidas pelos profissionais de enfermagem seguindo as três fases orientadas por Bardin: pré-análise, exploração do material, tratamento e interpretação dos dados obtidos(13). Os depoimentos foram recortados em unidades de registro, que foram reunidas, quantificadas e, depois, coligadas pela convergência dos significados. O passo seguinte foi à categorização, de onde surgiram duas categorias temáticas com base nos objetivos do estudo: Orientação para mães sobe o método canguru baseada no principal cuidado da equipe de enfermagem e medidas preventivas para o sucesso do Método Canguru fundamentado no objetivo de precauções a serem tomadas na primeira etapa do MC com vistas a se evitar falha do referido método. “...A mãe chega e a gente explica o método canguru, que é bom para o desenvolvimento da criança, que ela sai mais rápido daqui, que ele fica mais aquecidinho, no colo da mãe, que melhora a temperatura...” (E5). “...eu faço as orientações para mãe de como é feito e porque ser feito. Sinto-me feliz quando realizo isso, esse procedimento. É algo muito especial colocar o RN na posição canguru quando essa mãe aceita, quando ela entende é muito bom...” (E9). Considerando-se as questões éticas, previamente ao início da pesquisa foi solicitada à instituição em questão, dessa autorização do estudo, sendo este submetido à apreciação do Comitê de Ética de Pesquisa da Universidade Federal do Piauí. Recebeu parecer favorável com o Registro no CEP-UFPI (CAAE: 34780914.2.0000.5214) em 28 de novembro de 2014(14). METODOLOGIA O presente estudo é uma pesquisa qualitativa de natureza descritiva, não se preocupando com uma quantidade numérica, mas sim, com o aprofundamento da compreensão dos cuidados prestado pelos profissionais de enfermagem aos recém-nascidos de baixo peso ao nascer em uma Unidade de Cuidados Intermediário Neonatal Convencional(11). Em 1979, para o atendimento de RNs prematuros e os de baixo peso, situação que envolvia uma alta mortalidade, foi desenvolvido em Bogotá, na Colômbia, o Método Mãe Canguru - MMC como uma alternativa de cuidado, criado pelos doutores Reys Sanabria e Hector Matinez. O método não só conseguiu reduzir a mortalidade, mas melhorou o desenvolvimento dos bebês. Desde então, o MMC vem sendo desenvolvido em vários países, principalmente, naqueles que não dispõem de um número suficiente de incubadoras. O método adotou o nome da espécie dos marsupiais, os cangurus, porque as crias nascem antes de completar o seu desenvolvimento e migram para uma bolsa e, nesta „incubadora natural‟, os filhotes são mantidos aquecidos, mamando avidamente até completar a sua maturação(6). A pesquisa descritiva observa, registra, correlaciona e descreve fatos ou fenômenos de uma determinada realidade sem manipulá-los, procurando conhecer e entender as diversas situações e relações que ocorrem na vida social, política, econômica e demais aspectos que ocorrem na sociedade(12). A pesquisa descritiva observa, registra, correlaciona e descreve fatos ou fenômenos de uma determinada realidade sem manipulá-los, procurando conhecer e entender as diversas situações e relações que ocorrem na vida social, política, econômica e demais aspectos que ocorrem na sociedade(12). A pesquisa contou com a participação dos profissionais de enfermagem (enfermeiros e técnicos de enfermagem) que estavam de plantão no período da coleta, mês de dezembro de 2014 e que aceitaram participar do estudo após esclarecimento dos objetivos. Foram inclusos no estudo os profissionais Foi apenas em 1986 que finalmente conseguiu validar com sucesso os benefícios desta estratégia para recém-nascidos prematuros, por meio da “Fundação Canguru” que modificou o “Método Mãe 69 ISSN: 2238-7234 Mendes GVS, et al. ISSN: 2238-7234 Mendes GVS, et al. Mendes GVS, et al. Kangaroo Care Method at Neonatal Intensive Care Unit Kangaroo Care Method at Neonatal Intensive Care Unit de enfermagem que estão a mais de um ano na UCINco e excluídos os que trabalham em outros setores da maternidade e/ou que trabalham a menos de um ano na UCINco. profissional de enfermagem em cuidar, promover a manutenção e recuperação do recém nascido enquanto estiver internado. Portuguese Rev Enferm UFPI. 2015 Oct-Dec;4(4):68-74. METODOLOGIA Mas para realizar essas orientações de forma efetiva com vista na obtenção de alcançar os objetivos do referido método, a equipe se depara com algumas dificuldades como a falta de treinamento, funcionários e estrutura física. “...nem todos os profissionais fazem de maneira adequada, vamos dizer assim que alguns não sabem como chegar, não sabem como abordar a mãe, tanto que algumas mães ficam até mais resistentes...” (E4). Os participantes da pesquisa assinaram o TCLE e foram informados quanto ao uso de um gravador durante as entrevistas e esclarecidos quanto ao anonimato e a liberdade de poderem desistir do estudo sem qualquer prejuízo. “...Acredito que para evitar falhas deveria ter mais treinamento, se pelo menos os profissionais que já foram treinado, treinasse os demais...” (E8). Caracterização dos sujeitos Participaram do Estudo 10 profissionais da área de enfermagem efetivos da Unidade de Cuidados Intermediário Neonatal Convencional (UCINco) de uma maternidade de referência do Estado do Piauí, sendo 4 enfermeiras e 6 técnicas de enfermagem, com tempo de atuação entre 1 ano e 1 mês e 37 anos. Todas foram submetidas a treinamento de referido método com carga horária variando entre 48 e 90 horas. “Bom devido a pouca quantidade de funcionários que a gente tem aqui no berçário, a gente faz o método canguru, mas nem sempre com a atenção que merecia ser dada, a atenção que o método exige, a atenção que o método espera...” (E1). “...Ai, às vezes, uma mãe chama e ai a outra chama, ai não dá para a gente estar presente sempre quando elas pedem ajuda...” (E6). DISCUSSÃO O Método Canguru surgiu como alternativa de cuidado humanizado, contribuindo para minimizar os efeitos prejudiciais durante a hospitalização e evoluiu a partir da necessidade de reduzir as infecções e problemas respiratórios que os recém- nascidos de baixo peso eram acometidos; humanizar o atendimento ao RN significa, entre outros, ter segurança técnica de atuação profissional, eficácia na atenção ao RN, condições hospitalares compatíveis com o período neonatal e para a participação da família no processo assistencial. Tudo isso, aliado à necessidade da atenção individualizada(15). “Eu acho que a primeira coisa é a receptividade dessa mãe aqui no setor, está orientando, mantendo o primeiro contato, deixando que ela fique mais segura para realizar o método canguru sem temer, sem ficar achando que ela pode está influenciando de uma forma negativa, deixando bem claro para ela que o método canguru é um método eficaz e que a equipe esta ali para colaborar...” (E2). individualizada . A adesão ao Método Canguru implica em uma série de critérios necessários que devem ser cumpridos integralmente para que seja efetiva: informação e o suporte para os cuidadores e família; o treinamento da equipe de saúde, dimensionamento pessoal suficiente para que seja dada a atenção necessária a essas mães e uma estrutura física confortável e adequada. A participação materna ao uso do Método Canguru aponta a necessidade de as equipes de saúde estar atentas para as características individuais das mulheres na realização da posição canguru, para não generalizar e idealizar a participação materna no MC, bem como a importância de fortalecer orientações e fornecer materiais que viabilizem a prática no contexto hospitalar, fase significativa para a manutenção da posição canguru após a alta(16). Principalmente no caso de bebês que dependem muitas vezes da ventilação mecânica, que usam oxímetro e que estão com acesso venoso, a abordagem para realizar o método é de forma gradativa. “A abordagem não é feita assim que são admitidas na UCINco, é de forma gradativa, é ao longo da permanência deles aqui. Porque quando chega é muita informação, geralmente vem no oxigênio a gente vai explicar e tudo, elas se preocupam muito com a questão da amamentação, depois que passa a fase da amamentação que a gente vai falando aos poucos, tem as reuniões lá fora, a psicóloga tem uma cartilha...” (E6). Medidas Preventivas para o sucesso do Método Canguru O discurso da equipe de enfermagem evidenciou algumas medidas preventivas para alcance dos benefícios do Método canguru. Entre estas, destaca- se as orientações para as mães de recém nascido que estão internados: ISSN: 2238-7234 Mendes GVS, et al. ISSN: 2238-7234 Mendes GVS, et al. Kangaroo Care Method at Neonatal Intensive Care Unit profissional, sem colocar dificuldades, sem criar mitos ou tabus, a grande maioria teme essa questão. Então precisamos estar ali do lado, esta orientando, fazendo da forma correta...” (E3). “...eu sinceramente mudaria todo o espaço físico do setor para um lugar mais apropriado...” (E8). Medidas Preventivas para o sucesso do Método Canguru Orientação para mães sobe o Método Canguru “...na minha opinião para realizar o método canguru temos que ter espaço, as mães que ficam aqui tem essas cadeiras que são muito desconfortável...” (E4). A conscientização das mães é um fator de extrema relevância, pois amplia o entendimento a respeito do método, tornando-o mais frequente e aproveitando as vantagens que ele proporciona para a instituição. Nesse âmbito surge a importância do 70 DISCUSSÃO A alta demanda assistencial na UCINco evidenciada muitas vezes por algumas particip da pesquisa que ressaltaram a sobrecarg trabalho, a falta de profissionais no plant número reduzido de recursos humanos, ac prejuízo na qualidade das atividades assiste prestada na unidade e na supervisão da m Trabalhar em UTIn de alto e médio risco im cuidar de RN que apresenta um quadro c instável, oscilante diuturnamente entre estad melhora e de piora. Tal situação expõe o profis de enfermagem a uma gama de estímulos emoc nocivos à sua saúde por lidar com o desafio d cotidiano de trabalho permeado por experiê ligadas à dor, ao sofrimento e a constante amea morte(24). A área física adequada é de extrema import uma vez que o estabelecimento à saúde d proporcionar condições para a permanênci tempo integral de um dos pais ou respons Atualmente a maior parte da UCINco trabalha um espaço físico inferior do que o recomendado proporcionando a mãe conforto que o método su Quando as necessidades do cuidado humaniza neonato são atendidas, a família se sente respe pela equipe, pois percebe a abordagem carin competente e responsável com que ela e o beb cuidados. Por outro lado, quando a equipe considera suas necessidades, deixando-a for cuidados prestados, faz com que se menosprezada e descuidada, criando um espaço desconfiança e medo, agravando ainda m contexto de sofrimento vivenciado(25). CONCLUSÃO O Método Mãe Canguru foi criado com o ob de diminuir a mortalidade neonatal, tra benefícios para os recém-nascidos, visan assistência na humanização, proporcionando vínculo com seus pais, melhorando na qualida desenvolvimento neurocomportamental psicoafetivo, incentivando o aleitamento ma permitindo controle térmico adequado, contrib para redução de infecção hospitalar, proporcio melhor relacionamento com da família e equi saúde. Os resultados evidenciaram que a equip enfermagem que participou do estudo apes orientar as mães sobre a importância de coloca ISSN: 2238-7234 Mendes GVS, et al. ISSN: 2238-7234 Mendes GVS, et al. Kangaroo Care Method at Neonatal Intensive Care Unit aprofundando os cuidado até a assistência integral(23). portanto, um novo pensar na equipe e proporciona maior autonomia materna(18). O profissional precisa estar atento para os problemas que podem ser vivenciados por essas mães, pois eles associados podem gerar profunda angústia, acrescidos de afastamento do local de internação e consequentemente do bebê interferindo na flexibilidade e na espontaneidade de praticar o método canguru(19). Portuguese Rev Enferm UFPI. 2015 Oct-Dec;4(4):68-74. DISCUSSÃO A alta demanda assistencial na UCINco, foi evidenciada muitas vezes por algumas participantes da pesquisa que ressaltaram a sobrecarga de trabalho, a falta de profissionais no plantão, o número reduzido de recursos humanos, acarreta prejuízo na qualidade das atividades assistenciais prestada na unidade e na supervisão da mesma. Trabalhar em UTIn de alto e médio risco implica cuidar de RN que apresenta um quadro clínico instável, oscilante diuturnamente entre estados de melhora e de piora. Tal situação expõe o profissional de enfermagem a uma gama de estímulos emocionais nocivos à sua saúde por lidar com o desafio de um cotidiano de trabalho permeado por experiências ligadas à dor, ao sofrimento e a constante ameaça de morte(24). Deve sempre ser comunicado a família as peculiaridades de seu filho e assegurado que a equipe estar disponível para os familiares do recém- nascido. Também estar adequando o cuidado de acordo com as necessidades individuais expressadas pelo bebe. A mímica facial deve ser conhecida pelos cuidadores(20). A área física adequada é de extrema importância, uma vez que o estabelecimento à saúde deverá proporcionar condições para a permanência em tempo integral de um dos pais ou responsáveis. Atualmente a maior parte da UCINco trabalha com um espaço físico inferior do que o recomendado, não proporcionando a mãe conforto que o método sugere. Quando as necessidades do cuidado humanizado ao neonato são atendidas, a família se sente respeitada pela equipe, pois percebe a abordagem carinhosa, competente e responsável com que ela e o bebê são cuidados. Por outro lado, quando a equipe não considera suas necessidades, deixando-a fora dos cuidados prestados, faz com que se sinta menosprezada e descuidada, criando um espaço para desconfiança e medo, agravando ainda mais o contexto de sofrimento vivenciado(25). DISCUSSÃO Durante o período de hospitalização do neonato a mãe demonstra insegurança, sofrimento, medo, incompreensão, desespero em relação ao quadro clínico do seu bebê, necessitando constantemente de informações. A enfermagem no seu processo de cuidar tem o papel de mediar essa ligação entre mãe e filho, promovendo a recuperação do desenvolvimento do recém-nascido para melhor funcionamento pessoal. O profissional precisa estabelecer formas de comunicação e interação com os familiares dos bebês internados na UCINco, incentivando a participação dos pais na assistência, orientando e estimulando o toque em seus filhos(17). Como medidas de prevenção, encontrou-se ainda a parceria com outros profissionais, o dimensionamento de pessoal, capacitação pessoal e empatia para com a mãe. “...porque se todos não falarem a mesma língua, vai ficar difícil a gente está praticando o método canguru, principalmente nos setores...” (E2). “...porque se todos não falarem a mesma língua, vai ficar difícil a gente está praticando o método canguru, principalmente nos setores...” (E2). “Com certeza a quantidade de profissionais deixa a desejar, às vezes a gente até começa orientar o método...” (E7). A promoção do cuidado materno emancipatório ao bebê prematuro deve fazer parte da práxis da equipe multidisciplinar no cuidado ao neonato. Ao apropriar- se dos códigos de comunicação da equipe, a mãe do prematuro habilita-se, da sua forma, para poder participar das decisões sobre as condutas terapêuticas direcionadas a seu filho. Assim, ao mesmo tempo em que o empoderamento materno causa certa estranheza e desconforto na equipe, a mãe redefine seu espaço na unidade neonatal. Uma vez que troca a imagem de fragilidade para uma posição mais assertiva em relação ao filho, a mãe passa a assumir seu lugar, capacitando-se a maternar seu filho, estimulando a interação. O MC estimula, “...acredito que fazer curso de aperfeiçoamento da equipe continuamente, o método parece ser simples, mas ainda existe muito mitos tipo, o bebê não pode ir para o canguru dependendo do halo, entende? Ai acho que um treinamento da equipe seria o ideal para evitar falhas...” (E10). “...a disponibilidade de estar se colocando com as mães, esta disponível para que elas tenha mais segurança, que elas consigam desenvolver o método com o apoio 71 ISSN: 2238-7234 Mendes GVS, et al. Kangaroo Care Method at Neonatal Intensive Care portanto, um novo pensar na equipe e proporciona maior autonomia materna(18). DISCUSSÃO O profissional precisa estar atento para os problemas que podem ser vivenciados por essas mães, pois eles associados podem gerar profunda angústia, acrescidos de afastamento do local de internação e consequentemente do bebê interferindo na flexibilidade e na espontaneidade de praticar o método canguru(19). Deve sempre ser comunicado a família as peculiaridades de seu filho e assegurado que a equipe estar disponível para os familiares do recém- nascido. Também estar adequando o cuidado de acordo com as necessidades individuais expressadas pelo bebe. A mímica facial deve ser conhecida pelos cuidadores(20). Mesmo sabendo-se que a presença da mãe no espaço assistencial é uma das formas de sua participação no cuidado ao recém-nascido e de ser apoiada pela equipe multiprofissional, apenas permitir o seu livre trânsito e permanência na UCINco não garante uma assistência humanizada, baseada na qualidade de vida, capacitação e empoderamento da mãe. É importante que se considere e respeite a singularidade das necessidades dessa mãe no ambiente hospitalar e que a metodologia do cuidado à mãe seja incorporada à assistência neonatal como parte do tratamento ao recém-nascido pré-termo, sendo assim, reconhece-se a importância da criação de espaços onde as mães possam expressar as demandas relacionadas a si mesmas e ao momento vivido, favorecendo a diminuição do estresse psicológico durante a internação e a formação de uma rede de apoio com outras mães que vivenciam a mesma situação e dessas com os profissionais de saúde. Além disso, esses espaços podem ser utilizados para promover ações de educação em saúde, contribuindo para um cuidado humanizado(21). Ressalta-se que a implementação do método requer o treinamento de toda a equipe que precisa saber lidar com as emoções, adquirindo sensibilidade e capacidade de escuta. O profissional tem que estar compromissado em atingir os objetivos do método, buscando melhorar sua prática, influenciando diretamente na recuperação do recém nascido. Preparar a família para o acolhimento do RN e atentar para as solicitações, expectativas e sentimentos, são ações da equipe de enfermagem que promove o fortalecimento do vínculo afetivo, favorecendo também o desenvolvimento físico e psíquico do RN, na recuperação da saúde, por meio do atendimento humanizado(22). Essa capacitação consiste em habilitar esses aprofundando os cuidado até a assis integral(23). REFERÊNCIAS 14. Ministério da Saúde (BR). Conselho Nacional de Saúde. Resolução nº 466 de 12 de dezembro de 2012 – Diretrizes e Normas Regulamentadoras da Pesquisa Envolvendo Seres Humanos. Brasília, 2012. 1. Ministério da Saúde (BR). Secretaria de Politicas de Saúde. Área de Saúde da Criança. Atenção humanizada ao recém-nascido de baixo peso: Método Mãe-Canguru: manual do curso. 2. ed. Brasília: Ministério Da Saúde; 2011. 1. Ministério da Saúde (BR). Secretaria de Politicas de Saúde. Área de Saúde da Criança. Atenção humanizada ao recém-nascido de baixo peso: Método Mãe-Canguru: manual do curso. 2. ed. Brasília: Ministério Da Saúde; 2011. 15. Silva RA, Barros MC, Nascimento MHM. Conhecimento técnico de enfermagem sobre o Método Canguru na Unidade Neonatal. Rev. Brasileira em Promoção da Saúde. 2014; 27(1):124-130. 2. Ministério da Saúde (BR). Rede Interagencial de Informações para a Saúde. Indicadores de mortalidade: IDB 2012. Brasília: RIPSA, 2012. Disponível em 16. Spear MC, Seild EMF. Percepção Materna no Método Canguru: contato pele a pele, amamentação e autoeficácia. Rev. Eletronic Library Online. 2013; 18(4):647- 656. <http://tabnet.datasus.gov.br/cgi/idb2012/matriz.h tm> Acesso em 07 de jul de 2014. 3. BRASIL. Ministério da Saúde. Portaria nº 1.683, de 12 de julho de 2007. Normas de Orientação para a Implantação do Método Canguru. Diário Oficial da União, Poder Executivo. Brasília: DF; 2010. 17. Silva ARE, Garcia RN, Guarigua DA. Método Canguru e os Benefícios para o recém nascido. Revista Horus. 2013;7(2):1-11. 4. Ministério do Planejamento, Orçamento e Gestão (BR). Instituto Brasileiro de Geografia e Estatística. Contagem Populacional. Disponivel em: http://www.sidra.ibge.gov.br/bda/procurar/resulta do.asp?palavra=%C3%B3bito+infantil&o=1&esc=1. Acesso em: jul. 2014. 18. Nunes PN, Pessoa UML, Bucharles DG, Alverne M, Sá FE, Carvalho EM. Método Canguru: percepção materna acerca da vivência na Unidade de Terapia Intensiva Neonatal. Rev. Brasileira em Promoção à Saúde. 2015; 28(3):387-393. 19. Queiroz NA, Maranhão DG. Ações e cuidados de enfermagem na implementação do Método Mãe Canguru. Revista de Enfermagem UNISA. 2012; 13(2):121-125. 5. Ministério da Saúde (BR). Secretaria de Atenção à Saúde. Atenção à Saúde do Recém-nascido: Guia para Profissionais: Cuidados Gerais. 2ª ed. Vol. 1. Brasília: Ministério da Saúde; 2012. 20. Ministério da Saúde (BR). Secretaria de Politicas de Saúde. Área de Saúde da Criança. Atenção humanizada ao recém-nascido de baixo peso: Método Mãe-Canguru: manual do curso. 2. ed. Brasília: Ministério Da Saúde; 2011 p.23. 6. Cardoso ACA, Romiti R, Ramos JLA, Issler H, Grassiotto C, Sanches MTC. Método mãe-canguru: aspectos atuais. Pediatria 2006; 28(2):128-134. 7. CONCLUSÃO O Método Mãe Canguru foi criado com o objetivo de diminuir a mortalidade neonatal, trazendo benefícios para os recém-nascidos, visando à assistência na humanização, proporcionando maior vínculo com seus pais, melhorando na qualidade do desenvolvimento neurocomportamental e psicoafetivo, incentivando o aleitamento materno, permitindo controle térmico adequado, contribuindo para redução de infecção hospitalar, proporcionando melhor relacionamento com da família e equipe de saúde. Os resultados evidenciaram que a equipe de enfermagem que participou do estudo apesar de orientar as mães sobre a importância de colocar seu bebê na posição canguru ainda se depara com algumas falhas e dificuldades no cuidado e incentivo para a obtenção eficaz dos objetivos do Método Canguru. Essa capacitação consiste em habilitar esses profissionais para o atendimento humanizado, considerando as peculiaridades físicas e psicológicas de cada caso, as particularidades do psiquismo da mãe, da família e do recém-nascido, as características do ambiente, as interações e competência do recém-nascido e o desenvolvimento do apego. Faz- se necessário, entretanto, que as orientações sobre cuidado materno ao filho sejam diárias e contínuas, devendo a mãe ser introduzida gradativamente no processo de cuidado. Inicialmente, estimula-se a realização de cuidados mais simples, como higiene, banho, troca de fraldas, toque, alimentação da criança e vai gradativamente A carência de informação a respeito do método, a falta de treinamentos recorrentes para profissionais, o dimensionamento adequado e uma estrutura física satisfatória são lacunas que evidenciam que são necessárias mudanças na implementação no MC. 72 ISSN: 2238-7234 Mendes GVS, et al. ISSN: 2238-7234 Mendes GVS, et al. Kangaroo Care Method at Neonatal Intensive Care Unit Kangaroo Care Method at Neonatal Intensive Care Unit Portuguese Rev Enferm UFPI. 2015 Oct-Dec;4(4):68-74. REFERÊNCIAS Santos LM, Morais RA, Miranda JOF, Santana RCB, Oliveira VM. Percepção maternal sobre o contato pele a pele com prematura através da posição canguru. Rev. de Pesquisa: cuidado é fundamental online. Rio de Janeiro. 2013; 5(1):3504-3514. 21. Duarte DE, Ditz ES, Silva BCN, Rocha LLB. Grupos de apoio às mães de recém-nascidos internados em Unidade Neonatal. Rev. Rene. 2013;14(3):630- 638. 22. Rocha RS, Lucio IML, Lopes MMCO, Lima CRC, Freitas ASF. Promoção do cuidado humanizado à família pela equipe de enfermagem da Unidade Neonatal. Rev. Rene. 2011;12(3):502-509. 8. Ministério da Saúde (BR). Portaria nº 1.683, de 12 de julho de 2007. Normas de Orientação para a Implantação do Método Canguru. Diário Oficial da União, Poder Executivo. Brasília: DF; 2011. 23. Araújo BBM, Rodrigues BMRD, Pacheco STA. A promoção do cuidado materno ao neonato prematuro: a perspectiva da educação problematizadora em saúde. Rev. Enfermagem UERJ. 2014;23(1):128-131. 9. Freitas MIF, Xavier CC, Gontijo TL. Avaliação da Implantação do Método Canguru por gestores profissionais e mães de recém-nascidos. Cad. Saúde Pública. Rio de Janeiro. 2012; 28(5):935-944. 10. Oliveira TC, Ancântara KS, Mascarenhas MFP, Romão EF, Torres SMF. Implantação do Método Canguru em Unidade de Terapia Intensiva Neonatal: relato de experiência. Rev. de Enfermagem UFPE on line. Recife. 2014;8(7):2171-2174. 24. Oliveira EB, Silva AV, Junior EFP. Fatores de risco psicossocial em terapia intensiva neonatal: repercussões para saúde do enfermeiro. Rev. Enfermagem UERJ. 2013;21(4):490- 495. 25. Balbino FS, Meschinig GFG, Balieiro MMFG, Mandetta MA. Percepção do Cuidado Centrado na Família em Unidade Neonatal. Rev. Enfermagem da UFSM. 2016;6(1):84-92. 11. Minayo MCS. Pesquisa social; teoria, método e criatividade. Petrópolis/RJ: Vozes, 2010. 12. Paternez AC, Chinen JC. Normas para elaboração de trabalhos acadêmicos. Faculdades Integradas Coração de Jesus. Santo André. 2004. 13. Bardin L. Análise de conteúdo. Lisboa: Edições 70; 2009. 73 Portuguese Rev Enferm UFPI. 2015 Oct-Dec;4(4):68-74. Kangaroo Care Method at Neonatal Intensive Care Unit Kangaroo Care Method at Neonatal Intensive Care Unit Sources of funding: No Conflict of interest: No Date of first submission: 2015/04/06 Accepted: 2015/11/10 Publishing: 2015/12/01 Corresponding Address Gabrielle Visgueira Soares Mendes Endereço: Campus Universitário Ministro Petrônio Portella, s/n - Ininga, Teresina - PI, 64049-550 Universidade Federal do Piauí, Teresina. Telefone: (98) 982676829 E-mail: gabivisgueira@gmail.com 74
https://openalex.org/W3160913228	https://www.researchsquare.com/article/rs-74918/v1.pdf?c=1603128397000	English	null	Interpersonal trauma and its relation to childhood psychopathic traits: what does ADHD and ODD add to the equation?	Research Square (Research Square)	2,020	cc-by	7,154	Interpersonal trauma and its relation to childhood psychopathic traits: what does ADHD and ODD add to the equation? John Marshall (  johnj.marshall@nhs.net ) Glasgow Caledonian University School of Health and Life Sciences Karolina Sorman Karolinska Institutet Natalie Durbeej Karolinska Institutet Lucy Thompson University of Glasgow Institute of Health and Wellbeing Sebastian Lundström Goteborgs Universitet Helen Minnis University of Glasgow Institute of Health and Wellbeing Clara Hellner Karolinska Institutet Christopher Gillberg Goteborgs Universitet Results Linear regressions demonstrated statistically significant but clinically negligible effects of interpersonal trauma on total and subscale scores of parent-rated psychopathic traits. When exploring interaction effects of ADHD and ODD into the model, the effect increased. There were interaction effects between ODD and trauma in relation to psychopathic traits, suggesting a mediating role of ODD. Having been exposed to trauma before age 10 was significantly associated with higher parent rated psychopathy traits as measured by The Child Problematic Traits Inventory-Short Version (CPTI-SV), however the explained variance was small (0.3–0.9%). Conclusions The results challenge the notion of association between interpersonal trauma and youth psychopathic traits. They also highlight the need to gain an improved understanding of overlap between psychopathic traits, ADHD and ODD for clinical screening purposes and the underlying developmental mechanisms. Methods We examined to what degree childhood interpersonal trauma can predict parent-rated psychopathic traits in a large population based Swedish twin sample (N = 5057), using a stringent definition of interpersonal trauma occurring before age 10. Two hundred and fifty-one participants met the interpersonal trauma criteria for analysis. The study explored the additional impact of traits of attention-deficit hyperactivity disorder (ADHD) and oppositional defiant disorder (ODD). Background Childhood trauma has demonstrated associations with callous-unemotional traits (e.g., reflecting lack of remorse and guilt, unconcern about own performance). Less is known about associations between trauma and multiple domains of child psychopathic traits. There has also been limited focus on the role of co-occurring disorders to psychopathy traits among children, namely, attention-deficit hyperactivity disorder (ADHD) and oppositional defiant disorder (ODD) and how they interact with childhood trauma. Research article Keywords: ADHD, ODD, CPTI-SV License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Psychiatry on December 1st, 2021. See the published version at https://doi.org/10.1186/s12888-021-03610-7. Page 1/17 Trauma and psychopathic traits Childhood trauma or adversity can be among several factors predicting adult psychopathy.9 Even though research on childhood psychopathic traits has mainly focused on heritable pathways, risk trajectories, and neurobiological substrates,10 some research has explored associations between trauma and psychopathic traits. For example, a systematic review demonstrated how parenting styles may modulate the expression of CU traits.11 Risk factors in the parental style that may exacerbate CU traits include reduced quality of parent–child affective interactions, reduced eye contact, lower observed dyadic warmth and lower parental sensitivity.12 Harsh parenting experienced in early childhood,13,14 corporal punishment,15 psychological aggression, non-consistent discipline experienced in middle childhood,16 and poor parent–child communication during adolescence17 all predict higher levels of CU traits. Conversely, positive aspects of parenting may assist in the prevention of CU traits. In summary, problematic parenting may intensify CU traits, suggesting that childhood interpersonal trauma (CIT) may also have an amplifying effect on psychopathic traits. There are no studies concerning childhood interpersonal trauma and the full range of psychopathic traits. Background Research on youth psychopathic traits has mainly focused on callous unemotional (CU) traits reflecting a lack of remorse and guilt and unconcern about own performance.1 Factor analytic studies, however, have demonstrated the multi-dimensional nature of psychopathic traits in youth across measures.2 However, Page 2/17 Page 2/17 youth psychopathic traits are proposed to encompass several domains, including grandiose-manipulative (GM), and daring-impulsive (DI) traits.3 Antisocial outcomes are more likely to be predicted by the combination of the three domains of traits, rather than CU traits alone.4,5 Adult antisocial outcomes are more likely to be predicted by the combination of childhood interpersonal, affective, and behavioral traits than CU traits alone.4,5 For example, interpersonal psychopathic traits may be uniquely related to bullying, relational aggression, and delinquency, more so than CU traits.6 Moreover, studies concerned with cognitive functioning have found that GM traits within the psychopathy construct are critical to unprovoked aggression.7 Childhood studies are mainly concerned with CU traits and have not fully captured the construct of psychopathy, thereby potentially limiting risk prevention efforts. To understand psychopathic traits as a broad neurodevelopmental condition rooted in early childhood, it is important to investigate the manifestation and correlation of all three components of psychopathic traits (i.e., also including impulsive and grandiose traits). Childhood psychopathic traits manifesting in early or middle childhood are an important (albeit moderate) predictor of adolescent delinquency, general recidivism, and violent recidivism.8 Such traits also constitute a critical risk factor for adult psychopathy, antisocial behavior and substance abuse.1 Childhood interpersonal trauma and psychopathic traits CIT (e.g., physical, emotional, sexual abuse, physical and emotional neglect) is associated with a broad range of mental disorders in childhood and adulthood, including personality disorders (Waxman et al., 2014).18,19,20,21 Adult offenders consistently report higher levels of CIT compared to normative samples (Waxman et al., 2014),21 and meta-analytic data has demonstrated a strong relationship between adverse Page 3/17 Page 3/17 childhood experiences (ACES), including CIT-perpetrating adult interpersonal violence and a range of mental disorders.22 CIT has been proposed as a critical factor in the development of psychopathic traits; however, most studies have focused on the association between previous trauma and psychopathy in adulthood, asking participants to recall any previous CIT. In some retrospective studies, psychopathic adults have reported extensive CIT where the strongest associations are with physical abuse.23 In other forms of CIT (e.g., neglect), blunted affective expression, alexithymia and the unemotional features of psychopathy have been identified as concomitants, implying a direct link between aspects of psychopathy and CIT.24 Some studies indicate that putative CIT environmental factors such as parental rejection, neglect, and abuse are critical in the etiology of antisocial behavior and adult psychopathy.25,26,27 One study demonstrated that adults with psychopathy who had committed murder, rape, or child abuse were relatively more likely to report childhood interpersonal trauma than those who were not psychopathic.28 In particular, physical or emotional abuse and neglect have been associated with interpersonal callousness.29 In addition, research has identified prospective links between general psychosocial adversity and CU traits, including high levels of chaos in the home.30 Psychopathic traits and neurodevelopmental disorders (NDDs) Some youth with psychopathic traits present with a complex range of co-occurring neurodevelopmental disorders (NDDs). NDDs share symptomatology and etiology with each other and one diagnosis in childhood may be labeled as another disorder in adulthood.31 For example, psychopathic traits, in particular impulsivity, tend to co-occur with symptoms of attention-deficit hyperactivity disorder (ADHD), reflecting poor concentration, impulsivity, and overactivity.32,33,34 Psychopathic traits remain significantly higher in young offenders with ADHD after controlling for age, substance abuse, and early childhood adversities.35 Conversely, previous studies have demonstrated that children with conduct problems and co-occurring psychopathic traits have higher levels of fearlessness and ADHD symptoms, compared to children with conduct problems and CU traits alone.36 Research exploring the etiological underpinnings of co-occurring psychopathic traits and NDDs, particularly for interpersonal trauma, is notably lacking. Studies that are genetically sensitive, taking account of familial confounding on the effects of childhood traumatic experience on child and adult psychopathology, have revealed a shared liability in the causal role of traumatic events and NDDs. 37 Research exploring the etiological underpinnings of co-occurring psychopathic traits and NDDs, particularly for interpersonal trauma, is notably lacking. Studies that are genetically sensitive, taking account of familial confounding on the effects of childhood traumatic experience on child and adult psychopathology, have revealed a shared liability in the causal role of traumatic events and NDDs. 37 Dinkler et al 37 found that the co-occurrence of childhood maltreatment and NDD symptoms to a large extent was accounted for by a shared genetic liability, increasing both the risk of being maltreated and of having more co-occurring NDDs. Psychopathic traits in childhood could be at least partly transdiagnostic interacting with the complexity of other neurodevelopmental comorbidities. Whether CIT is associated with the several dimensions of childhood psychopathic traits, taking account of NDDs, has not been established with a child population sample as opposed to stand-alone CU traits. The role of childhood interpersonal trauma factors impacting childhood psychopathy traits via NDDs such as ADHD is Dinkler et al 37 found that the co-occurrence of childhood maltreatment and NDD symptoms to a large extent was accounted for by a shared genetic liability, increasing both the risk of being maltreated and of having more co-occurring NDDs. Psychopathic traits in childhood could be at least partly transdiagnostic, interacting with the complexity of other neurodevelopmental comorbidities. Participants In this study, data from the Child and Adolescent Twin Study in Sweden (CATSS) was used (see Anckarsäter et al 2011 38 for a description of the CATSS-study). Briefly, parents of all twins born in Sweden from the 1st of July 1992 are contacted in connection with the twins’ 9th birthday (the first three years of the study also included 12-year old’s) and asked to participate in a telephone interview (CATSS- 9). At age 15, the twins and their parents are contacted again (CATSS-15) and asked to log on to a web page and fill out questionnaires. The CATSS-15 includes individuals born 1st January 1994 and onwards. Among participants in CATSS-9, 50% also responded in CATSS-15. In this study, participants born from January 1st, 1994, to June 31st, 1998, were included (as one of the instruments used was excluded from the study protocol in 1998). At the assessment at age 15, 5075 individuals had complete responses to A- TAC, CPTI-SV, and trauma. Thus, the final sample comprised 5057 participants among which 2408 (49.4%) were boys and 2463 (50.6%) were girls. Psychopathic traits and neurodevelopmental disorders (NDDs) Whether CIT is associated with the several dimensions of childhood psychopathic traits, taking account of NDDs, has not been established with a child population sample as opposed to stand-alone CU traits. The role of childhood interpersonal trauma factors impacting childhood psychopathy traits via NDDs such as ADHD is Page 4/17 Page 4/17 unknown, though such problems could feasibly exacerbate psychopathy via NDDs given how inattention and impulsivity might interact with core psychopathy traits. unknown, though such problems could feasibly exacerbate psychopathy via NDDs given how inattention and impulsivity might interact with core psychopathy traits. This study examines to what degree childhood interpersonal trauma can predict parent-rated psychopathic traits in a large population-based Swedish twin sample. The study has two unique features: (i) a stringent definition of childhood interpersonal trauma occurring before age 10 and (ii) exploring the additional impact of traits of attention-deficit hyperactivity disorder (ADHD) and oppositional defiant disorder (ODD). This can further our knowledge of the pathways involved in the development of child psychopathic traits and may help inform the types of preventive interventions that are appropriate for this group of children. Measures The Child Problematic Traits Inventory-Short Version (CPTI-SV) was used to assess parent-rated psychopathic traits in CATSS-9. This is an abbreviated 12-item version of the 28-item Child Problematic Traits Inventory. 39 The CPTI was developed to assess all three dimensions of psychopathic traits in children, with three subscales: grandiose deceitful (GD), callous unemotional (CU), and impulsive need for stimulation (INS). The development of CPTI was theoretically based and guided by some prerequisites: included items have empirical support for being assessable in children and do not directly reflect rule breaking (e.g., conduct symptoms, antisocial behavior). CPTI has demonstrated adequate psychometric properties across studies, including excellent internal consistency and support for the three-factor structure using different informants (i.e., teachers and parents), genders and age groups. 39 Items of the CPTI-SV were selected by the test developer, based on the perceived degree of correspondence with the three domains of psychopathic traits (Andershed, personal communication). The CPTI-SV encompasses four items from the GD-subscale (e.g., “Is often superior and arrogant towards others”), four items from Page 5/17 the CU-subscale (e.g., “Usually does not seem to share others’ joy and sorrow”), and four items from the INS-subscale (e.g., “Often does things without thinking ahead”). In line with the original scale, items were rated based on the child’s typical behavior with the following 4-point Likert scale: 1 (does not apply at all), 2 (does not apply well), 3 (applies fairly well), and 4 (applies very well). The Life Stressor Checklist-Revised (LSC-R) 40 was used to assess CIT. The LSC-R is a screening measure of traumatic life events according to DSM-IV criteria for posttraumatic stress disorder and other seriously stressful life events. In this study, CIT refers to interpersonal victimization, the elements of malevolence, betrayal, injustice, and immorality are more likely to be factors than in accidents, diseases, and natural disasters.40 This definition of CIT did not include, for example, children, who were “victims” of accidents. To assess CIT at age 15, we used 9 dichotomous (i.e., yes/no) parent-reported questions (covering the following themes; emotional abuse; physical neglect; physical abuse; sexual touch; forced sex; witnessing physical violence in family; witnessing a threatening or violent incident; victim of hate crime; see appendix 1a for the questions). The Autism-Tics, ADHD and other Comorbidities Inventory (A-TAC) wa The Autism-Tics, ADHD and other Comorbidities Inventory (A-TAC) was used to assess symptoms of ADHD and ODD in CATSS-9. This measure has been validated in cross-sectional and longitudinal studies. 41 The A-TAC is a fully structured parental telephone interview measuring various domains of child and adolescent psychiatry. It was designed for use by laypeople over the phone. It consists of 96 symptom questions, which are asked from a lifetime perspective (Larson et al., 2014). Out of the 96 questions, 19 pertains to the ADHD-domain and five to the ODD-domain. All items in the A-TAC are coded ‘no’ (0), ‘yes, to some extent’ (0.5) and ‘yes’ (1). The ADHD domain displays an area under the curve of .93 and α of .92 in a population-based sample including individuals who were diagnosed with ADHD before the age of 10. 41 In the current study, a screening cut-off for ADHD symptoms (≥ 6) was used, with a sensitivity of .79 and a specificity of .90. ODD has been reported to have an AUC of .999 with α of .75. The cut-off for ODD was ≥ 3 with corresponding sensitivity and specificity of .51 and .55. Measures An open question was also asked, “Has the child ever been direct witness (not film, internet or TV) to any other serious incident that you have not mentioned?” Two authors (JM & KS) reviewed responses to this open question. A response that indicated an act directed towards the child or if the child was a direct witness was deemed as likely to have caused a traumatic response (e.g., seeing the mother being beaten, being exposed to mental abuse). Of the 419 answers on the open question, four were considered to reflect interpersonal trauma. If a parent endorsed a question about maltreatment, a follow-up question inquiring at what age the maltreatment had happened the first and/or the last time was asked. Only maltreatment reported to have occurred before the twins’ 10th birthday was included. In a subsequent step, participants who had not already reported trauma before age 10 but endorsed trauma at age 9 were added to the analysis. This assessment was based on three parameters: witnessing violence, being robbed, and trauma (including bullying, sexual abuse, and maltreatment, gauged through an open question). The responses to the open questions were reviewed by the two authors (JM & KS). Descriptive At age 15, a total of 193 individuals endorsed having been exposed to our stringent definition of CIT before age 10. At age 9, the number of individuals reporting at least one type of interpersonal trauma (not already included at age 15) was 58. In total, therefore, 251 participants had been exposed to CIT before age 10. A total of 306 participants (6.1%) reported symptoms of ADHD and 54 (1.1%) reported symptoms of ODD (see Table 1). Mean scores of the CPTI-SV scales in the sample and subgroups are demonstrated in Table 1. A total of 306 participants (6.1%) reported symptoms of ADHD and 54 (1.1%) reported symptoms of ODD (see Table 1). Mean scores of the CPTI-SV scales in the sample and subgroups are demonstrated in Table 1. ( ) p g p 1. Relations between trauma, psychopathic traits and NDDs Single linear regressions exploring associations between trauma and CPTI-SV scores are demonstrated in Table 2. Having been exposed to trauma before age 10 was significantly associated with higher CPTI-SV (across all scales); however, the explained variance was small (0.3 – 0.9%). Multiple linear regressions were computed to explore the impact of ADHD and ODD (Tables 3-5). As demonstrated, CPTI-SV total scores were predicted by trauma before age 10 and ADHD. The interaction between interpersonal trauma and ADHD was not significant (Table 3). Moreover, both trauma and ODD predicted higher CPTI-SV total scores, and there was a significant interaction between trauma and ODD. Both trauma and ADHD predicted higher CPTI-SV GD scores; however, there was no significant interaction between the two variables (Table 4). A corresponding pattern emerged when ODD was added to the model. When using CPTI-SV CU scores as an outcome, both trauma and ADHD predicted higher scores, but the interaction term did not (Table 5). In contrast, both trauma and ODD predicted higher scores as well as the interaction between the two variables. Finally, trauma, ADHD, and ODD all predicted higher CPTI-SV INS scores but there was no interaction between trauma and ADHD or ODD. Statistical analyses Page 6/17 Page 6/17 Descriptive statistics (means, standard deviations, frequencies and percentages) were used to describe the sample and paired sample t-tests were used to compare means. Relations between trauma and psychopathic traits were explored using single and multiple linear regressions. In the multiple linear regression analysis, we investigated the single contributions of and interactions between trauma, ADHD, and ODD. The CPTI-SV total and subscale scores were used as dependent variables. All analyses were computed using SPSS, version 23, and STATA, version 14. Discussion Page 7/17 This study set out to explore associations between childhood interpersonal trauma (CIT), all three domains of psychopathic traits, and NDDs. The main finding is that CIT had a significant but negligible association with CPTI-SV-assessed youth psychopathic traits. The prediction of psychopathic traits by interpersonal trauma was small, indirect, and mediated via NDDs, specifically ODD. The direct association between CIT and psychopathic traits was low, but there was an effect via the mediating role of NDDs. The absence of a childhood interpersonal trauma association with psychopathic traits in this study might indicate that maltreatment is a signal for the presence of neurodevelopmental disorders transmitted from adult to child via genetic factors. CU-traits are demonstrated to be moderately to strongly heritable.42 Therefore, children’s pre-existing psychopathic traits may evoke negative parenting responses. 43 Conversely, differential susceptibility indicates that positive parenting may buffer the emergence of genetically underpinned psychopathy traits. Studies concerned with maltreatment and childhood CU traits may highlight a complex reciprocal interplay between interpersonal trauma, genetic risk, and developmental status in emerging psychopathic traits. Maltreatment in the form of harsh parenting impacted younger children transitioning from toddlerhood to preschool more, albeit moderated by genetic risk for these traits. 14 It may be that there is a critical earlier developmental window for CIT to impact later emerging psychopathic traits and that more specific timing of CIT needs to be included in future research to better understand this. Alternatively, biological and underpinning temperamental drivers, such as fearlessness, for psychopathy traits may eclipse psychosocial factors, compared to a CIT-based etiology. This finding runs counter to several previous studies on adults and youths, demonstrating some relationship between maltreatment (or problematic parenting) and psychopathic traits. It is important to point out, however, that previous studies have generated mixed findings. In a sample of youth (11– 17 years old), Milone et al (2019) demonstrated associations between maltreatment and externalizing behaviors, but not CU traits. 44 A recent systematic review found that primary psychopathy may precede the effects of adverse childhood experiences.45 About 2% of our sample experienced CIT. In other population samples of similar-aged children, the incident rate of child abuse or maltreatment is reported at 2.5%. 46 The causal nature of CIT as an environmental factor impacting the propensity for psychopathic traits is difficult to determine without behavioral genetic methodologies such as co-twin designs to account for genetic and shared environmental confounding. Discussion Furthermore, there are parental effect causal assumptions underlying association studies.37 Dinkler et al37 hypothesize that the parents’ genes related to their own neurodevelopmental traits are correlated with their children’s genetically influenced NDDs and at the same time may increase the risk of the parents maltreating their child. Moreover, Dinkler et al found a small increase in symptoms of ADHD and Autism Spectrum Disorder in maltreated MZ twins as compared with their non-maltreated co-twins.37 Page 8/17 Page 8/17 The results demonstrated a potential mediating role of ODD, in the associations between psychopathic traits and trauma. ODD is characterized by defiant, disobedient, and hostile behavior towards authority figures. Where children present with ODD and symptoms of ADHD, this may put greater strain on parents, adversely affect their sense of competence, and child ODD behaviors, in particular, may be more influential in engendering hostile negative parenting behaviors than vice versa. 47 Parents are known to use harsh parenting where children have callous-unemotional traits along with conduct problems or other externalising behavior problems, such as ODD, compared to CU traits alone. 48 Additional layers of oppositionality among children with psychopathic traits may evoke greater risk for parental maltreatment. Core deficits in children with psychopathic traits, such as their ability to recognize others’ distress cues43 and pervasive lack of concern for others’ feelings,44 are likely to engender negative behaviors from peers and parents, particularly where the latter are also experiencing NDDs, compromising parenting capacity. This study did not find an important relationship between CIT and psychopathy traits perhaps because of causal models involving a developmental cascade from genetic factors to early trait/temperament precursors and their interactions with parental factors across different developmental periods. Causal mechanisms can only be elucidated by genetically informed research designs. Given the mediating role of NDDs such as ODD, it is important to study different combinations of neuro- developmental problems along with psychopathic traits. For example, the combination of ADHD symptoms may be linked to particularly high-risk pathways of callousness, impulsivity, or narcissism. 49,50 ODD is also often co-morbid with ADHD and can be associated with poorer life outcomes. Such combinations of ODD, ADHD, and psychopathic traits may pose a greater parenting challenge than psychopathic traits alone and increase the likelihood of maltreatment, leading to CIT. The early identification of combinations of overlapping NDDs among children with psychopathic traits may, therefore, be critical for prevention. Discussion Treatment for co-occurring NDDs such as ODD and ADHD may be important in deflecting negative outcomes for children with psychopathic trait trajectories. Further behavioral genetic research using co-twin design methods is needed to disentangle etiological gene x environment factors with the inclusion of CIT. Abbreviations ADHD: Attention Deficit Hyperactivity Disorder Conclusion CIT confers negligible variance for psychopathy traits among children in this study, contrary to retrospective evidence among psychopathic adults and to studies of hostile parenting among younger children with CU traits who are at risk of developing psychopathy. The variance of psychopathy traits explained by CIT in the context of co-occurring ADHD and ODD remains low. The need for genetically informed studies that consider CIT at various stages of childhood risk for psychopathy traits is critical along with how psychopathy traits interact with cascade in combination with other disorders such as ADHD and ODD. Strengths And Limitations This study has several strengths, including a prospective design, large sample size, assessment of all three dimensions of psychopathic traits, and the stringent definition of childhood interpersonal trauma. Moreover, the community sample used in the present study also should make the results more generalizable than association studies based on clinical, incarcerated, or offending samples. The study is also hampered by several limitations. In contrast to the other scales used, the CPTI-SV has not been formally validated (i.e., no factor analysis has been conducted). Therefore, subscale scores should be interpreted with caution. Future studies should apply a genetically informed research design to infer the causal mechanisms with regards to CIT in population cohorts. Studies should also apply the broad conceptualisation of psychopathic traits rather than CU traits alone and consider the differential impacts of primary versus secondary psychopathy in the context of CIT. Given that the LSC-R is parent reported in Page 9/17 Page 9/17 this study, biases might have been introduced (see Dinkler et al 2016 37 for a discussion of validity and reliability of the LSC-R in previous studies.) this study, biases might have been introduced (see Dinkler et al 2016 37 for a discussion of validity and reliability of the LSC-R in previous studies.) Consent for publication Ethical approval from the Swedish Ethical Review Authority (DNR: 2010/597-31/1; 2016/2135- 31;2009/739-31-5) included consent to publish. Ethics approval and consent to participate The parents were informed about the study in connection with their twins 9th and 15 the birthday. The parents were informed about the study in connection with their twins 9th and 15 the birthday. Participating in interviews, after being informed about the study, is regarded as verbal informed consent (age 9) and, at age 15 the parents agreed to participate via a web-questionnaire and digitally consented after being informed about the study. Both the CATSS-9 and 15 have ethical approval from the Swedish Ethical Review Authority (DNR: 2010/597-31/1; 2016/2135-31;2009/739-31-5) in which the procedures above were described and approved. Competing interests The authors have no competing interests. Funding There was no funding provided for this study. Authors' contributions Page 10/17 Dr John Marshall conceived the idea for the study, Dr’s John Marshall and Karolina Sörman co-wrote the manuscript. Dr Natalie Durbeej advised on data analysis and methods. Dr Lucy Thompson reviewed the manuscript. Dr Sebastian Lundström conducted data analysis and wrote the results section. Dr Clara Hellner supported data analysis. Professors Helen Minnis and Christopher Gillberg supervised the project. Acknowledgements Thank you to the parents and young people who participated in the CATSS-9 studies. Thank you to the parents and young people who participated in the CATSS References 1. Frick PJ, Ray J, Thornton L, Kahn RE. Can callous-unemotional traits enhance the understanding, diagnosis, and treatment of serious conduct problems in children and adolescents? A comprehensive review. Psychological Bulletin.2014;140:1-57. 2. Salekin RT, Andershed H, Batky, BD, Bontemps AP. Are callous unemotional (CU) traits enough? Journal of Psychopathology and Behavioral Assessment. 2018;40:1-5. 3. Salekin RT. Review: What do we know about psychopathic traits in children? 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CPTI-SV scores (mean values) in the total sample, and subgroups with and without trauma before age 10 Page 14/17 CPTI-SV total M (SD) GD M (SD) CU M (SD) INS M (SD) N = 5057 1.36 (.30) 1.13 (.28) 1.14 (.31) 1.82 (.57) Subsample without trauma before age 10 (n = 4807) 1.36 (.30) 1.12 (.27) 1.14 (.30) 1.81 (.56) Subsample with trauma before age 10 (n = 250) 1.49 (.40) 1.21 (.40) 1.22 (.43) 2.02 (.66) Note. CPTI-SV = Child Problematic Traits Inventory. GD = Grandiose Deceitful. CU = Callous Unemotional. INS = Impulsivity Need for stimulation. Note. CPTI-SV = Child Problematic Traits Inventory. GD = Grandiose Deceitful. CU = Callous Unemotional. INS = Impulsivity Need for stimulation. atic Traits Inventory. GD = Grandiose Deceitful. CU = Callous Unemotional. able 2. Single linear regressions exploring the relations between trauma before age 10 (independent ariable) and CPTI-SV total and subscale scores (dependent variables)) (n = 5057) Standardized β (95% CI) P R2 Trauma before age 10 CPTI-SV GD .073 (.059-.129) <.001 .005 CPTI-SV CU .058 (.043.121) <.001 .003 CPTI-SV INS .082 (.144-.288) <.001 .007 CPTI-SV Total .095 (.095-.171) <.001 .009 ote. CPTI-SV = Child Problematic Traits Inventory. GD = Grandiose Deceitful. CU = Callous Unemotional. S I l i it N d f ti l ti Table 2. Single linear regressions exploring the relations between trauma before age 10 (independent variable) and CPTI-SV total and subscale scores (dependent variables)) (n = 5057) Table 2. Single linear regressions exploring the relations between trauma before age 10 (independent variable) and CPTI-SV total and subscale scores (dependent variables)) (n = 5057) Standardized β (95% CI) P R2 Trauma before age 10 CPTI-SV GD .073 (.059-.129) <.001 .005 CPTI-SV CU .058 (.043.121) <.001 .003 CPTI-SV INS .082 (.144-.288) <.001 .007 CPTI-SV Total .095 (.095-.171) <.001 .009 Note. CPTI-SV = Child Problematic Traits Inventory. GD = Grandiose Deceitful. CU = Callous Unemotional. Note. CPTI-SV = Child Problematic Traits Inventory. GD = Grandiose Deceitful. CU = Callous Unemotional. INS = Impulsivity Need for stimulation. Table 3. Prediction of CPTI-SV total scores by multiple linear regressions with impact of trauma, ADHD and ODD, as well as interactions between trauma, ADHD or ODD. Tables Standardized beta coefficients (β), p- values, 95% confidence intervals (CI), and R2 values are shown (n= 5057). Page 15/17 Model Independent variables Standardized β p 95 % CI Lower 95 % CI Upper R2 1 Trauma before age 10 .073 <.001 .035 .112 .143 ADHD .430 <.001 .397 .463 Trauma before age 10 x ADHD .086 .087 -.012 .185 2 Trauma before age 10 .097 <.001 .059 .135 .075 ODD > dsm cut-off .644 <.001 .566 .722 Trauma before age 10 x ODD > dsm cut-off .186 .044 .005 .367 Table 4. Prediction of CPTI-SV GD scores by multiple linear regressions with impact of trauma, ADHD and ODD, as well as interactions between trauma, ADHD or ODD. Standardized beta coefficients (β), p-values, 95% confidence intervals (CI), and R2 values are shown (n= Table 4. Prediction of CPTI-SV GD scores by multiple linear regressions with impact of trauma, ADHD and ODD, as well as interactions between trauma, ADHD or ODD. Standardized beta coefficients (β), p-values, 95% confidence intervals (CI), and R2 values are shown (n= Model Variables Standardized β p 95 % CI Lower 95 % CI Upper R2 1 Trauma before age 10 .055 .004 .018 .093 .057 ADHD > cut-off low .238 <.001 .207 .269 Trauma before age 10 x ADHD > cut-off low .082 .089 -.013 .177 2 Trauma before age 10 .064 <.001 .029 .099 .075 ODD > dsm cut-off .622 <.001 .550 .693 Trauma before age 10 x ODD > dsm cut-off .088 .298 -.077 .253 5057). Page 16/17 Page 16/17 Table 5. Prediction of CPTI-SV CU scores by multiple linear regressions with impact of trauma, ADHD and ODD, as well as interactions between trauma, ADHD or ODD. Standardized beta coefficients (β), p-values, 95% confidence intervals (CI), and R2 values are shown (n= 5057). Model Variables Standardized β p 95 % CI Lower 95 % CI Upper R2 1 Trauma before age 10 .051 .017 .009 .093 .045 ADHD > cut-off low .242 <.001 .208 .277 Trauma before age 10 x ADHD > cut-off low .038 .482 -.068 .143 2 Trauma before age 10 .046 .022 .007 .085 .043 ODD > dsm cut-off .458 <.001 .377 .539 Trauma before age 10 x ODD > dsm cut-off .340 <.001 .154 .527 Table 6. Tables Prediction of CPTI-SV INS scores by multiple linear regressions with impact of trauma, ADHD and ODD, as well as interactions between trauma, ADHD or ODD. Standardized beta coefficients (β), p- values, 95% confidence intervals (CI), and R2 values are shown (n= Model Variables Standardized β p 95 % CI Lower 95 % CI Upper R2 1 Trauma before age 10 .111 .003 .038 .185 .139 ADHD > cut-off low .805 <.001 .743 .866 Trauma before age 10 x ADHD > cut-off low .125 .187 -.061 .310 2 Trauma before age 10 .176 <.001 .103 .248 .038 ODD > dsm cut-off .859 <.001 .709 1.01 Trauma before age 10 x ODD > dsm cut-off .109 .536 -.237 .455 5057). Page 17/17
https://openalex.org/W4289866275	https://www.researchsquare.com/article/rs-1663684/latest.pdf	English	null	The pancreas-to-muscle signal intensity ratio on T1-weighted MRI as a predictive biomarker for postoperative pancreatic fistula after distal pancreatectomy: a single-center retrospective study	World journal of surgical oncology	2,022	cc-by	7,285	The pancreas-to-musc -weighted MRI as pred postoperative pancrea pancreatectomy: A sin Masahiro Fukada (  flyhighvb@yahoo.co. Gifu University Hospital Katsutoshi Murase Gifu University Hospital Toshiya Higashi Gifu University Hospital Seito Fujibayashi Gifu University Hospital Masashi Kuno Gifu University Hospital Itaru Yasufuku Gifu University Hospital Yuta Sato Gifu University Hospital Shigeru Kiyama Gifu University Hospital Yoshihiro Tanaka Gifu University Hospital Naoki Okumura Gifu University Hospital Nobuhisa Matsuhashi Gifu University Hospital Takao Takahashi Gifu University Hospital The pancreas-to-muscle signal intensity ratio on T 1 -weighted MRI as predictive biomarker for postoperative pancreatic fistula after distal pancreatectomy: A single-center retrospective study Seito Fujibayashi Gifu University Hospital Masashi Kuno Gifu University Hospital Masashi Kuno Gifu University Hospital Itaru Yasufuku Gifu University Hospital Itaru Yasufuku Gifu University Hospital Yuta Sato Gifu University Hospital Yuta Sato Gifu University Hospital Shigeru Kiyama Gifu University Hospital Yoshihiro Tanaka Gifu University Hospital Keywords: Signal intensity ratio on T1-weighted MRI, postoperative pancreatic fistula, distal pancreatectomy Keywords: Signal intensity ratio on T1-weighted MRI, postoperative pancreatic fistula, distal pancreatectomy Page 1/26 Page 1/26 Abstract Background: Postoperative pancreatic fistula (POPF) is the most serious complication in pancreatic surgery. When POPF occurs and becomes severe, it causes secondary complications and leads to a longer treatment period. We previously reported a correlation between pancreatic fibrosis and magnetic resonance imaging (MRI) findings, and MRI may have potential to predict for POPF. This study aimed to assess the diagnostic ability of the pancreas-to-muscle signal intensity ratio on T1-weighted MRI (SIR on T1-w MRI) for POPF after distal pancreatectomy (DP). Methods: This retrospective, single-institution study comprised 117 patients who underwent DP for pancreatic disease between 2010 and 2021 at the Gifu University Hospital. We statistically analyzed pre-, intra-, and post-operative factors to assess the correlation with POPF. Results: According to the definition and grading of the International Study Group of Pancreatic Fistula (ISGPF), 29 (24.8%) of 117 patients had POPF grades B and C. In the univariate analysis, POPF was significantly associated with the pancreas-to-muscle SIR on T1-w MRI, the drainage fluid amylase concentration (D-Amy) levels on postoperative day (POD)1 and 3, white blood cell count on POD1 and 3, C-reactive protein level on POD3, and heart rate on POD3. In multivariate analysis, only the pancreas-to- muscle SIR on T1-w MRI (>1.37; odds ratio [OR] 23.25; 95% confidence interval [CI] 3.93 – 454.03; p < 0.01) and D-Amy level on POD3 (>737 U/L; OR 3.91; 95% CI 1.02 – 16.36; p = 0.046) were identified as independent predictive factors. In addition, the pancreas-to-muscle SIR on T1-w MRI showed significant positive correlations with D-Amy levels both on POD1 and 3(p < 0.01), and was significantly higher in non- pancreatic ductal adenocarcinoma cases (p < 0.01) and soft pancreas cases (p=0.04). Conclusions: The pancreas-to-muscle SIR on T1-w MRI was able to diagnose the development of POPF after DP as well as postoperative D-Amy levels. Furthermore, the pancreas-to-muscle SIR on T1-w MRI also showed significantly correlated with pancreatic disease and pancreatic texture. The pancreas-to- muscle SIR on T1-w MRI may be a potential objective biomarker reflecting pancreatic status. Posted Date: May 24th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1663684/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: May 24th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1663684/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/26 Patients In this single-center retrospective study, we enrolled 134 consecutive patients who underwent DP for pancreatic disease at Gifu University Hospital between January 2010 and December 2021. All procedures were conducted by expert surgeons who had qualified through the board certification system of the Japanese Society of Hepato-Biliary-Pancreatic Surgery (JSHBPS). We excluded 17 patients in total (simultaneous resection of other organs), so 117 patients were included in this study (Fig. 1). We conducted our study in accordance with the World Medical Association Declaration of Helsinki and the study was approved by the Ethics Committee of Gifu University (approval number: 2021-026). Patient characteristics were classified into three categories: pre-, intra-, and post-operative factors (Fig. 2). The 9 preoperative factors were age, sex, body mass index (BMI), diabetes mellitus, serum albumin level, pancreatic disease (pancreatic ductal adenocarcinoma (PDAC) or non-PDAC), tumor size, tumor location, and the pancreas-to-muscle SIR on T1-w MRI. The 6 intraoperative factors included operative time, blood loss, surgical procedure (i: open or laparoscopic surgery, ii: spleen preserving or non-preserving), Patient characteristics were classified into three categories: pre-, intra-, and post-operative factors (Fig. 2). The 9 preoperative factors were age, sex, body mass index (BMI), diabetes mellitus, serum albumin level, pancreatic disease (pancreatic ductal adenocarcinoma (PDAC) or non-PDAC), tumor size, tumor location, and the pancreas-to-muscle SIR on T1-w MRI. The 6 intraoperative factors included operative time, blood loss, surgical procedure (i: open or laparoscopic surgery, ii: spleen preserving or non-preserving), pancreatic resection procedure (hand-sewn or stapler), pancreas texture (soft or hard), and pancreas thickness measured intraoperatively on resection site. Finally, the 6 postoperative factors included the amylase concentration levels of drainage fluid and serum (D-Amy and S-Amy), the white blood cell (WBC) count, C-reactive protein (CRP) level, body temperature, and heart rate on postoperative day (POD) 1 and 3. Body temperature was defined as the maximum value and heart rate was defined as the average value on the measurement day. Background Postoperative pancreatic fistula (POPF) is the most serious complication of pancreatic surgery for pancreatic disease. POPF causes secondary complications, such as abdominal abscess, delayed gastric emptying, and postoperative bleeding, and may lead to not only prolonged hospital stay but also surgery- related death [1–3]. Although standardization of surgical procedures and development of surgical devices have been achieved in pancreatic surgery, the incidence of POPF has been reported to range from 3 to 50% even at high-volume centers [4–7]. Furthermore, POPF still occurs at as high a rate as 24–39% even after distal pancreatectomy (DP) without pancreaticoenteral anastomosis [8–13]. Therefore, POPF is considered to be caused by not only surgery-related factors, but also pancreas-related factors. Page 3/26 Page 3/26 Page 3/26 Pancreatic parenchyma becomes hardened because of fibrosis, and the hardness of the pancreatic parenchyma, the so-called soft pancreas, is known to be associated with POPF [14, 15]. We previously reported a significant correlation between the pathological classification of the pancreatic fibrosis grade and the development of POPF [16, 17]. We also reported the use of the pancreas-to-muscle signal intensity ratio on T1-weighted MRI (SIR on T1-w MRI) in evaluating pancreatic fibrosis and predicting POPF [16, 17]. The aim of this study was to assess i) the potential of the pancreas-to-muscle SIR on T1-w MRI as a predictive factor for POPF after DP, and ii) the correlation between the pancreas-to-muscle SIR on T1-w MRI and other pancreas-related factors. The pancreas-to-muscle signal intensity ratio on T1- weighted MRI The pancreas-to-muscle signal intensity ratio on T1- weighted MRI Previously, we studied the potential value of preoperative MRI in evaluating pancreatic properties [16, 17] and reported that the pancreas-to-muscle SIR on T1-w MRI significantly correlated with pancreatic fibrosis, and that it may be a potential biomarker for predicting POPF for pancreatic surgery. The signal intensity (SI) of the pancreatic parenchyma on the portal vein and the paraspinal muscle was measured using fat-suppressed axial T1-weighted imaging (Fig. 3). The pancreas-to-muscle SIR on T1-w MRI was calculated using the following equation: [SI of the pancreatic parenchyma] / [SI of the paraspinal muscle]. Definition of POPF In this study, we included only clinically symptomatic POPF. Therefore, only grades B and C pancreatic fistulas were defined as POPF (Grade B, symptomatic fistula requiring therapeutic intervention such as antibiotics and percutaneous drainage; Grade C, symptomatic fistula associated with a severe general condition of patients, sepsis, and multiorgan failure requiring aggressive treatment in the intensive care unit and surgical intervention), based on International Study Group of Pancreatic Fistula (ISGPF) definitions [19]. Diagnosis day of POPF was defined as the date when intra-abdominal fluid collection with positive cultures was identified by ultrasonography (US) or computed tomography (CT). Perioperative management In cases of DP for PDAC, regional lymph node dissection with splenectomy in accordance with the classification of pancreatic carcinoma of the Japan Pancreas Society [18], and pancreatic resection on the portal vein were performed. In the case of DP for non-PDAC, systematic lymph node dissection was Page 4/26 omitted, and pancreatic resection was performed with sufficient margin from the tumor. Pancreatic resection was performed with hand-sewn closure or using a linear stapler. In the hand-sewn closure group, the pancreas was resected after the identification of the main pancreatic duct, and the main pancreatic duct ligated with a 3 − 0 silk suture. The stump of the remnant pancreas was closed with vertical mattress suture using 5 − 0 polypropylene. Among the group that underwent pancreatic resection using a linear stapler, and the pancreas resected using Endo GIA™ Tri-Staple or Signia™ stapling system (Medtronic plc., Dublin, Ireland) with a purple or black cartridge. The closure jaw was clamped carefully and slowly, taking 5 minutes at a fixed speed. The firing was performed at a speed of 1 cm per minute by firmly fixing the stapler. After firing, the jaws of the stapler were held shut for 1 minute. One 19Fr. Blake silicon drain (Johnson and Johnson Inc. New Brunswick, NJ, USA) was placed near the stump of the remnant pancreas. The drain was to be removed on POD 4–5 when the drainage fluid was clear and the postoperative course could pose no problem. The D-Amy and S-Amy levels were measured on POD 1, 3, and 5. All patients received prophylactic antibiotics (cefmetazole) only intraoperatively or for 2 days postoperatively. In the hand-sewn closure group, the pancreas was resected after the identification of the main pancreatic duct, and the main pancreatic duct ligated with a 3 − 0 silk suture. The stump of the remnant pancreas was closed with vertical mattress suture using 5 − 0 polypropylene. Among the group that underwent pancreatic resection using a linear stapler, and the pancreas resected using Endo GIA™ Tri-Staple or Signia™ stapling system (Medtronic plc., Dublin, Ireland) with a purple or black cartridge. The closure jaw was clamped carefully and slowly, taking 5 minutes at a fixed speed. The firing was performed at a speed of 1 cm per minute by firmly fixing the stapler. After firing, the jaws of the stapler were held shut for 1 minute. One 19Fr. Perioperative management Blake silicon drain (Johnson and Johnson Inc. New Brunswick, NJ, USA) was placed near the stump of the remnant pancreas. The drain was to be removed on POD 4–5 when the drainage fluid was clear and the postoperative course could pose no problem. The D-Amy and S-Amy levels were measured on POD 1, 3, and 5. All patients received prophylactic antibiotics (cefmetazole) only intraoperatively or for 2 days postoperatively. Comparison of clinical outcomes between patients with and without POPF In total, 117 patients underwent DP for pancreatic disease. Symptomatic POPF occurred in 29 (24.8 %) patients. Patients’ clinical outcomes after DP are summarized in Table 1. The median time at which POPF was confirmed was POD 7 (range, 3-25 days). In the patients with POPF, the median time until hospital discharge was 35 days postoperatively (range, 12-121 days), and 2 patients had died within 30 postoperative days. A comparison between patients with and without POPF indicated that there were significant differences in hospital days (p < 0.01). Comparison of pre-, intra-, and post-operative status between patients with and without POPF Table 2 shows a summary of the 21 factors (classified into three categories) compared between patients with and without POPF. Among pre-operative factors, the pancreas-to-muscle SIR on T1-w MRI was significantly higher in the POPF group than in the non-POPF group (p < 0.01). Among intra-operative factors, there was no significant difference between two groups. Among post-operative factors, D-Amy level on POD 1 and 3 (p = 0.02 and p < 0.01, respectively), WBC on POD1 and 3 (p < 0.01 and p = 0.048, respectively), CRP level on POD3 (p < 0.01), and heart rate on POD3 (p < 0.01) were significantly higher in the POPF group than in the non-POPF group. Statistical analysis Continuous variables are expressed as median (range) values, and categorical variables are expressed as frequencies (percentages). For comparisons of variables between the POPF and non-POPF groups, a Fisher’s exact test was used for categorical variables, and a Mann-Whitney U test was used for Page 5/26 Page 5/26 continuous variables. The predictive ability for POPF after DP was assessed by calculating the area under the receiver operating characteristic (ROC) curve. Youden’s index was used to determine the optimal cut- off value to calculate both specificities and sensitivities in the ROC curve analysis. The variables identified as potentially significant by univariate analysis were selected for multivariate analysis with a logistic regression model to identify the independent predictors of POPF after DP. The correlation analysis was performed using Spearman’s rank correlation coefficient to examine the correlation between the pancreas-to-muscle SIR on T1-w on MRI and D-Amy levels. The limit of statistical significance for all analyses was defined as a 2-sided p-value of 0.05. All statistical analyses were performed using JMP software (SAS Institute Inc., Cary, NC, USA). Uni- and multivariate analysis of prediction for POPF after DP In univariate logistic regression analysis, POPF after DP was significantly associated with the pancreatic- to-muscle SIR on T1-w MRI (p < 0.01), D-Amy level on POD1 and 3 (both p < 0.01), WBC on POD1 and 3 (p < 0.01 and p = 0.04, respectively), CRP level on POD3 (p < 0.01), and heart rate on POD3 (p=0.02). A multivariate logistic regression analysis revealed that the pancreas-to-muscle SIR on T1-w MRI (>1.37; odds ratio [OR] 23.25; 95% confidence interval [CI] 3.93 – 454.03; p < 0.01) and D-Amy level on POD3(>737 U/L; OR 3.91; 95% CI 1.02 – 16.36; p = 0.046), were independent predictive factors of POPF after DP (Table 3). Correlation analysis between the pancreas-to-muscle SIR on T1-w MRI and other pancreas-related factors Next, we evaluated the correlation between the pancreas-to-muscle SIR on T1-w MRI and other pancreas- related factors, such as pancreatic disease, pancreatic texture, and D-Amy levels. There was a significant positive correlation between the pancreas-to-muscle SIR on T1-w MRI and D-Amy levels on both POD1 and POD3 (both p < 0.01) (Figure 5a and 5b). In addition, the pancreas-to-muscle SIR on T1-w MRI was significantly higher in non-PDAC cases (p< 0.01, non-PDAC: median 1.57 [range 0.74 – 2.68] vs PDAC: 1.31[0.82 – 1.86]) and soft pancreatic cases (p=0.04, soft: 1.56 [0.74 – 2.01] vs hard: 1.32[0.86 – 2.68]) (Figure 5c and 5d). Page 6/26 Page 6/26 The ROC curves for generating cut-off values of the pancreas-to-muscle SIR on T1-w MRI, D-Amy level on POD 1 and 3 are shown in Figure 4. The cut-off value of the pancreas-to-muscle SIR on T1-w MRI was +1.37, with an area under the curve (AUC) of 0.741, a sensitivity of 96.3%, and specificity of 52.0% (Figure 4a). The cut-off value of D-Amy level on POD1 was 7238 U/L, with an AUC of 0.729, a sensitivity of 55.2%, and specificity of 80.5% (Figure 4b). The cut-off value of D-Amy level on POD3 was 737 U/L, with an AUC of 0.721, a sensitivity of 72.4%, and specificity of 65.5% (Figure 4c). Discussion A high incidence of POPF is still reported in pancreatic surgery despite ongoing attempts to reduce the frequency of POPF with the development of surgical techniques and devices [4–13]. The clinical nuisance of POPF is that delayed therapeutic intervention for POPF can lead to secondary complications [1–3]. This can lead to severe disease and prolonged treatment. In this study, patients with POPF showed increase in both hospital days and mortality. Furthermore, we have previously reported that in PDAC cases, the onset of POPF leads to a delay in the initiation of postoperative adjuvant chemotherapy. Thus, POPF may affect not only short-term but also long-term prognosis. Therefore, early and accurate Page 7/26 prediction and diagnosis of POPF and the promptest possible intervention are required. However, the median time for POPF diagnosis was 7 days (range, 3–25) in this study, making early diagnosis difficult with only routine postoperative examination. We identified two predictive factors for POPF: i) the pancreas-to-muscle SIR on T1-w MRI > 1.37, and ii) D-Amy level on POD3 > 737U/L. D-Amy levels are one of the most established predictive and diagnostic factors for POPF. Therefore, the definition of POPF according to the ISGPF offers the standard diagnosis according to the D-Amy level on POD3. In this study, D-Amy levels were also significantly correlated with POPF. There is no doubt that amylase in the drainage fluid is useful in the diagnosis of POPF, as has been reported many times [20– 41]. However, the following remain somewhat unclear: (1) the optimal timing of measurement, (2) the optimal cutoff value, (3) the optimal drain placement site, and (4) whether drainage fluid concentration or total amount of amylase is more reliable. In addition, postoperative drain obstruction due to fibrin or clots and drain misalignment often occur, which can interfere with accurate D-Amy level measurements. The nature of the pancreas itself is thought to play a profound role in the development of POPF. In particular, the texture of pancreatic parenchyma, (soft pancreas) has been reported to be an important risk factor for POPF. However, the problem is that the pancreatic texture is very subjective and cannot be quantified. Conclusions We found that the pancreas-to-muscle SIR on T1-weighted MRI has excellent predictive and diagnostic ability for POPF as well as D-Amy level. The pancreas-to-muscle SIR is an objective and quantitative biomarker reflecting pancreatic characteristics. Postoperative management based on the pancreas-to-muscle SIR on T1-weighted MRI may contribute to shortened hospital stay. Discussion In this study, no significant correlation was found between surgical- related factors, especially surgical technique (open or lap, spleen preserving or non-preserving, hand sewn or stapler) and POPF. Therefore, the pancreas-to-muscle SIR on T1-w MRI may be effective in predicting POPF after PD and gastrectomy. caused a selection bias. This limitation should be considered when evaluating our study results. A prospective, multi-centered study is needed involving a larger number of patients in the future. Second, this study was limited to DP, for which the surgical technique is relatively simple compared to pancreaticoduodenectomy (PD). In this study, no significant correlation was found between surgical- related factors, especially surgical technique (open or lap, spleen preserving or non-preserving, hand sewn or stapler) and POPF. Therefore, the pancreas-to-muscle SIR on T1-w MRI may be effective in predicting POPF after PD and gastrectomy. Abbreviations BMI, body mass index; CI, confidence interval; CT, computed tomography; D-Amy, drainage fluid amylase; DP, distal pancreatectomy; MRI, magnetic resonance imaging; OR, odds ratio; ISGPF, International Study Group of Pancreatic Fistula; PD, pancreaticoduodenectomy; PDAC, Pancreatic ductal adenocarcinoma; POD, postoperative day; POPF, Postoperative pancreatic fistula; ROC, receiver operating characteristic; S- Amy, serum amylase; SIR on T1-w MRI, signal intensity ratio on T1-weighted MRI; US, ultrasonography Ethics approval and consent to participate: The present study was conducted in accordance with the World Medical Association Declaration of Helsinki and was approved by the Ethics Committee of Gifu University (approval number ‘2021-26’). As this study was a retrospective study and did not include any potentially identifiable patient data, informed consent was not obtained from the enrolled patients. This retrospective study was approved by our Institutional Review Board. Discussion To solve this problem, we previously investigated the correlation between preoperative pancreatic MRI features and the histopathological pancreatic fibrosis grade of surgical specimens (fibrosis was graded as follows: F0 = normal pancreatic parenchyma, no fibrotic changes; F1 = mild fibrosis with thickening of periductal fibrosis tissue; F2 = moderate fibrosis with marked sclerosis of interlobular septa and no evidence of architectural changes; and F3 = severe fibrosis with detection of architectural destruction) [16, 17]. We found that the pancreas-to-muscle SIR on T1-w MRI had a significantly negative correlation with the pancreatic fibrosis grade. This is because normal pancreatic parenchyma exhibits hyperintensity on T1-w MRI, as pancreatic juice is rich in glycoproteins, and the endoplasmic reticulum within the pancreatic cells contributes to the T1 shortening effect. However, the signal intensity gradually decreases with progression of pancreatic atrophy, fibrosis, interstitial edema, or fat deposition [42, 43]. In our previous study, the mean pancreas-to-muscle SIR on T1-w MRI values for F0 and F1, which correspond to soft pancreas, were 1.51 and 1.48, respectively. Furthermore, the pancreas- to-muscle SIR on T1-w MRI in the patients with POPF was significantly higher than that in patient without POPF. Based on these findings, we hypothesized that the pancreas-to-muscle SIR on T1-w MRI might be a potential biomarker for predicting POPF and calculated the cutoff value of 1.41 [16]. Yoon et al conducted a similar study and also reported the mean pancreas-to-muscle SIR on T1-w MRI values for F0 and F1 and the cut-off value for predicting POPF were 1.51, 1.48, and 1.40, respectively [44]. Interestingly, the calculated cut-off value for predicting POPF (1.37) in this study is very close to that in the previous studies. Furthermore, the pancreas-to-muscle SIR on T1-w MRI also showed a significant correlation with not only POPF, but also pancreatic disease, texture of pancreatic parenchyma, and D-Amy levels. This study had some limitations. First, it was retrospective in design and undertaken at a single institution, and involved a small number of study patients. The relatively small sample size may have Page 8/26 Page 8/26 caused a selection bias. This limitation should be considered when evaluating our study results. A prospective, multi-centered study is needed involving a larger number of patients in the future. Second, this study was limited to DP, for which the surgical technique is relatively simple compared to pancreaticoduodenectomy (PD). Acknowledgements: The authors thank the medical staff of the Department of Gastroenterological Surgery at Gifu University Hospital for their participation in this study. We could not have completed this study without their diligence and support. We would also like to thank Editage (www.editage.jp) for English language editing. Funding: Not applicable Consent for publication: Not applicable Availability of data and materials: Page 9/26 The datasets used during this study are available from the corresponding author upon reasonable request. Authors’ contributions: MF conceived the study concept and planned the design as the principal investigator. MF interpreted the results and wrote the manuscript draft. KM and TH revised the manuscript draft through adding intellectual content and providing critical advice. MF, KM, TH, SF, MK, IY, YS, SK, YT, NO, NM, and TT obtained the data and provided critical comments to improve the manuscript and gave final approval for submission. Competing interests: T. Takahashi received honoraria for lectures from Takeda Pharmaceutical Co., Ltd. The remaining authors declare that they have no conflicts of interest. References 1. Fujino Y. Perioperative management of distal pancreatectomy. 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World J Gastroenterol. 2014;20:8535–44. https://doi.org/10.3748/wjg.v20.i26.8535, PMID: 25024608 Page 13/26 Page 13/26 40. Fukami Y, Saito T, Osawa T, Hanazawa T, Kurahashi T, Kurahashi S, et al. Which is the best predictor of clinically relevant pancreatic fistula after pancreatectomy: drain fluid concentration or total amount of amylase? Ann Gastroenterol Surg. 2021;5:844–52. https://doi.org/10.1002/ags3.12471, PMID: 34755016 41. Matar A, Meares T, Fisher OM, Gauci C, Rao A, Alshahrani M, et al. Postoperative pancreatic fistula after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy: an update on incidence, risk factors, management, and clinical sequelae in 1,141 patients. Anticancer Res. 2021;41:5577–84. https://doi.org/10.21873/anticanres.15371, PMID: 34732428 41. Matar A, Meares T, Fisher OM, Gauci C, Rao A, Alshahrani M, et al. Postoperative pancreatic fistula after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy: an update on incidence, risk factors, management, and clinical sequelae in 1,141 patients. Anticancer Res. 2021;41:5577–84. https://doi.org/10.21873/anticanres.15371, PMID: 34732428 42. Mitchell DG, Vinitski S, Saponaro S, Tasciyan T, Burk DL Jr, Rifkin MD. Liver and pancreas: improved spin-echo T1 contrast by shorter echo time and fat suppression at 1.5 T. Radiology. 1991;178:67–71. https://doi.org/10.1148/radiology.178.1.1984328, PMID: 1984328 42. Mitchell DG, Vinitski S, Saponaro S, Tasciyan T, Burk DL Jr, Rifkin MD. Liver and pancreas: improved spin-echo T1 contrast by shorter echo time and fat suppression at 1.5 T. Radiology. 1991;178:67–71. https://doi.org/10.1148/radiology.178.1.1984328, PMID: 1984328 43. Winston CB, Mitchell DG, Outwater EK, Ehrlich SM. Pancreatic signal intensity on T1-weighted fat saturation MR images: clinical correlation. J Magn Reson Imaging. 1995;5:267–71. https://doi.org/10.1002/jmri.1880050307, PMID: 7633102 43. Winston CB, Mitchell DG, Outwater EK, Ehrlich SM. Pancreatic signal intensity on T1-weighted fat saturation MR images: clinical correlation. J Magn Reson Imaging. 1995;5:267–71. https://doi.org/10.1002/jmri.1880050307, PMID: 7633102 44. Yoon JH, Lee JM, Lee KB, Kim SW, Kang MJ, Jang JY, et al. Pancreatic steatosis and fibrosis: quantitative assessment with preoperative multiparametric MR imaging. Radiology. 2016;279:140– 50. https://doi.org/10.1148/radiol.2015142254 44. Yoon JH, Lee JM, Lee KB, Kim SW, Kang MJ, Jang JY, et al. Table 2 Table 2 Comparison of pre-, intra-, and post-operative status between patients with and without POPF after distal pancreatectomy. Table 1 Comparison of clinical outcomes between patients with and without POPF after distal pancreatectomy. Comparison of clinical outcomes between patients with and without POPF after distal pancreatectomy. Page 14/26 Page 14/26 Patients with POPF (n=29) Patients without POPF (n=88) p- value Diagnosis days of POPF (day) 7 (3-25) - - Grade of POPF¶ Grade B Grade C 27(93.1) 2 (6.9) - - - Treatment for POPF ・Drain replacement and irrigation ・Endoscopic transgastric drainage ・Antibiotics and octreotide 19 (65.5) 7 (24.1) 3(10.4) - - Postoperative death within 30 days 2 (6.9) 0 (0.0) 0.06 Hospital days (days) 35 (12-121) 13 (7-23) <0.01✝ Data are expressed as median (range) or number of patients (percentage) Data are expressed as median (range) or number of patients (percentage) Data are expressed as median (range) or number of patients (percentage) ata are expressed as median (range) or number of patients (percentage) ¶: International Study Group (ISGPS) definition and grading of POPF as follow: Grade B, symptomatic fistula requiring therapeutic intervention such as antibiotics and percutaneous drainage; Grade C, symptomatic fistula associated with a severe general condition of patients, sepsis, and multiorgan failure requiring aggressive treatment in the intensive care unit and surgical intervention. §: Period until the start of postoperative adjuvant chemotherapy was calculated from the date of the surgery. §: Period until the start of postoperative adjuvant chemotherapy was calculated from the date of the surgery. ✝: p<0.05 Comparison of pre-, intra-, and post-operative status between patients with and without POPF after distal pancreatectomy. Data are expressed as median (range) or number of patients. POPF: postoperative pancreatic fistula Data are expressed as median (range) or number of patients. POPF: postoperative pancreatic fistula Page 15/26 Patients with POPF (n=29) Patients without POPF (n=88) p-value Pre- operative Age (years) 67 (40-82) 67 (11-84) 0.87 Sex Male Female 18 (62.1) 11 (37.9) 50 (56.8) 38 (43.2) 0.67 BMI (kg/m2) 23.6 (17.6-32.2) 22.4 (16.2-32.2) 0.37 Diabetes mellitus 7 (24.1) 29 (33.0) 0.37 Serum albumin level (g/dl) 4.1 (3.3-5.0) 4.3 (2.7-4.9) 0.38 Pancreatic ductal adeno carcinoma 12 (41.4) 43 (48.9) 0.53 Tumor size (mm) 22.5 (10-88) 27 (4-120) 0.61 Location Pb Pt 14 (48.3) 15 (51.7) 54 (61.4) 34 (38.6) 0.28 Pancreas-to-muscle SIR on T1-w MRI 1.64 (1.25-2.68) 1.35 (0.74-2.16) <0.01✝ Intra- operative Operative time (min) 284 (174-537) 264 (143-564) 0.13 Blood loss (ml) 190 (10-1910) 260 (0-1840) 0.84 Surgical procedure Open Laparoscopic 23 (79.3) 6 (20.7) 60 (68.2) 28 (31.8) 0.35 Spleen preserving Non spleen preserving 3 (10.3) 26 (89.7) 15 (17.1) 73 (82.9) 0.56 Resection procedure Hand-sewn Stapler 11 (37.9) 18 (62.1) 36 (40.9) 52 (59.1) 0.83 Pancreas texture 0.37 Hard 8 (27.6) 34 (38.6) Pancreas thickness (mm) 11 (8-17) 12 (3-24) 0.46 Post- operative D-Amy levels (U/l) POD1 POD3 7652 (108- 34076) 1290 (42- 16515) 1899 (42-61075) 403 (35-43873) 0.02✝ <0.01✝ S-Amy levels (U/l) POD1 POD3 113 (47-1108) 40 (11-223) 163 (35-1921) 50 (15-663) 0.24 0.10 WBC (×103/µl) POD1 POD3 12.6 (7.5-26.5) 13.3 (5.9-26.9) 11.1 (5.3-18.2) 11.3 (3.9-23.9) <0.01✝0.048✝ CRP (mg/dl) POD1 POD3 9.3 (1.8-15.4) 20.1 (8.0-33.5) 8.3 (0.2-17.5) 14.5 (0.3-26.5) 0.16 <0.01✝ Body temperature (℃) POD1 POD3 38.0 (37.1-39.4) 37.4 (36.1-38.9) 38.0 (36.9-39.3) 37.4 (36.2-39.4) 0.55 0.30 Heart rate (bpm) POD1 POD3 98 (81-142) 88 (72-111) 92 (61-122) 82 (56-119) 0.07 <0.01✝ BMI: body mass index Hard Hard Page 17/26 Pancreas-to-muscle SIR on T1-w MRI: The pancreas-to-muscle signal intensity ratio on unenhanced T1- weigthed magnetic resonance imaging S-Amy: Serum amylase concentration D-Amy: Drainage fluid amylase concentration POD: postoperative day WBC: white blood cell Page 17/26 Page 17/26 CRP: c-reactive protein ✝: p< 0.05 Uni- and multivariate predictive factors of POPF after DP. POPF: postoperative pancreatic fistula OR: odds ratio 95%CI: 95% confidence interval BMI: body mass index Pancreas-to-muscle SIR on T1-w MRI: The pancreas-to-muscle signal intensity ratio on unenhanced T1- weigthed magnetic resonance imaging D-Amy: Drainage fluid amylase concentration S-Amy: Serum amylase concentration POD: postoperative day WBC: white blood cell CRP: c-reactive protein ✝: p< 0.05 Figures Page 18/26 n Univariate Multivariate OR 95%CI p- value OR 95%CI p- value e Age (years) >70 <70 45 72 0.79 1 0.32- 1.89 0.61 Sex Male Female 68 49 1.24 1 0.53- 3.01 0.62 BMI (kg/m2) >24 <24 39 77 2.07 1 0.85- 5.00 0.10 Diabetes mellitus yes no 36 81 0.65 1 0.23- 1.63 0.36 Serum albumin level (mg/dl) >3.6 <3.6 10 107 0.46 1 0.12- 1.90 0.27 Pancreatic ductal adeno carcinoma yes no 55 62 0.74 1 0.31- 1.72 0.48 Tumor size (mm) >20 <20 70 43 0.93 1 0.39- 2.29 0.88 Location Pb Pt 68 49 0.59 1 0.25- 1.37 0.22 SIR on T1-w MRI >1.37 <1.37 61 41 29.7 1 23.25 1 Intra- operative Operative time (min) >300 <300 38 78 1.36 1 0.55- 3.24 0.50 Blood loss (ml) >400 <400 39 77 0.86 1 0.33- 2.07 0.73 Surgical procedure Open Laparoscopic 83 34 1.79 1 0.69- 5.28 0.24 Spleen preserving Non spleen preserving 18 99 0.56 1 0.12- 1.87 0.37 Resection procedure Hand-sewn Stapler 47 70 0.88 1 0.36- 2.07 0.78 Pancreas texture Soft Hard 75 42 1.65 1 0.68- 4.35 0.28 Pancreas thickness (mm) >12 <12 56 61 0.71 1 0.30- 1.64 0.42 Post- operative D-Amy levels (U/l) – POD1 >7238 <7238 33 83 5.07 1 2.07- 12.78 <0.01✝ 1.21 1 0.31- 4.75 0.78 D-Amy levels (U/l) – POD3 51 4.99 2.04- 13.25 <0.01✝ 1.02- 16.36 0.046✝ <737 1 S-Amy levels (U/l) – POD1 >100 <100 85 32 0.51 1 0.21- 1.28 0.15 S-Amy levels (U/l) - POD3 >100 <100 16 100 1 1 0.26- 3.17 1.00 WBC (×103/µl) – POD1 >1.20 <1.20 48 69 3.16 1 1.34- 7.75 <0.01✝ 2.44 1 0.69- 9.12 0.17 WBC (×103/µl) - POD3 >1.40 <1.40 34 83 2.59 1 1.07- 6.29 0.04✝ 1.03 1 0.27- 3.73 0.96 CRP (mg/dl) – POD1 >10 <10 40 77 1.51 1 0.63- 3.58 0.35 CRP (mg/dl) - POD3 >20 <20 34 83 3.89 1 1.61- 9.60 <0.01✝ 2.75 1 0.75- 10.70 0.13 Body temperature (℃) – POD1 >38.0 <38.0 71 46 0.74 1 0.32- 1.75 0.49 Body temperature (℃) - POD3 >38.0 <38.0 14 103 0.47 1 0.07- 1.87 0.31 Heart rate (bpm) – POD1 0.85- 4.88 0.11 1 <737 >100 <100 79 1 Heart rate (bpm) - POD3 >100 <100 13 104 4.35 1 1.32- 14.81 0.02✝ 3.84 1 0.77- 21.40 0.10 Figure 1 Figure 1 Exclusion criteria Figure 2 Analysis flow chart for identifying predictive factors for postoperative pancreatic fistula (POPF) after distal pancreatectomy (DP) Page 23/26 Figure 3 The pancreas-to-muscle signal intensity ratio on fat-suppressed axial T1-weighted MRI was calculated by [Signal intensity of the pancreatic parenchyma] (arrow) / [Signal intensity of the paraspinal muscle] (arrowhead). Figure 3 The pancreas-to-muscle signal intensity ratio on fat-suppressed axial T1-weighted MRI was calculated by [Signal intensity of the pancreatic parenchyma] (arrow) / [Signal intensity of the paraspinal muscle] (arrowhead). Page 24/26 Page 24/26 Figure 4 Receiver operating characteristics (ROC) curve analysis of pancreas-to-muscle SIR on T1-weighted MRI and D-Amy level on POD 1 and 3 for discriminating to POPF. Figure 4 Figure 4 Receiver operating characteristics (ROC) curve analysis of pancreas-to-muscle SIR on T1-weighted MRI and D-Amy level on POD 1 and 3 for discriminating to POPF. Receiver operating characteristics (ROC) curve analysis of pancreas-to-muscle SIR on T1-weighted MRI and D-Amy level on POD 1 and 3 for discriminating to POPF. Receiver operating characteristics (ROC) curve analysis of pancreas-to-muscle SIR on T1-weighted MRI and D-Amy level on POD 1 and 3 for discriminating to POPF. Page 25/26 Figure 5 (a) (b) Correlation between the pancreas-to-muscle SIR on T1-weighted MRI and D-Amy levels on POD1 and 3. Pearson’s rank correlation coefficient (r) is shown. Figure 5 (a) (b) Correlation between the pancreas-to-mu and 3. Pearson’s rank correlation coefficient (r) Figure 5 (a) (b) Correlation between the pancreas-to-muscle SIR on T1-weighted MRI and D-Amy levels on POD1 and 3. Pearson’s rank correlation coefficient (r) is shown. (a) (b) Correlation between the pancreas-to-muscle SIR on T1-weighted MRI and D-Amy levels on POD1 and 3. Pearson’s rank correlation coefficient (r) is shown. (a) (b) Correlation between the pancreas-to-muscle SIR on T1-weighted MRI and D-Amy levels on POD1 and 3. Pearson’s rank correlation coefficient (r) is shown. (c) The pancreas-to-muscle SIR on T1-weighted MRI between non-PDAC cases (n = 62) and PDAC cases (n = 55). The Wilcoxon signed-rank test was performed for comparisons between groups. (c) The pancreas-to-muscle SIR on T1-weighted MRI between non-PDAC cases (n = 62) and PDAC cases (n = 55). The Wilcoxon signed-rank test was performed for comparisons between groups. (d) The pancreas-to-muscle SIR on T1-weighted MRI between soft pancreas (n = 75) and hard pancreas (n = 42). The Wilcoxon signed-rank test was performed for comparisons between groups. (d) The pancreas-to-muscle SIR on T1-weighted MRI between soft pancreas (n = 75) and hard pancreas (n = 42). The Wilcoxon signed-rank test was performed for comparisons between groups. Page 26/26
https://openalex.org/W2811014042	https://pure.eur.nl/files/47911944/Rop_et_al-2018-Journal_of_Computer_Assisted_Learning.pdf	English	null	Effects of task experience and layout on learning from text and pictures with or without unnecessary picture descriptions	Journal of computer assisted learning	2,018	cc-by	13,126	KEYWORDS cognitive load, extraneous processing, eye tracking, multimedia learning, study strategies This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. © 2018 The Authors. Journal of Computer Assisted Learning Published by John Wiley & Sons, Ltd. Effects of task experience and layout on learning from text and pictures with or without unnecessary picture descriptions Gertjan Rop1 \| Anne Schüler2 \| Peter P.J.L. Verkoeijen1,3 \| Katharina Scheiter2,5 \| Tamara van Gog4,5 Gertjan Rop1 \| Anne Schüler2 \| Peter P.J.L. Verkoeijen1,3 \| Katharina Scheiter2,5 \| Tamara van Gog4,5 1Department of Psychology, Education, and Child Studies, Erasmus University Rotterdam, The Netherlands 2Leibniz‐Institut für Wissensmedien, Germany 3Learning and Innovation Center, Avans University of Applied Sciences, The Netherlands 4Department of Education, Utrecht University, The Netherlands 5University of Tübingen, Germany Correspondence Gertjan Rop, Erasmus School of Social and Behavioural Sciences, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. Email: rop@fsw.eur.nl 1Department of Psychology, Education, and Child Studies, Erasmus University Rotterdam, The Netherlands Abstract The presentation of extraneous (i.e., irrelevant or unnecessary) information may hamper learning with multimedia. The present study examined whether people can learn to ignore unnecessary information with increasing experience with the task and whether this depends on the layout of that information. In two experiments, participants learned about the process of mitosis from a multimedia slideshow, with each slide presenting a combination of expository text and a picture on one of the stages in the process. Slides either contained no unnecessary text (control condition) or unnecessary text (i.e., merely describing the picture) either integrated in the picture (integrated condition) or presented underneath the picture (separated condition). Knowledge about the studied mitosis phase was tested immediately after each slide using a cloze test. Across Experiments 1 and 2, we did not find a reliable negative effect of the unnecessary text on cloze test performance. As a result, the question of whether task experience would reduce or eliminate that negative effect could not be answered. The eye movement data did confirm, however, that participants attended less to the unnecessary information with increasing task experience, suggesting that students can adapt their study strategy and learn to ignore unnecessary information. O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E wileyonlinelibrary.com/journal/jcal 1 Received: 18 December 2017 Revised: 4 May 2018 Accepted: 25 May 2018 Received: 18 December 2017 Revised: 4 May 2018 Accepted: 25 May 2018 Received: 18 December 2017 Revised: 4 May 2018 Accepted: 25 May 2018 DOI: 10.1111/jcal.12287 1.2 \| Task experience Eye‐tracking studies already showed that participants may learn to ignore task‐irrelevant information as a consequence of task experi- ence (Haider & Frensch, 1999) or explicit instruction (Canham & Hegarty, 2010; Hegarty, Canham, & Fabrikant, 2010), and the results of Rop et al. (2018) indicate that this effect can be generalized to learning with multimedia materials. Indeed, their results suggest that when learners gain more experience with the learning materials, they adapt their study strategy and start to ignore irrelevant information, thereby diminishing the negative effect of irrelevant information on learning. Thus, task experience may be a boundary condition for the negative effect of extraneous information on learning, because par- ticipants stop allocating attention to this information. It is important to establish potential boundary conditions as they describe the limits of generalizability of scientific theories (Busse, Kach, & Wagner, 2016; Whetten, 1989). However, as the extraneous information in the study by Rop et al. (2018; see also Rop et al., 2017) was picto- rial, obviously irrelevant (i.e., it mismatched the verbal information that participants had to remember), and not integrated with other information (i.e., integrated extraneous information seems to hamper learning more; Chandler & Sweller, 1991), it is an open question whether task experience would have similar effects when the extra- neous information is textual (e.g., a text describing the elements of a picture), unnecessary rather than irrelevant (i.e., in the sense that the information provided by the text is relevant for the learning task but not necessary as it can also be inferred from the picture), and integrated with relevant information (e.g., unnecessary text inte- grated with a picture). The negative effect of extraneous information processing on learning has been demonstrated with a variety of materials and types of extraneous information and has been labelled the coherence princi- ple (cf. Mayer & Fiorella, 2014) and/or the redundancy principle (cf. Kalyuga & Sweller, 2014). The coherence principle states that people learn more deeply from a multimedia message when unnecessary or irrelevant material is excluded rather than included. The redundancy principle suggests that presenting redundant material (e.g., the same information in two different formats, making one format unnecessary for learning) interferes with rather than facilitates learning. In effect, both principles entail that the presentation of extraneous information should be avoided, because it hampers learning compared to instruc- tional materials in which this information has been eliminated. 1 \| INTRODUCTION diagrams are accompanied by textual explanations (Bobis, Sweller, & Cooper, 1993; Chandler & Sweller, 1991) and when unnecessary details and examples are added to learning materials (e.g., Mayer, Bove, Bryman, Mars, & Tapangco, 1996; Reder & Anderson, 1982). to ignore the pictures based on their content, and not their location (Rop, Verkoeijen, & Van Gog, 2017). diagrams are accompanied by textual explanations (Bobis, Sweller, & Cooper, 1993; Chandler & Sweller, 1991) and when unnecessary details and examples are added to learning materials (e.g., Mayer, Bove, Bryman, Mars, & Tapangco, 1996; Reder & Anderson, 1982). However, because the extraneous information was pictorial and obviously irrelevant (i.e., it mismatched the verbal information that participants had to remember), it is an open question whether task experience would have similar effects when the extraneous information is textual (e.g., a text describing the elements of a picture) and unnecessary rather than irrelevant (i.e., in the sense that the information provided by the text is relevant for the learning task but not necessary as it can also be inferred from the picture). The present study addressed this question. In all these studies, the extraneous information was either irrelevant or unnecessary, depending on its relation with the learning goal. Irrelevant information is unrelated to the learning goal and would adhere more to the coherence principle (e.g., seductive details, information about related systems, and mismatching information). Unnecessary information, on the other hand, is related to the learning goal, but not necessary for learning because the information is presented twice (e.g., the same spoken and written text accompanying an illustration, self‐contained diagrams with unnecessary textual explanations) or is unnecessarily elaborate (e.g., unnecessary details and examples). The negative effect of unnecessary information therefore more closely resembles the redundancy principle and is addressed in the present study. 1.1 \| Effects of extraneous information on learning While learning from multimedia materials, that is, materials in which text (either spoken or written) and pictures (either static or dynamic) are combined (Mayer, 2014), a learner first has to select the relevant information from the text and picture (by attending to it). Subse- quently, this information has to be organized into a coherent cognitive structure in working memory and has to be integrated with prior knowledge from long‐term memory (Mayer, 2014). When either one of these processes (i.e., selection, organization, or integration) is disrupted, learning is hampered. The presentation of extraneous infor- mation in multimedia learning materials may hamper learning when it captures attention, because working memory capacity is limited (e.g., Baddeley, 2000; Cowan, 2001; Miller, 1956) and processing this extraneous information that is not conducive to learning reduces the working memory resources available for the selection, organization, and integration of information that is essential for learning. As mentioned above, both the coherence and the redundancy principle entail that presenting extraneous information hampers learning because it captures learners' attention, and learners spend valuable cognitive resources on processing this information that is not conducive to learning. However, recently, evidence emerged that people might learn to ignore extraneous information with increasing task experience. 1 \| INTRODUCTION De Nooijer, Verkoeijen, & Van Gog, 2018). In this study on word learn- ing, participants who were presented with pictures that depicted the to‐be‐learned action words performed better initially (i.e., on the first set of words) than participants who were presented with pictures showing a different action. However, this difference between relevant and irrelevant picture conditions disappeared as learners gained more task experience (i.e., on later sets of words). Eye‐tracking data sug- gested that the initial negative effect on learning disappeared because learners started to ignore the irrelevant pictures with task experience. Moreover, a subsequent study provided evidence that learners started According to well‐known multimedia design principles, presenting extraneous (i.e., irrelevant or unnecessary) information in study mate- rial should be avoided, as it hinders learning (for reviews, see Kalyuga & Sweller, 2014; Mayer & Fiorella, 2014). A recent study suggested, however, that task experience (i.e., familiarity with the design of the material) may be a boundary condition for the negative effect of extra- neous information on learning, at least when this information is picto- rial and irrelevant for the learning task (i.e., Rop, Van Wermeskerken, - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. © 2018 The Authors. Journal of Computer Assisted Learning Published by John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jcal 1 J Comput Assist Learn. 2018;1–13. 2 ROP ET AL. to ignore the pictures based on their content, and not their location (Rop, Verkoeijen, & Van Gog, 2017). 1.2 \| Task experience For instance, a negative effect of extraneous information on learning has been shown to occur when multimedia learning materials are enriched with interesting and entertaining information (i.e., seductive details; Harp & Mayer, 1998; Lehman, Schraw, McCrudden, & Hartly, 2007; Mayer, Heiser, & Lonn, 2001; Moreno & Mayer, 2000; Rey, 2014; Sanchez & Wiley, 2006), when information on related systems is presented when learning about a specific system (Mayer, DeLeeuw, & Ayres, 2007), or when mismatching pictorial information is provided when learning word definitions (De Nooijer, Van Gog, Paas, & Zwaan, 2013; Hald, Van den Hurk, & Bekkering, 2015; Rop et al., 2018, 2017). Moreover, the effect has been demonstrated when text accompanying pictures or animation is presented in both spoken and written form (e.g., Craig, Gholson, & Driscoll, 2002; Mayer et al., 2001; but see Mayer & Johnson, 2008; Yue, Bjork, & Bjork, 2013), when self‐containing There are several reasons why task experience might not have a similar effect (i.e., might not help students to learn to ignore ROP ET AL. 3 extraneous information) under those circumstances. First, textual information may be harder to ignore than pictorial information as learners often focus more quickly and more strongly on text than on the associated pictures (Cromley, Snyder‐Hogan, & Luciw‐Dubas, 2010; Hannus & Hyönä, 1999; Schmidt‐Weigand, Kohnert, & Glowalla, 2010). Second, unnecessary information might be harder to ignore than irrelevant information, as it is likely less obvious for learners that unnecessary information is extraneous to their learning process. Finally, whereas extraneous information that is presented separated from the relevant information might be relatively easy for participants to ignore, that might be more difficult when it is inte- grated with relevant information. The present study addressed these questions. the slide (control condition; see Figure 1). In this condition, we did not expect any effect of task experience on learning, as there was no information that could be ignored. However, this condition was added to assess whether the unnecessary text indeed led to lower learning outcomes. the slide (control condition; see Figure 1). In this condition, we did not expect any effect of task experience on learning, as there was no information that could be ignored. However, this condition was added to assess whether the unnecessary text indeed led to lower learning outcomes. 1.2 \| Task experience Experiment 1 investigated, by measuring learning immediately after each slide, whether an initial negative effect of unnecessary information would occur; whether this negative effect would decrease (or even disappear) as participants gained task experience; and whether this decrease would be stronger when the unnecessary text was presented separated from the picture (i.e., separated unnecessary text would be easier to ignore than integrated unnecessary text). Because we expected that the processing of unnecessary information would increase cognitive load, participants were asked to rate how much mental effort they invested in learning the materials immedi- ately after each slide (as an indicator of how much cognitive load par- ticipants experienced: Paas, Tuovinen, Tabbers, & Van Gerven, 2003). We also asked participants to rate how much mental effort they invested during the test phase after each slide, as participants who gained more knowledge during the learning phase should be able to attain higher test performance with less investment of mental effort (Van Gog & Paas, 2008). We expected that participants in the unnec- essary‐information conditions would initially invest more mental effort during the learning and test phase than participants in the control condition, while this difference should decrease (or even disappear) as participants gained task experience (at least in the separated condition). Experiment 2 was a direct replication of Experiment 1, apart from the fact that eye tracking was employed to directly study attention allocation processes. 1.3 \| The present study The present study aimed to answer two questions: (a) Do students learn to ignore unnecessary textual information with increasing task experience, and (b) is the unnecessary textual information more diffi- cult to ignore when it is integrated with relevant information? We conducted two experiments in which participants learned about the process of mitosis using a multimedia slideshow. The slides consisted of a text explaining the process of mitosis (relevant text), and a picture of the visuo‐spatial appearance of the cell in that particular stage of mitosis. In two conditions, a description of the picture components (unnecessary text) was added to the slide, either separated from (separated condition; see Figure 1) or integrated in (integrated condition; see Figure 2) the pictorial information. The design of the integrated condition (i.e., text boxes that were integrated into the picture using lines) was similar to integrated conditions used in other studies on multimedia learning (e.g., Cierniak, Scheiter, & Gerjets, 2009; Mayer & Johnson, 2008) and allowed full processing of the pic- ture. The textual information was relevant for the learning goals, but it was unnecessary as it provided a description of the picture, while a picture is generally a better representation of visuo‐spatial content (Levie & Lentz, 1982; Schmidt‐Weigand & Scheiter, 2011). In the third condition, only the relevant text and the picture were presented on 2.1.1 \| Participants and design Initially, 96 individuals participated in the study, recruited via the university's online recruitment systems. Due to an error in one of FIGURE 1 Example slide for the separated and control conditions with the areas of interest used in Experiment 2 (green: phase title, red: relevant text, pink: picture, and blue: unnecessary text). The description of the components in the picture (i.e., unnecessary text) is presented underneath the picture. In the control condition, this description is not present [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 1 Example slide for the separated and control conditions with the areas of interest used in Experiment 2 (green: phase title, red: relevant text, pink: picture, and blue: unnecessary text). The description of the components in the picture (i.e., unnecessary text) is presented underneath the picture. In the control condition, this description is not present [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 1 Example slide for the separated and control conditions with the areas of interest used in Experiment 2 (green: phase title, red: relevant text, pink: picture, and blue: unnecessary text). The description of the components in the picture (i.e., unnecessary text) is presented underneath the picture. In the control condition, this description is not present [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 1 Example slide for the separated and control conditions with the areas of interest used in Experiment 2 (green: phase title, red: relevant text, pink: picture, and blue: unnecessary text). The description of the components in the picture (i.e., unnecessary text) is presented underneath the picture. In the control condition, this description is not present [Colour figure can be viewed at wileyonlinelibrary.com] 4 ROP ET AL. FIGURE 2 Example slide for the integrated condition with the areas of interest used in Experiment 2 (green: phase title; red: relevant text, pink: picture, and blue: unnecessary text). The description of the components in the picture is integrated in the picture [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 2 Example slide for the integrated condition with the areas of interest used in Experiment 2 (green: phase title; red: relevant text, pink: picture, and blue: unnecessary text). 2.1.1 \| Participants and design The description of the components in the picture is integrated in the picture [Colour figure can be viewed at wileyonlinelibrary.com] On average, the relevant text consisted of 77 words on each slide (range 72–82), whereas the unnecessary text consisted of 39 words per slide (range 28–46). The location of the picture and the unneces- sary text was varied (left‐ or right‐hand side of the screen) between subjects to control for potential bias in attention as a result of reading direction. The learning materials were system‐paced, and the available time was the same in each condition, so participants could not com- pensate for time spent on processing the unnecessary text by investing more time on the materials overall. A small user‐paced pilot (n = 8) was used to determine the presentation time per slide. We cal- culated the average time those eight participants spent on each slide and used this as the presentation time of the corresponding slide in the current experiment (Slide 1: 65 s, Slide 2: 120 s, Slide 3: 110 s, Slide 4: 97 s, Slide 5: 107 s, and Slide 6: 77 s). It was not possible for participants to go back to a previously presented slide. those systems, it turned out that two participants had already graduated and therefore they were excluded from the sample. The final sample comprised 94 undergraduate students from a Dutch University (Mage = 21.74 years, SD = 2.55 years; 65 female), consisting of psychology students who participated for course credit (n = 37) or other students (mostly from the economic faculty) who participated for a financial compensation of 5 euro (n = 57). They were randomly assigned to one of the three conditions: control (n = 32), integrated (n = 31), and separated (n = 31). Cloze test Participants' prior knowledge was tested with four multiple‐choice questions about the process of mitosis (e.g., what is mitosis?) with four possible answers (e.g., the correct alternative: “A process in which the nucleus and duplicated chromosomes of a cell divide and are evenly distributed”). During this test, and all other tests used in this experiment, participants had to guess when they did not know an answer, as the program would not progress unless they answered a question. Knowledge about the studied mitosis phase was tested immediately after each slide, using a cloze test in which participants were pre- sented with four short sentences from the relevant text they had just studied, with one or two keyword (s) omitted (e.g., The nucleus of the newly formed cell is bound by the __). Participants were asked to fill in the blanks by typing the correct answer into the answer box. To minimize the possibility that participants recognized that only their knowledge of the relevant text was tested, the sentences were all presented on a different slide and used slightly different wording on some occasions. For example, while the slide read: “The newly formed cell contains a nucleus which is bound by the nuclear envelope,” the corresponding question was “The nucleus of the newly formed cell is bound by the __.” Learning materials The learning materials consisted of a slideshow in which six slides described and depicted the process of mitosis. During mitosis, the nucleus and chromosomes of a cell are duplicated and are divided over two new daughter cells. This process consists of six phases: inter- phase, prophase, prometaphase, metaphase, anaphase, and telophase. Each phase in the process was described on a separate slide, accompa- nied by a drawing depicting that phase. That the drawings were rele- vant for learning had been established in prior research with these learning materials, which showed that the expository text accompa- nied by pictures led to better learning outcomes than the text alone (Scheiter, Schüler, Gerjets, Huk, & Hesse, 2014; Schüler, Scheiter, & Gerjets, 2013). 2.1.2 \| Materials The materials were designed and presented using E‐Prime 2.0 (Psy- chology Software Tools, Pittsburgh, PA). 2.2.2 \| Cloze test We also estimated the Cronbach's alpha for the cloze test, which was relatively high (α = 0.80). The average performance on the first three (low task experience) and last three cloze tests (high task experience) is presented in Table 2. We performed a 3 × 2 × 2 mixed ANOVA with between‐subjects factors condition (separated, integrated, or control) and PUT location (left or right) and within‐subjects factor task experi- ence (low or high) on these data. The analysis revealed a main effect of condition, F (2, 88) = 5.06, p = 0.008, ηp 2 = 0.10, with follow‐up tests showing that performance in the separated condition (M = 1.19, SD = 0.62) was significantly lower than performance in the control condition (M = 1.71, SD = 0.65), p = 0.005, d = 0.82. Performance in the integrated condition (M = 1.45, SD = 0.64) did not significantly dif- fer from performance in the control condition, p = 0.349, d = 0.39, or the separated condition p = 0.319, d = 0.42. Moreover, the analysis revealed a main effect of task experience, F (1, 88) = 10.20, p = 0.002, ηp 2 = 0.10, indicating that cloze test performance was higher on the first three slides, when participants had less task experi- ence (M = 1.55, SD = 0.74), than on the last three slides, when they had more task experience (M = 1.36, SD = 0.74). We found no main 2.1.4 \| Scoring For all multiple‐choice questions, participants received 1 point when they gave the correct answer and no points when they gave the wrong answer. Thus, participants could score a maximum of 4 points on the pretest, and 7 points on the picture test. For the cloze test questions, participants were awarded 1 point if the correct answer was given, 0.5 points when the answer was partially correct, and 0 points when they did not provide an answer or if it was completely wrong. Thus, partic- ipants could score 0 to 4 points per cloze test after each slide. A ran- dom subset of the cloze test data (10.4%) was scored by a second rater, and interrater reliability was high (κ = 0.91). 1We took the midpoint to make the division between low and high task experience. Invested mental effort Participants were asked to indicate how much effort they invested in learning the content of each preceding slide on a 9‐point rating scale (Paas, 1992), ranging from 1 (extremely low effort) to 9 (extremely high effort). Moreover, participants were asked to indicate how much effort they invested in answering the cloze test after each slide, using the same 9‐point scale. 2.1.3 \| Procedure Participants were tested either individually or with two participants simultaneously. First, the prior knowledge test was administered, and participants were asked to fill in their age and gender. After this test, participants learned about mitosis under one of the three conditions. After each slide, participants first had to indicate how much effort they invested in studying the preceding slide, then fill in the cloze test, and then indicate how much effort they invested in answering the cloze test questions. After the learning phase, participants had to fill in the picture test. In total, the experiment took approximately 20 to 30 min, and it was administered without breaks. Picture test Because processing the unnecessary text might have gone at the expense of processing the pictures, we also tested participants' knowl- edge of the pictures at the end of the experiment. To do so, we used a multiple‐choice test consisting of seven items. In six items (present in a random order), participants were presented with a picture of one of the phases and had to choose which phase it depicted, from six alter- natives. In the seventh item, participants saw all six phases depicted on the screen and had to indicate the correct order of the pictures (i.e., according to the phases of mitosis) from six possible answers. We first estimated the Cronbach's alpha of the prior knowledge test (although it should be noted that this estimate has a high degree of imprecision because of the small sample it is based upon), which was relatively low, α = 0.28. This was to be expected as participants likely had low prior knowledge and as a result resorted to guessing. Perfor- mance on the prior knowledge test is presented in Table 1 and was analysed with a 3 × 2 analysis of variance (ANOVA) with condition (separated, integrated, or control) and PUT location (left or right) as between‐subjects factors. The analyses revealed no effect of condi- tion, F < 1, no effect of PUT location, F (1, 88) = 3.00, p = 0.087, ηp 2 = 0.03, and no interaction, F (2, 88) = 1.81, p = 0.169, ηp 2 = 0.04. Hence, there were no significant differences in prior knowledge among conditions. 2.2.1 \| Prior knowledge ROP ET AL. ROP ET AL. 5 Note. PUT: picture and unnecessary text. 2.2.3 \| Mental effort Self‐reported invested mental effort during the learning phase and the cloze test are presented in Tables 3 and 4. We performed 3 × 2 × 2 mixed ANOVAs with between‐subjects factors condition (separated, integrated, or control) and PUT location (left or right) and within‐sub- jects factor task experience (low or high) on the invested mental effort data obtained during the learning phase and the cloze test. For the invested mental effort during the learning phase, this analysis revealed no main effect of condition, F < 1, and no main effect of PUT loca- tion, F (1, 88) = 2.41, p = 0.124, ηp 2 = 0.03. The analysis did reveal a small main effect of task experience, F (1, 88) = 6.16, p = 0.015, ηp 2 = 0.07, indicating that participants invested more mental effort when they had lower task experience, (M = 6.73, SD = 1.09), than when they had more task experience, (M = 6.52, SD = 1.21). There were no significant interactions, all F s < 1. Note. PUT: picture and unnecessary text. effect of PUT location, F < 1, but the analysis did reveal an interaction between task experience and PUT location, F (1, 88) = 11.11, p = 0.001, ηp 2 = 0.11. Two‐tailed independent samples t tests showed that the location of the unnecessary information (left, M = 1.51, SD = 0.73; right, M = 1.59, SD = 0.75) did not influence cloze test per- formance when participants had low task experience, t(92) = 0.53, p > 0.999, d = 0.11, whereas participants with more task experience seemed to perform better when the unnecessary text was presented left (M = 1.52, SD = 0.78) than when it was presented right (M = 1.20, SD = 0.66), t(92) = 2.18, p = 0.064, d = 0.45. We found no interaction between PUT location and condition, F < 1. More rel- evant for our hypotheses, we found no interaction between task expe- rience and condition, F (2, 88) = 1.65, p = 0.198, ηp 2 = 0.04. However, we did find a small three‐way interaction between condition, task experience, and PUT location, F (2, 88) = 3.51, p = 0.034, ηp 2 = 0.07. 2.2.3 \| Mental effort This three‐way interaction presumably arose because initial perfor- mance differences between the control and separated conditions diminished when participants gained task experience, but only when the unnecessary information was presented on the right‐hand side of the screen (see Figure 3). We did no predict this three‐way For the invested mental effort during the cloze test, this analysis revealed no main effect of condition, F < 1, no main effect of PUT location, F (2, 88) = 1.82, p = 0.181, ηp 2 = 0.02, and no main effect of task experience, F < 1. The analysis revealed no interaction between PUT location and condition, F (2, 88) = 2.29, p = 0.107, ηp 2 = 0.05, PUT location and task experience, F (2, 88) = 1.93, p = 0.168, ηp 2 = 0.02, task experience and condition, F < 1, nor a three‐way interaction condition × PUT location × task experience, F (2, 88) = 1.15, p = 0.323, ηp 2 = 0.03. 2.2 \| Results This is because performance in the control condition diminished while reasoning from our theoretical framework one would predict the performance level to remain constant in the control condition, whereas it ought to increase in the separated condition. interaction based on our theoretical framework, and the pattern of results was not in line with it. This is because performance in the control condition diminished while reasoning from our theoretical framework one would predict the performance level to remain constant in the control condition, whereas it ought to increase in the separated condition. Experiment 1 Experiment 2 Task experience Task experience Low High Low High PUT left Control 1.72 (0.71) 1.76 (0.69) 1.48 (0.64) 1.31 (0.70) Integrated 1.48 (0.71) 1.67 (0.77) 1.43 (0.65) 1.38 (0.71) Separated 1.30 (0.74) 1.09 (0.73) 1.50 (0.50) 1.39 (0.59) Total 1.51 (0.73) 1.52 (0.78) 1.47 (0.59) 1.36 (0.66) PUT right Control 2.02 (0.72) 1.32 (0.75) 1.58 (0.83) 1.34 (1.06) Integrated 1.48 (0.72) 1.18 (0.61) 1.29 (0.73) 1.32 (0.61) Separated 1.28 (0.66) 1.09 (0.64) 1.56 (0.77) 1.60 (0.87) Total 1.59 (0.75) 1.20 (0.66) 1.48 (0.77) 1.43 (0.86) Total Control 1.86 (0.72) 1.56 (0.74) 1.53 (0.73) 1.32 (0.89) Integrated 1.48 (0.70) 1.43 (0.73) 1.36 (0.69) 1.35 (0.66) Separated 1.29 (0.69) 1.09 (0.67) 1.53 (0.64) 1.50 (0.74) Total 1.55 (0.74) 1.36 (0.74) 1.47 (0.69) 1.39 (0.76) Note. PUT: picture and unnecessary text. 2.2 \| Results TABLE 1 Mean (and SD) performance on the prior knowledge test (max. = 4) and picture test (max. = 7) as a function of condition and PUT location in Experiments 1 and 2 As mentioned in the materials section, we controlled for reading direc- tion by counterbalancing the location of the picture and the unneces- sary text (hereafter called PUT). As a check revealed that PUT location seemed to influence the dependent variables, we included it as a fac- tor in the analyses. In the analyses of the effects of task experience (i.e., on cloze test performance and invested mental effort during learning and in the cloze test), we made the distinction between low (Slides 1 to 3) and high task experience (Slides 4 to 6).1 When the sphericity assumption was violated, we report the results after Greenhouse–Geisser correction. We used partial eta‐squared and Cohen's d as measures of effect size; both can be interpreted in terms of small (ηp 2 ~ 0.01, d ~ 0.2), medium (ηp 2 ~ 0.06, d ~ 0.5), and large (ηp 2 ~ 0.14, d ~ 0.8) effect sizes (Cohen, 1988). Moreover, when post hoc follow‐up tests were performed, we used a Bonferroni correction (i.e., multiplying the p value with the number of tests performed). Prior knowledge test Picture test Exp. 1 Exp. 2 Exp. 1 Exp. 2 PUT left Control 2.76 (1.09) 2.26 (1.10) 4.59 (1.77) 4.89 (1.70) Integrated 2.19 (0.98) 2.50 (1.05) 4.69 (1.82) 4.95 (1.79) Separated 2.80 (1.08) 2.23 (1.23) 4.67 (1.99) 5.32 (1.43) Total 2.58 (1.07) 2.33 (1.12) 4.65 (1.82) 5.07 (1.62) PUT right Control 2.13 (1.13) 2.37 (0.96) 4.07 (1.22) 5.79 (1.44) Integrated 2.40 (1.06) 2.55 (1.19) 5.20 (2.01) 5.40 (1.64) Separated 2.06 (1.12) 2.41 (1.14) 4.81 (1.56) 5.23 (1.66) Total 2.20 (1.09) 2.44 (1.09) 4.70 (1.66) 5.46 (1.58) Total Control 2.47 (1.14) 2.32 (1.02) 4.34 (1.54) 5.34 (1.62) Integrated 2.29 (1.01) 2.53 (1.11) 4.94 (1.90) 5.18 (1.71) Separated 2.42 (1.15) 2.32 (1.18) 4.74 (1.75) 5.27 (1.53) Total 2.39 (1.09) 2.39 (1.10) 4.67 (1.73) 5.26 (1.60) Note. PUT: picture and unnecessary text. 6 6 ROP ET AL. ROP ET AL. TABLE 2 Mean (and SD) cloze test performance (max. = 4) as a function of condition, PUT location, and task experience in Experi- ments 1 and 2 interaction based on our theoretical framework, and the pattern of results was not in line with it. 2.2.4 \| Picture test Performance on the picture test is shown in Table 1 and was analysed with a 3 × 2 ANOVA with condition (separated, integrated, or control) and PUT location (left or right) as between‐subjects factors. The analy- sis revealed no effect of condition, F (2, 88) = 1.02, p = 0.366, ηp 2 = 0.02, no effect of PUT location, F < 1, nor an interaction effect, F < 1. FIGURE 3 Mean cloze test performance in Experiment 1 (max. = 4) as a function of condition and task experience, when the picture and the unnecessary text (PUT) location was right (Figure 3a) or left (Figure 3b) [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 3 Mean cloze test performance in Experiment 1 (max. = 4) as a function of condition and task experience, when the picture and the unnecessary text (PUT) location was right (Figure 3a) or left (Figure 3b) [Colour figure can be viewed at wileyonlinelibrary.com] ROP ET AL. TABLE 3 Mean (and SD) invested mental effort (max. = 9) during the learning phase as a function of condition, PUT location, and task experience in Experiments 1 and 2 essential for learning, it was also not immediately apparent that the added textual information was not essential to the learning goal. Therefore, our participants might still have attempted to integrate the unnecessary information with the essential information (at least during the first few phases), which led to unnecessary visual search in the separated condition. essential for learning, it was also not immediately apparent that the added textual information was not essential to the learning goal. Therefore, our participants might still have attempted to integrate the unnecessary information with the essential information (at least during the first few phases), which led to unnecessary visual search in the separated condition. 3 \| EXPERIMENT 2 Experiment 1 Experiment 2 Task experience Task experience Low High Low High PUT left Control 6.88 (1.25) 6.86 (1.17) 6.26 (1.39) 6.37 (1.46) Integrated 6.90 (0.84) 6.89 (0.84) 5.77 (1.21) 5.62 (1.18) Separated 6.02 (1.07) 6.02 (1.06) 5.91 (1.46) 5.82 (1.07) Total 6.62 (1.12) 6.72 (1.06) 5.97 (1.36) 5.92 (1.26) PUT right Control 6.04 (1.01) 6.47 (1.27) 5.98 (1.09) 5.74 (1.11) Integrated 6.09 (1.46) 6.09 (1.46) 6.02 (1.39) 6.08 (1.45) Separated 6.40 (1.30) 6.44 (1.27) 6.05 (1.05) 5.98 (0.98) Total 6.18 (1.25) 6.33 (1.34) 6.02 (1.16) 5.94 (1.18) Total Control 6.49 (1.21) 6.68 (1.21) 6.12 (1.24) 6.05 (1.32) Integrated 6.51 (1.23) 6.44 (1.40) 5.89 (1.29) 5.85 (1.32) Separated 6.22 (1.19) 6.45 (1.02) 5.98 (1.26) 5.90 (1.02) Total 6.40 (1.20) 6.52 (1.21) 5.99 (1.26) 5.93 (1.21) Note. PUT: picture and unnecessary text. In Experiment 2, we employed eye tracking to investigate how much attention participants devoted to the unnecessary information in the separated and integrated conditions and whether they would start to ignore it over time. We hypothesized that the unnecessary text would initially attract attention, but with increasing task experience, partici- pants would start to ignore the unnecessary text and allocate less attention to it, especially in the separated condition. This should result in (a) shorter fixation duration on the unnecessary text and longer fix- ation duration on the relevant text and picture with increasing task experience; and (b) more transitions between relevant information sources and less transitions between relevant and unnecessary infor- mation sources with increasing task experience. 2.2.4 \| Picture test Experiment 1 Experiment 2 Task experience Task experience Low High Low High PUT left Control 7.00 (0.89) 6.86 (1.17) 6.58 (1.05) 6.61 (1.36) Integrated 6.88 (1.11) 6.80 (1.11) 6.40 (1.16) 6.10 (1.24) Separated 6.78 (0.72) 6.47 (0.88) 6.15 (1.05) 6.09 (1.03) Total 6.89 (0.91) 6.72 (1.06) 6.37 (1.08) 6.26 (1.22) PUT right Control 6.62 (0.77) 6.47 (1.27) 6.60 (1.09) 6.19 (1.21) Integrated 6.44 (1.52) 6.07 (1.62) 6.50 (1.24) 6.30 (1.39) Separated 6.65 (1.36) 6.44 (1.17) 6.58 (0.89) 6.45 (0.85) Total 6.57 (1.24) 6.33 (1.34) 6.56 (1.06) 6.32 (1.15) Total Control 6.82 (0.85) 6.68 (1.21) 6.59 (1.05) 6.40 (1.29) Integrated 6.67 (1.32) 6.44 (1.40) 6.45 (1.18) 6.20 (1.30) Separated 6.71 (1.08) 6.45 (1.02) 6.36 (0.99) 6.27 (0.95) Total 6.73 (1.09) 6.52 (1.21) 6.46 (1.07) 6.29 (1.18) Note. PUT: picture and unnecessary text. The main effect of separated unnecessary text was qualified by a three‐way interaction between condition, task experience, and PUT location, suggesting that initial performance differences between the control and separated condition diminished when participants gained task experience, but only when the unnecessary information was presented on the right‐hand side of the screen. In contrast to our expectations, however, the reduced difference between the condi- tions seemed to result from a decline in performance in the control condition rather than an increase in performance in the separated con- dition. In sum, our hypotheses regarding task experience were not confirmed in Experiment 1. To get more insight into how students process the unnecessary text, whether the separated condition leads to unnecessary visual search, and whether this changes over time (i.e., with increasing task experience), Experiment 2 replicated Experiment 1, using eye‐tracking methodology. Note. PUT: picture and unnecessary text. TABLE 4 Mean (and SD) Invested mental effort (max. = 9) during the cloze test as a function of condition, PUT location, and task experience in Experiments 1 and 2 3.1 \| Method Note. PUT: picture and unnecessary text. 3.2 \| Results The data on prior knowledge, cloze test performance, invested mental effort, and picture test performance are analysed with the same ANOVAs as in Experiment 1. 3.1.2 \| Apparatus and materials To measure integration of the different sources of information (i.e., relevant text, picture, unnecessary text), we used transitions between the different AoIs. We defined three types of transitions: rel- evant‐picture transitions, which are transitions between the picture and relevant text and vice versa; unnecessary‐relevant transitions, which are transitions between the unnecessary and relevant text and vice versa; and unnecessary‐picture transitions, which are transitions between the unnecessary text and the picture and vice versa. The unnecessary‐relevant and unnecessary‐picture transitions can only be calculated for the two unnecessary text conditions. To control for differences in presentation duration between slides, we divided the number of transitions by the total fixation time on that slide in seconds (i.e., the same value we used in the fixation time measure). The materials were identical to those of Experiment 1. The materials were presented in SMI Experiment Center (Version 3.6; SensoMotoric Instruments), on a monitor with a resolution of 1920 × 1080 pixels. Participants' eye movements were recorded using SMI RED 250 Mobile eye trackers (SensoMotoric Instruments) that record binocu- larly at 250 Hz using SMI iView software (Version 2.8; SensoMotoric Instruments). The data were subsequently analysed using BeGaze software (Version 3.7; SensoMotoric Instruments). 2.3 \| Discussion Participants were 133 German University students (Mage = 21.28 years, SD = 2.34 years; 107 female) who participated for course credit or a small fee of 5 euro. All participants had normal or corrected‐to‐normal vision. One participant indicated after completing the experiment that s/he wanted to retract his/her data. For six participants, the data on how long they spend on each slide indicated that they had skipped parts of the learning phase. Furthermore, due to a randomization error, four participants participated in two conditions of the Experiment (i.e., they saw each slide twice, in two different conditions). The data of these 11 participants were excluded from all future analyses, resulting in a sample of 122 participants (Mage = 21.06 years, SD = 2.28 years; 98 female). Participants were randomly assigned to one of the three conditions: control (n = 38), separated (n = 44), and integrated (n = 40). Again, within conditions, PUT location was varied: For half Surprisingly, we did not find a consistent negative effect of unneces- sary information on (initial) learning. Unnecessary information only had a negative effect on cloze test performance when it was pre- sented separated from the relevant information, not when it was integrated. This unexpected finding may be due to a combination of a negative effect of unnecessary information and a kind of split‐ attention effect (as the integrated condition, without the need to split attention between the PUT, scored in between the control and sepa- rated conditions). Indeed, the split‐attention effect states that learning is hampered when mutually referring information sources that are both essential for learning are presented separately, compared with when they are physically integrated (Ayres & Sweller, 2014). Although the sources of information in the present study were not both of the participants, the PUT was presented at the right, whereas for the other half, the PUT was presented at the left. by that AoI to co value by the tota 8 ROP ET AL. ROP ET AL. 8 by that AoI to control for the size of the AoI. We then divided this value by the total fixation time on that slide in seconds (i.e., the sum of all fixations on the different AoIs and white space), to control for the differences in presentation duration and tracking ratio. by that AoI to control for the size of the AoI. 3.1.3 \| Procedure The procedure was similar to that of Experiment 1, only in Experiment 2 participants were tested individually, or in groups of up to three participants simultaneously, and their eye movements were recorded during the learning phase. At the start of the experiment, participants were seated in front of a mobile eye tracker, with their head approxi- mately 60 cm from the monitor. After a short introduction and the prior knowledge test, the eye tracker was calibrated using a 13‐point calibration plus 4‐point validation procedure, and participants were instructed to move as little as possible (although they were allowed to move somewhat). The experiment lasted around 20 min and was administered without breaks. 3.2.2 \| Cloze test Cronbach's alpha of the cloze test was again relatively high, α = 0.80. On the cloze test performance (see Table 3), the analyses revealed no main effect of condition, F < 1, no main effect of PUT location, F < 1, and no main effect of task experience, F (2, 116) = 2.50, p = 0.116, ηp 2 = 0.02. Furthermore, we found no interaction between condition and task experience, F (2, 116) = 1.35, p = 0.262, ηp 2 = 0.02, between task experience and PUT location, F < 1, or between condition and PUT location, F < 1. Finally, we did not find any evidence for the three‐way interaction observed in Experiment 1, F < 1. For the eye‐tracking analyses, we defined fixations using a 40°/s velocity threshold and a minimal duration of 100 ms (cf. Holmqvist et al., 2011). On each slide in each condition, we created areas of interest (AoIs) for the picture, for the relevant text, and for the title (see Figure 1). In the separated condition, we defined one extra AoI for the unnecessary text (see Figure 1), whereas in the integrated con- dition, we created additional AoIs for each text block (see Figure 2). In this condition, in the first three phases, there were four unnecessary text blocks, whereas the slides in the last three phases had three unnecessary text blocks. 3.1.4 \| Data analysis For the eye tracking analyses, we first checked the accuracy of calibra- tion, which was sufficient for all participants (i.e., no deviations from the four validation points of more than 1° visual angle). We then checked the tracking ratio (i.e., the percentage of time for which the eye tracker actually measured the eye movements) for each partici- pant. We had to exclude 22 participants (control: n = 8; separated: n = 5; integrated n = 9) because their tracking ratio was below 70%. The final sample (n = 100) had an average tracking ratio of 92.03% (SD = 6.75%), with a mean calibration accuracy of 0.28° (SD = 0.15o) and was distributed across the conditions as follows: control (n = 30), separated (n = 39), and integrated (n = 31). 3.2.1 \| Prior knowledge Cronbach's alpha of the prior knowledge test was again relatively low, α = 0.21, and performance on this test is presented in Table 1. The analysis revealed no effect of condition, F < 1, no effect of PUT location, F < 1, nor an interaction, F < 1. 2.3 \| Discussion We then divided this value by the total fixation time on that slide in seconds (i.e., the sum of all fixations on the different AoIs and white space), to control for the differences in presentation duration and tracking ratio. of the participants, the PUT was presented at the right, whereas for the other half, the PUT was presented at the left. 3.2.4 \| Picture test Regarding the picture test (see Table 1), the analysis showed no effect of condition, F < 1, no effect of PUT location, F (2, 116) = 2.04, p = 0.156, ηp 2 = 0.02, or an interaction, F < 1. For the fixation time on the relevant text (seeTable 5), our analysis revealed a significant main effect of condition, F (2, 94) = 31.93, p < 0.001, ηp 2 = 0.41. Follow‐up tests showed that participants in the integrated condition (M = 49.31, SD = 7.56; p < 0.001, d = 1.81) and separated condition (M = 50.20, SD = 7.51; p < 0.001, d = 1.68) fixated less on the relevant text than participants in the control condi- tion (M = 61.90, SD = 6.33). Time spent fixating on the relevant text did not differ significantly between the integrated and separated con- ditions, p > 0.999, d = 0.12. We found a significant effect of task expe- rience, F (2, 94) = 40.56, p < 0.001, ηp 2 = 0.30, indicating that participants spent more time fixating the relevant text after they gained task experience (M = 56.40, SD = 10.26), compared with when they had lower task experience (M = 50.46, SD = 10.11). Furthermore, the analysis revealed a main effect of PUT location, F (2, 94) = 17.47, p < 0.001, ηp 2 = 0.16, indicating that participants attended more to the relevant text when the PUT was on the left (M = 56.64, SD = 8.44) than when it was on the right side of the screen (M = 50.48, SD = 8.64). There were no interaction effects, smallest p = 0.070, ηp 2 = 0.06. 3.2.3 \| Invested mental effort The invested mental effort during learning is presented in Table 3. The analysis revealed no main effect of condition, F < 1, and no main effect of PUT location, F < 1. However, we did find a small main effect of task experience, F (2, 116) = 4.45, p = 0.037, ηp 2 = 0.04, indi- cating that invested mental effort during learning was higher on the first three slides, when participants had less task experience (M = 6.46, SD = 1.07) than on the last three slides when they had more task experience (M = 6.29, SD = 1.18). We found no interactions, all F s < 1. As a measure of attention to the different AoIs, we used fixation time. Because presentation time between slides was different (see the Learning materials section of Experiment 1), and the size of the AoIs was different between conditions (see Figures 1 and 2), we had to calculate a relative measure of fixation time. We did so by dividing the fixation time on each AoI by the percentage of the screen covered The analysis on the invested mental effort during the cloze test (see Table 4) revealed no main effect of condition, F < 1, no main effect of PUT location, F < 1, and no main effect of task experience, F < 1. Furthermore, we found no interactions between condition and ROP ET AL. task experience, F < 1, between PUT location and task experience, F < 1, between condition and PUT location, F (2, 116) = 1.27, p = 0.258, ηp 2 = 0.02, nor a three‐way interaction, F (2, 116) = 1.16, p = 0.317, ηp 2 = 0.02. (M = 28.62, SD = 11.53). Moreover, the analysis revealed a significant effect of task experience, F (1, 66) = 6.98, p = 0.010, ηp 2 = 0.10, indi- cating that participants spent less time fixating on the unnecessary text on the last three slides, after they gained task experience (M = 21.84, SD = 14.59), compared with on the first three slides, when they had lower task experience (M = 25.60, SD = 11.30). The analysis showed no effect of PUT location, F (1, 66) = 3.58, p = 0.063, ηp 2 = 0.05, and no interaction effects, smallest p = 0.056, ηp 2 = 0.05. 3.2.5 \| Eye movement data The eye movement data were analysed in two steps. First, we tested whether the presence and layout of unnecessary information leads to differences in attention towards unnecessary and relevant informa- tion. Second, we tested whether the presence and layout of unneces- sary information would lead to differences in integration of text and pictures. To do so, we performed a 3 × 2 × 2 mixed ANOVA with between‐subjects factors condition (separated, integrated, or control) and PUT location (left or right) and within‐subjects factor task experi- ence (low or high) on the fixation time on the relevant text and the picture. On the fixation time on the unnecessary text, we performed a 2 × 2 × 2 mixed ANOVA with between‐subjects factors condition (separated or integrated) and PUT location (left or right) and within‐ subjects factor task experience (low or high). The data on the rele- vant‐picture, unnecessary‐picture, and unnecessary‐relevant transi- tions are analysed with nonparametric tests as the assumptions of normality were violated. Regarding the fixation time on the picture (see Table 5), we found no significant effect of condition, F (2, 94) = 1.69, p = 0.189, ηp 2 = 0.04, or task experience, F (2, 94) = 1.67, p = 0.200, ηp 2 = 0.02. However, the analysis revealed a main effect of PUT location, F (2, 94) = 8.11, p = 0.005, ηp 2 = 0.08, indicating that participants fixated more on the picture when the PUT was presented on the right (M = 6.77, SD = 3.12) than when it was presented on the left side of the screen (M = 5.14, SD = 2.98). The analyses revealed no interactions, smallest p = 0.116, ηp 2 = 0.05. Note. PUT: picture and unnecessary text. 3.3 \| Discussion The results of Experiment 2 are mixed. Although the eye‐tracking measures supported our hypotheses, indicating that attention towards the unnecessary text waned with increasing task experience, this did not affect cloze test performance. More surprisingly, it seems that unnecessary text attracts less attention (as measured by fixation time) when it is integrated than when it is separated, although integrated unnecessary text leads to more integration attempts (as measured by transitions). It might have been more easy for participants to identify the function of the unnecessary text when it is integrated than when it is separated, leading to more attention towards the separated text. At the same time, the decreased spatial distance might have induced more integration attempt when the unnecessary text is integrated (cf. Ballard, Hayhoe, & Pelz, 1995; Gray & Fu, 2004). It should be noted that, although these differences are statically significant, the actual differences are quite small on some occasions. In contrast to our hypothesis, presentation of unnecessary text did not initially hamper learning about the process of mitosis, regardless of whether the unnecessary text was presented integrated in, or separated from the picture. Because we found no initial negative effect of the unnecessary text on learning, the question of whether task experi- ence would reduce or eliminate that negative effect could not be answered. Regarding the unnecessary‐relevant transitions (i.e., transitions between the unnecessary text and the relevant text; see Table 6), the analysis revealed a main effect of condition, U = 353.00, p = 0.003, indicating that participants in the integrated condition (Mdn = 0.021, Range = 0.07) made more unnecessary‐relevant transi- tions than participants in the separated condition (Mdn = 0.013, Range = 0.13). We again found a main effect of task experience, Z = 3.64, p < 0.001, indicating that participants made fewer unneces- sary‐relevant transitions after they gained task experience (Mdn = 0.013, Range = 0.10) than when they had lower task experi- ence (Mdn = 0.018, Range = 0.16). Furthermore, the analysis revealed a main effect of PUT location, U = 301.00, p < 0.001, showing that participants made more unnecessary‐relevant transitions when the PUT was presented at the right (Mdn = 0.023, Range = 0.13) than when it was presented at the left (Mdn = 0.011, Range = 0.04). Transitions relevant‐picture transitions, U = 549.00, p > 0.999. We found a signif- icant effect of task experience, Z = 3.36, p = 0.001, indicating that par- ticipants made more relevant‐picture transitions after they gained task experience (Mdn = 0.09, Range = 0.79), compared with when they had lower task experience (Mdn = 0.07, Range = 0.51). Finally, the analysis revealed an effect of PUT location, U = 904.00, p = 0.018, showing that participants made more relevant‐picture transitions when the PUT was presented at the right (Mdn = 0.09, Range = 0.45) than when it was presented at the left (Mdn = 0.5, Range = 0.37). relevant‐picture transitions, U = 549.00, p > 0.999. We found a signif- icant effect of task experience, Z = 3.36, p = 0.001, indicating that par- ticipants made more relevant‐picture transitions after they gained task experience (Mdn = 0.09, Range = 0.79), compared with when they had lower task experience (Mdn = 0.07, Range = 0.51). Finally, the analysis revealed an effect of PUT location, U = 904.00, p = 0.018, showing that participants made more relevant‐picture transitions when the PUT was presented at the right (Mdn = 0.09, Range = 0.45) than when it was presented at the left (Mdn = 0.5, Range = 0.37). On the unnecessary‐picture transitions (i.e., transitions between the unnecessary text and the picture; see Table 6), a Mann–Whitney test revealed a main effect of condition, U = 314.00, p = 0.001, indicating that participants in the integrated condition (Mdn = 0.11, Range = 0.34) made more unnecessary‐picture transitions than participants in the separated condition (Mdn = 0.06, Range = 0.34). A Wilcoxon Signed Ranks test showed a significant effect of task experience, Z = 4.75, p < 0.001, indicating that participants made fewer unnecessary‐picture transitions after they gained task experience (Mdn = 0.07, SD = 0.49) than when they had little task experience (Mdn = 0.11, Range = 0.52). Finally, a Mann–Whitney test revealed no effect of PUT location, U = 558.00, p = 0.556. Fixation time The data on the fixation time (corrected for AoI size and total fixation time, see Section 6.1.4) on the unnecessary text are presented in Table 5. The analysis revealed a significant main effect of condition, F (1, 66) = 21.14, p < 0.001, ηp 2 = 0.24, indicating that participants in the integrated condition (M = 17.57, SD = 7.80) spent less time fixating on the unnecessary text than participants in the separated condition TABLE 5 Mean (and SD) fixation duration on the different areas of interest as a function of condition, PUT location, and task experience Unnecessary text Relevant text Picture Task experience Task experience Task experience Low High Low High Low High PUT left Control 63.63 (6.90) 66.02 (6.02) 5.09 (3.45) 5.91 (4.86) Integrated 17.49 (4.79) 13.63 (9.13) 49.44 (5.99) 57.64 (8.99) 5.43 (2.81) 4.87 (2.99) Separated 30.60 (11.87) 20.82 (14.18) 46.41 (7.54) 57.12 (9.27) 4.69 (2.67) 4.93 (3.23) Total 24.86 (11.43) 17.68 (12.58) 53.03 (10.23) 60.24 (9.06) 5.04 (2.94) 5.24 (3.74) PUT right Control 56.92 (7.28) 60.17 (7.37) 7.74 (3.26) 7.47 (4.10) Integrated 20.52 (10.65) 17.92 (8.54) 42.83 (8.40) 48.80 (9.04) 6.41 (3.09) 8.33 (3.98) Separated 30.85 (9.81) 31.37 (17.16) 46.46 (7.61) 51.27 (10.27) 5.50 (3.63) 5.98 (3.11) Total 26.23 (11.32) 25.36 (15.38) 48.09 (9.49) 52.86 (10.10) 6.40 (3.42) 7.15 (3.75) Total Control 60.50 (7.74) 63.29 (7.20) 6.33 (3.57) 6.63 (4.51) Integrated 19.15 (8.53) 15.99 (8.93) 45.82 (8.02) 52.79 (9.93) 5.97 (2.96) 6.77 (3.92) Separated 30.73 (10.66) 26.50 (16.53) 46.44 (7.48) 53.97 (10.13) 5.12 (3.21) 5.50 (3.17) Total 25.60 (11.30) 21.84 (14.59) 50.46 (10.11) 56.40 (10.26) 5.75 (3.26) 6.23 (3.85) Note. PUT: picture and unnecessary text. TABLE 5 Mean (and SD) fixation duration on the different areas of interest as a function of condition, PUT location, and task experience xation duration on the different areas of interest as a function of condition, PUT location, and task experience BLE 5 Mean (and SD) fixation duration on the different areas of interest as a function of condition, PUT locat ROP ET AL. 10 4 \| GENERAL DISCUSSION According to well‐known principles in multimedia learning, the presen- tation of extraneous information (i.e., irrelevant or unnecessary) should be avoided, because it hinders learning. These are the coher- ence principle (cf. Harp & Mayer, 1998; Mayer & Fiorella, 2014) and the redundancy principle (cf. Chandler & Sweller, 1991; Kalyuga & Sweller, 2014). Although the coherence principle mostly entails the negative effect of irrelevant information (not related to the learning goal), the redundancy principle mostly concerns the negative effect of unnecessary information (related to the learning goal, but not nec- essary for learning). Recent research with irrelevant pictorial informa- tion demonstrated that students may learn to ignore such information when they gain experience with the task, at which point it no longer negatively affects their learning (Rop et al., 2018). The present study aimed to examine whether these findings would extend to extraneous information that is textual and unnecessary rather than irrelevant. Moreover, we investigated the role of the layout of the unnecessary textual information: We expected that it would be harder for students to (learn to) ignore unnecessary text when it is presented spatially integrated in a relevant picture, as compared with spatially separated from the picture. Another potential explanation might lie in the amount of time that learners had available. We based the time per slide on the average study time of eight participants in a pilot study, which can therefore be assumed to have been sufficient for most of our participants. It is possible that processing unnecessary information would start to ham- per learning when there is time pressure. When there is little time available for processing, any time spent on the unnecessary informa- tion goes at the expense of thoroughly processing essential informa- tion, and as a result, learning is hampered. In the present study, learners may have had sufficient time for processing all sources of information, which would explain why their attention to the unneces- sary text (as demonstrated in Experiment 2) did not significantly increase experienced cognitive load and did not negatively affect learning as measured by either the cloze tests or the picture test. Sys- tematically varying the presentation time in future research could shed some light on this issue. The eye‐movement data collected in Experiment 2 showed that the unnecessary textual information was processed by students and, more interestingly, that they seemed to start ignoring the unnecessary information with increasing task experience. 3.3 \| Discussion A possible explanation is that this might be due to the fact that participants looked at the centre of the screen at the beginning of each new slide because the mental effort question on the preceding slide was presented in the centre. When attention is centrally located, one may be inclined (because of Western reading direction) to process information on the right‐hand side of the screen first. However, this explanation makes the implicit assumption that participants directly attend to (relevant) information on a new slide, without first making a saccade from the centre to the (top)‐left part of the screen, and is therefore speculative. Future research should replicate these findings before any theoretical or practical conclusions can be drawn. strategy and start to focus less on extraneous and more on essential information once they gain experience with a task, which is relevant information for instructional designers. Although the results also implied that more attention was paid to the essential text on the later slides, this change in study strategy did not lead to improvements in test performance (improvements that were observed by Rop et al., 2018). Surprisingly, the presentation of unnecessary text did not consistently hamper learning about the pro- cess of mitosis in the two experiments (i.e., only a small negative effect of separated unnecessary text in Experiment 1, but not in Experiment 2; no negative effect of integrated unnecessary text in both experi- ments). Because we did not reliably find an initial negative effect of the unnecessary text on learning, the question of whether task expe- rience would reduce or eliminate that negative effect could not be answered. A possible explanation for why the unnecessary informa- tion did not initially have a negative effect on learning even though it was processed might lie in the nature of the extraneous information, that is, in whether it is irrelevant or unnecessary. It is possible that the negative effects of irrelevant information on learning would be larger than the effects of unnecessary information. That is, processing irrele- vant information not only takes up working memory capacity but also might actively interfere with learning the essential information, by disrupting the processing, organization, and integration of essential information. Processing unnecessary information on the other hand (which is identical in content to the essential information) does take up working memory capacity but may interfere less with learning the essential information. 3.3 \| Discussion Regarding the relevant‐picture transitions (i.e., transitions between the relevant text and the picture; see Table 6), a Kruskall–Wallis test revealed a significant effect of condition, χ2(2) = 11.56, p = 0.003. Fol- low‐up Mann–Whitney tests showed that both participants in the integrated (Mdn = 0.05, Range = 0.45; U = 247.00, p = 0.006) and sep- arated conditions (Mdn = 0.07, Range = 0.39; U = 359.00, p = 0.018) made significantly fewer relevant‐picture transitions than participants in the control condition (Mdn = 0.12, Range = 0.40). Participants in the integrated and separated conditions did not differ in the number of Next to the main finding of diminishing attention to the unneces- sary text in favour of attention to the essential textual information, the eye‐tracking analyses suggested an effect of screen location of TABLE 6 Median (and range) number of transitions between the different areas of interest as a function of condition, PUT location, and task experience BLE 6 Median (and range) number of transitions between the different areas of interest as a function of cond i BLE 6 Median (and range) number of transitions between the different areas of interest as a function of c erience Relevant‐picture Unnecessary‐picture Unnecessary‐relevant Task experience Task experience Task experience Low High Low High Low High PUT left Control 0.12 (0.44) 0.11 (0.45) Integrated 0.03 (0.09) 0.04 (0.25) 0.11 (0.19) 0.07 (0.16) 0.018 (0.03) 0.012 (0.05) Separated 0.03 (0.35) 0.05 (0.25) 0.11 (0.52) 0.05 (0.13) 0.008 (0.05) 0.000 (0.03) Total 0.04 (0.47) 0.05 (0.46) 0.11 (0.52) 0.06 (0.18) 0.012 (0.05) 0.009 (0.05) PUT right Control 0.13 (0.29) 0.11 (0.69) Integrated 0.08 (0.49) 0.11 (0.78) 0.13 (0.20) 0.10 (0.48) 0.034 (0.11) 0.018 (0.08) Separated 0.08 (0.38) 0.07 (0.40) 0.07 (0.42) 0.07 (0.26) 0.016 (0.16) 0.016 (0.10) Total 0.08 (0.51) 0.10 (0.78) 0.10 (0.42) 0.08 (0.49) 0.025 (0.16) 0.017 (0.10) Total Control 0.12 (0.44) 0.11 (0.75) Integrated 0.05 (0.51) 0.06 (0.79) 0.12 (0.20) 0.08 (0.48) 0.024 (0.11) 0.017 (0.08) Separated 0.06 (0.38) 0.06 (0.42) 0.08 (0.52) 0.07 (0.26) 0.010 (0.16) 0.009 (0.10) Total 0.07 (0.51) 0.09 (0.79) 0.11 (0.52) 0.07 (0.49) 0.018 (0.16) 0.013 (0.10) ROP ET AL. 11 the unnecessary text and picture. When the relevant text was on the right‐hand side of the screen (and the picture + unnecessary text on the left), participants attended more to it than when it was on the left‐hand side of the screen. 4.1 \| Limitations and future research It is interesting that we replicated the finding that attention to extra- neous information wanes with the present materials, as these are more ecologically valid and more complex than the word learning materials in the studies by Rop and colleagues (2018, 2017). However, a possible limitation of the present study, which might perhaps also explain the lack of effects on learning outcomes, is that the different phases of the process of mitosis are not fully independent of each other. As each phase is building on the information that was provided in the previous phase, the processing of later slides might have been dependent on how well information from the previous slides had been learned. Moreover, some phases might be more complex than others, which is also suggested by the differences in processing time per slide. Another potential limitation is that the cloze test mostly tested retention of the essential text while disruption of relevant learning processes (i.e., selection, organization, or integration; Mayer, 2014) might be more reflected in outcome measures that reflect deeper pro- cessing (such as a measure of transfer). Therefore, it is possible that the results regarding learning outcomes would be different when the test would assess understanding (e.g., by means of inference ques- tions). Future studies should try to rule out this possibility by including measures of both retention and transfer. Moreover, because students were tested in between, we should be cautious in concluding that students learned to ignore unnecessary information spontaneously; they may have been aided by the cloze tests, which gave clues regard- ing the essential information. 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Contemporary Educa- tional Psychology, 35, 59–74. https://doi.org/10.1016/j.cedpsych. 2009.10.002 Concluding, the results of this study are interesting in that they provide evidence that learners adapt their study strategy and start to ignore unnecessary information with increasing task experience. However, this does not seem to lead to a change in learning outcomes, presumably because we found no initial negative effect of such unnec- essary information on learning. Therefore, our results call for further research aiming to pinpoint conditions under which extraneous information presentation negatively affects learning, and employing eye‐tracking methodology to study the attention allocation processes during learning may help accomplish this (see also Van Gog & Scheiter, 2010). 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(2018), who showed that learners start to ignore pictorial, obviously irrelevant, and separated extraneous information. The present study shows that these results also apply when the extraneous information is textual, unnecessary rather than irrelevant, and when it is integrated or separated with relevant infor- mation. This provides further evidence that learners adapt their study The finding that students adapt their study strategy with increas- ing task experience is interesting in light of the expertise reversal effect (for a review, see Kalyuga, 2014), which states that learning materials that are essential and nonredundant for novices become redundant when learners gain or have more prior knowledge, at which point they will no longer aid, and might even hinder learning. 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https://openalex.org/W4205908365	https://figshare.com/articles/journal_contribution/Development_of_a_Remote_Online_Collaborative_Medical_School_Pathology_Curriculum_with_Clinical_Correlations_across_Several_International_Sites_through_the_Covid-19_Pandemic/21597846/1/files/38278320.pdf	English	null	Development of a remote online collaborative medical school pathology curriculum with clinical correlations, across several international sites, through the covid-19 pandemic	Research Square (Research Square)	2,020	cc-by	5,999	Item type Journal Contribution Journal Contribution Development of a Remote Online Collaborative Medical School Pathology Curriculum with Clinical Correlations, across Several International Sites, through the Covid-19 Pandemic Gerardo E. Guiter, Sandra Sapia, Alexander I. Wright, Gordon G. A. Hutchins, Thurayya Arayssi Development of a Remote Online Collaborative Medical School Pathology Curriculum with Clinical Correlations, across Several International Sites, through the Covid-19 Pandemic Gerardo E. Guiter, Sandra Sapia, Alexander I. Wright, Gordon G. A. Hutchins, Thurayya Arayssi Development of a Remote Online Collaborative Medical School Pathology Curriculum with Clinical Correlations, across Several International Sites, through the Covid-19 Pandemic Terms of use This work is licensed under a CC BY 4.0 license This version is available at https://manara.qnl.qa/articles/journal_contribution/Development_of_a_Remote_Online_Collaborative_Medical_School_Pathology_C 19 P d i /21 9 846/1 This version is available at This version is available at https://manara.qnl.qa/articles/journal_contribution/Development_of_a_Remote_Online_Collaborative_Medical_School_Pathology_C 19 Pandemic/21597846/1 https://manara.qnl.qa/articles/journal_contribution/Development_of_a_Remote_Online_Collaborative_Med 19 Pandemic/21597846/1 _ Access the item on Manara for more information about usage details and recommended citation. Access the item on Manara for more information about usage details and recommended citation. * Gerardo E. Guiter geg2003@qatar-med.cornell.edu Posted on Manara – Qatar Research Repository on Posted on Manara – Qatar Research Repository on 2022-11-22 https://doi.org/10.1007/s40670-021-01212-2 Medical Science Educator (2021) 31:549–556 ORIGINAL RESEARCH 1 Division of Medical Education, Weill Cornell Medicine-Qatar, 445 East 69 Street, RM 432, New York, NY 10021, USA Abstract Students conveyed high levels of satisfaction about the elective’s overall quality, their pathology learning and online interactions, with minimal challenges related to the remote nature of the course. Discussion and Conclusions Technological innovations mitigate sudden disruptions in medical education. A remote curriculum allows instruction at any distance, at any time, from anywhere, enhancing educational exchanges, flexibility and globalization in medical education. Keywords Pathology . Online . Remote . Curriculum . Medical . School Keywords Pathology . Online . Remote . Curriculum . Medical . School 4 Leeds Teaching Hospitals NHS Trust/University of Leeds. Histopathology and Molecular Pathology, St James’ University Hospital, Beckett Street, Leeds LS9 7TF, UK Development of a Remote Online Collaborative Medical School Pathology Curriculum with Clinical Correlations, across Several International Sites, through the Covid-19 Pandemic Gerardo E. Guiter1 & Sandra Sapia2 & Alexander I. Wright3 & Gordon G. A. Hutchins4 & Thurayya Arayssi1 Gerardo E. Guiter1 & Sandra Sapia2 & Alexander I. Wright3 & Gordon G. A. Hutch Accepted: 8 January 2021 # The Author(s) 2021 / Published online: 20 January 2021 Abstract Introduction Due to the Covid-19 social distancing restrictions, in March 2020, Weill Cornell Medicine-Qatar decid- ed to replace students’ clinical instruction with novel online electives. Hence, we implemented an innovative online and remote pathology curriculum, anchored on virtual microscopy and Zoom videoconferencing: ideal tools to support online teaching. g a new curriculum implementation at Weill Cornell Medicine-Qatar. pp g Objective To assess a new curriculum implementation at Weill Cornell Medicine-Qatar. j p Q Materials and Methods This for-credit, 2-week elective included 6 synchronous Zoom sessions where complex clinico- pathological cases were discussed in small groups. We used open access digital microscopy slides from the University of Leeds’ Virtual Pathology Library (http://www.virtualpathology.leeds.ac.uk/slides/library/). Students independently prepared for these sessions by reviewing cases, slides, readings, and questions in advance (asynchronous self-directed learning anchored on a flipped classroom model), and wrote a final review of a case. An assessment and feedback were given to each student. Results Four elective iterations were offered to a total of 29 students, with learners and faculty spread over 4 countries. During the Zoom sessions, students controlled the digital slides and offered their own diagnoses, followed by group discussions to strength- en autonomy and confidence We surveyed learners about the elective’s performance (program evaluation) Students conveyed Results Four elective iterations were offered to a total of 29 students, with learners and faculty spread over 4 countries. During the Zoom sessions, students controlled the digital slides and offered their own diagnoses, followed by group discussions to strength- en autonomy and confidence. We surveyed learners about the elective’s performance (program evaluation). Students conveyed high levels of satisfaction about the elective’s overall quality, their pathology learning and online interactions, with minimal Results Four elective iterations were offered to a total of 29 students, with learners and faculty spread over 4 countries. During the Zoom sessions, students controlled the digital slides and offered their own diagnoses, followed by group discussions to strength- en autonomy and confidence. We surveyed learners about the elective’s performance (program evaluation). Students conveyed high levels of satisfaction about the elective’s overall quality, their pathology learning and online interactions, with minimal challenges related to the remote nature of the course. en autonomy and confidence. We surveyed learners about the elective’s performance (program evaluation). 3 Section of Pathology, Leeds Institute of Medical Research, University of Leeds, 4.11 Wellcome Trust Brenner Building, St James’s University Hospital, Beckett Street, Leeds LS9 7TF, UK 2 Division of Medical Education, Weill Cornell Medicine- Qatar, Qatar Foundation - Education City, P.O. Box 24144, Doha, Qatar 1 Division of Medical Education, Weill Cornell Medicine-Qatar, 445 East 69 Street, RM 432, New York, NY 10021, USA 2 Division of Medical Education, Weill Cornell Medicine- Qatar, Qatar Foundation - Education City, P.O. Box 24144, Doha, Qatar 3 Section of Pathology, Leeds Institute of Medical Research, University of Leeds, 4.11 Wellcome Trust Brenner Building, St James’s University Hospital, Beckett Street, Leeds LS9 7TF, UK 4 Leeds Teaching Hospitals NHS Trust/University of Leeds. Histopathology and Molecular Pathology, St James’ University Hospital, Beckett Street, Leeds LS9 7TF, UK Design of the Elective As a result, our institution faced a sudden challenge: to adapt to the new situation and enable our students to progress in the curriculum safely. Consequently, WCM-Q decided to replace our learners’ clinical work with a portfolio of novel electives to be delivered entirely online starting in mid-April 2020, covering various fields of expertise. The strategy in- cluded offering 2- or 4-week electives to provide students with enhanced flexibility if they needed to return to the clinical space on short notice. We designed a 2-week online elective with the overarching goal that learners, by focusing on the study of several malig- nant tumors and their precursor lesions, would acquire an enhanced knowledge of malignant diseases and therefore the basis for robust understandings of oncologic pathology and oncology. The elective was available for any student who had successfully completed our foundational sciences curriculum. Students passing the elective would gain appropriate credit as determined by institutional policy. At that challenging time, the authors of this project be- lieved that pathology teaching offered a superb solution for our institutional needs, based on the use of virtual microscopy and digital slides. These are ideally suited for delivering an undergraduate pathology curriculum [9], and many institu- tions in North America and Europe have already switched to this approach [10–12]. Indeed, WCM-Q has been using virtu- al slides for pathology and histology instruction throughout all basic science courses since 2005, and the American Board of Pathology certification exam began using virtual microscopy in 2003 [13]. Large academic medical centers have ample experience with virtual microscopy and employing digital slides for undergraduate teaching [14] and clinical diagnostic purposes [15], and most recently, during the Covid-19 pan- demic [16, 17]. In the US, a corresponding relaxation of gov- ernment regulations has allowed pathologists to review and report pathology specimens from remote, non-Clinical Laboratory Improvement Amendments (CLIA) certified facil- ities [17], expanding the use of digital slides with remote reviews. The elective included a total of 6 interactive, synchronous live sessions using Zoom videoconferencing (3 sessions per week). We decided to accept a maximum of 10 students per elective iteration, believing that an instructional approach based on small groups best suited our program, supporting increased student participation and positive learning outcomes. Introduction * Gerardo E. Guiter geg2003@qatar-med.cornell.edu Weill Cornell Medicine-Qatar (WCM-Q) is an international site of Weill Cornell Medicine. Established in 2001 as a part- nership between Cornell University and Qatar Foundation, WCM-Q is part of Cornell University in Ithaca, New York, and shares the tripartite mission of Weill Cornell Medicine- New York (WCM-NY): a dedication to excellence in educa- tion, patient care, and research. It offers an integrated program of pre-medical and medical studies leading to the Cornell University MD degree. 1 Division of Medical Education, Weill Cornell Medicine-Qatar, 445 East 69 Street, RM 432, New York, NY 10021, USA WCM-Q medical school curriculum is guided by key prin- ciples of enhancing the integration between the foundational Med.Sci.Educ. (2021) 31:549–556 550 restrictions imposed by the Covid-19 pandemic, our project sought to develop and implement an undergraduate innovative pathology collaborative learning experience. Students and in- structors would connect remotely and share––synchronously and asynchronously––virtual microscopy, digital slides, and other images, as well as discussions, questions, and answers through the Zoom videoconferencing system, currently avail- able at WCM-Q as an institutional subscription. We targeted WCM-Q medical students that had already completed their foundational sciences courses. The theoretical framework for this new curriculum was anchored on the flipped classroom model, with students initially learning and constructing new knowledge on their own, and faculty subsequently utilizing online classroom time for problem solving, formative teach- ing, and answering students’ questions. sciences and clinical learning, and introducing students to pa- tient care from the first day of their studies. The curriculum is divided into 3 phases. During Phase 1, which extends over 18 months, students complete their foundational sciences courses. Phase 2 is dedicated to 12 months of clinical clerk- ships at primary and tertiary care academic centers in Doha, Qatar. Phase 3 focuses on advanced clinical experiences and the completion of a scholarly capstone project. Due to the global Covid-19 pandemic, in March 2020, WCM-Q suspended all clinical instruction in a move aligned with the Association of American Medical Colleges (AAMC) and the Liaison Committee on Medical Education (LCME) recommendations [1–3] and similar decisions by other educa- tional institutions in North America, Asia, and Europe [4–7]. Introduction This approach to protecting students was sensible particularly in light of past experiences, such as when 16 medical students exposed to severe acute respiratory syndrome (SARS) patients in a hospital during the Hong Kong 2003 outbreak unfortu- nately contracted the disease [8]. Design of the Elective Two WCM-Q and Department of Pathology and Laboratory Medicine WCM-NY faculty members developed 9 clinicopathological cases which included links to access remotely corresponding digital slides from the University of Leeds Virtual Pathology Slide Library (http://www. virtualpathology.leeds.ac.uk/slides/library/). This open access collection of slides is digitized and curated by trained pathologists and stored with anonymized clinical information, used for teaching and training. Slides can be viewed on standard computer screens and mobile devices and are globally accessible. All cases included an initial clinical vignette describing a patient presentation and diagnostic imaging and laboratory results, followed by biopsy and/or surgical excision histopathological findings. The cases also presented illustrations of gross specimens, immunohistochem- ical images, and for some, descriptions and results of However, virtual microscopy and digital slides in medical school education have traditionally accompanied face-to-face “in-situ” interactions, with very few remote applications and mostly targeting post-graduate training [18, 19]. To commu- nicate with our learners within the social distancing Med.Sci.Educ. (2021) 31:549–556 551 images, and other documents, and assessment of students after completion of the elective. advanced molecular studies. The cases focused on uterine cervical, colonic, pulmonary, and mammary oncologic pa- thology, and included a list of differential diagnoses, with the University of Leeds Slide Library virtual images illustrat- ing alternative but meaningful and related pathological entities. Students were assessed through direct faculty observa- tions of their preparation for case discussions, in class participation and contributions, responses to basic and USMLE-type questions, and also by writing a final 500- word summary review of a case discussed during the elec- tive. This review had to address all significant pathologi- cal and related clinical issues. It was open-ended, and no written rubric was provided to the students; instead, they were given verbal instructions at the beginning and end of the elective. The weight of this assignment was 50% of the overall final grade. As our foundational sciences curriculum at WCM-Q in- cludes general and systemic pathology, our students had al- ready acquired a basic knowledge of this discipline. We ac- cordingly created cases that were somewhat more complex than those our learners had already studied, so they would continue incorporating new information and constructing new understandings in pathology throughout this elective. We included in all cases 2 types of questions aimed at topics addressed during the sessions: first order basic queries and more complex United Stated Medical Licensing Examination (USMLE) type items. Design of the Elective Students were expected to answer these questions to the best of their knowledge drawn from readings and general preparation. We included the USMLE items with the following 3 objectives: first and most importantly, to ensure students had learned their materials and increased their knowledge; second, to strengthen their self- directed learning (SDL) and subsequent self-assessment skills; and third, to contribute to their preparation for the USMLE Step 1, an exam taken by most of our learners prior to graduation. A final written assessment of each student’s performance included several items graded on a 5-point Likert type rating scale, including formative and summative comments. In addi- tion, the faculty provided oral formative feedback to the stu- dents through one-on-one Zoom meetings. The final course grade was assigned as Pass/Fail. This project was submitted to the WCM-Q Institutional Review Board (IRB) and determined IRB exempt. In addition, the institutional leadership was fully supportive of our initia- tive, quickly approving the elective for credit recognition and ensuring all the resources were readily accessible to the teach- ing faculty. We decided to interactively teach and connect with the students via the Zoom videoconferencing platform. Zoom al- lows for screen sharing and control at a distance by any par- ticipant––a significant plus, we believed, that could enhance learner autonomy and self-discovery skills by granting stu- dents full remote control to point out specific areas while describing virtual pathology or radiology images. In addition, we also chose to make use of the built-in Zoom polling as another way to increase student contributions during case discussions. Implementation of the Elective This questions and answers section also allowed faculty to assess learners’ prior pre- paratory SDL effectiveness, identify any knowledge gaps, make all necessary corrections, and provide formative feedback (Fig. 1). Finally, we asked the students to describe the most and the least effective aspects of their remote learning experiences. Virtual pathology slides, ability to communicate with peers and engage more in the activities through online learning rath- er than when it is face-to-face, easier communication with the professors, more relaxed environments at home, enhanced time management with no time lost in transportation to/from the college, lack of distraction by peers, solving the cases prior to the sessions and inclusion of USMLE-type questions were all reported as highly effective. Indeed, some students men- tioned they preferred remote instruction over face-to-face teaching. No comments were received on the least effective aspects of the course. We did not experience any difficulties with the Zoom plat- form, which worked very well during all sessions, and en- countered no “Zoombombing” [19]. Implementation of the Elective Four elective iterations were offered to a total of 29 students between April 19 and June 25. The first iteration included 10 students, the second included 5 students, and the third and fourth iterations included 7 students each. Learners and facul- ty were spread over 4 different countries (Qatar, Saudi Arabia, Peru and Argentina). As we planned the elective, we decided that students would be expected to independently prepare for these Zoom sessions by reviewing the cases and digital slides, completing assigned readings, and answering all basic and USMLE-type questions in advance (asynchronous SDL). Accordingly, students were provided with access to all materials, including the virtual pathology slides, 72 h before each Zoom meeting. This inde- pendent preparation was factored into the students’ assess- ment (please see below). Therefore, in order to support our students’ success, the program comprised two components of approximately equal importance: a preparatory asynchronous SDL step followed by synchronous interactive sessions with live Zoom discussions. The 2 participating WCM-Q/WCM-NY faculty facilitat- ed the Zoom instruction. Following the program design, we met with students 3 times per week for sessions lasting 90 to 120 min. While discussing cases, the students had many opportunities to take control of the virtual slides, moving the images and zooming in and out at their discre- tion, and selecting areas of interest to share with the group. They also described their initial preliminary pathological diagnoses based on their evaluation of the virtual slides. In so doing, the learners “acted” as pathologists in control of their cases, similar to what happens at pathology depart- ments during consensus meetings where difficult cases are shown to colleagues. In general, learners took turns to offer their preliminary and differential diagnoses, with faculty facilitating active participation and contributions by each student. In addition, towards the end of each case The elective was hosted on the WCM-Q Canvas Learning Management System (LMS). Each iteration included a unique centralized site on Canvas, allowing for faculty and learner interactions, student submissions, access to teaching files, 552 Med.Sci.Educ. (2021) 31:549–556 in this course. Only 1 out of 23 (4.35%) respondents answered that they had, noting “some lag in the communications.” discussion, students answered the basic and USMLE-type questions anonymously, through the Zoom polling. Subsequently, responses were analyzed by the group, in order to enrich the discussions. Discussion The program was very well received by the students. Based on their written and oral feedback, the remote nature of the course presented minimal challenges. Learners reported high satisfaction with the opportuni- ties to interact online with peers and faculty, and with their learning of pathology and related topics. Indeed, some students mentioned they preferred this mode of teaching over face-to-face instruction. Reasons for this predilection included, among others, as mentioned be- fore, enhanced time management and a lack of distrac- tion by peers, as sometimes happens during instruction at the college. In addition, we as faculty did not en- counter any difficulties with the remote delivery of our curriculum. This project involved the development and implementation of a novel undergraduate pathology curriculum at WCM-Q, built on the use of virtual microscopy, with learners and faculty interacting remotely by Zoom videoconferencing––a notably original approach in pathology teaching in medical school education. Fortunately, we were able to design and implement this elective swiftly, as our appraisal of existing infrastructure con- firmed that all the resources needed were already available. These included Zoom videoconferencing, Canvas, Information Technology assistance, secretarial support, OED collaboration, and the University of Leeds virtual slides. In addition, we obtained crucial WCM-Q leadership support, as evidenced by their quick approval of the elective for credit recognition and by ensuring that all the resources were readily accessible. Our program was a response to the sudden need to interact with students at a distance. We switched from a traditional teaching paradigm of face-to-face interactions with our learners to a new ideal approach of 100% remote exchanges. This experience parallels similar needs and responses in the clinical arena. For example, in the US as a result of this pandemic, hospital pathol- ogists have been allowed to temporarily switch reporting of cases to remote locations, following guidance by the College of American Pathologists (CAP) and the During our needs assessment, we also considered the characteristics of our targeted learners. First, they are tech savvy, critical in this elective, which is heavily reliant on technology. Second, they are familiar with flipped classrooms and therefore were well prepared for our elective entailing many aspects of the flipped classroom model. Assessment of the Elective and Students’ Feedback (2021) 31:549–556 Table 1 Survey of students about the elective’s performance (program assessment)-Part A Table 1 Survey of students about the elective’s performance (program assessment)-Part A Meana Standard Deviation Elective’s overall quality 4.9 0.34 Clarity of elective’s goals and objectives 4.9 0.34 Elective’s organization (syllabus, schedule, learning arrangements, support mechanisms) 4.8 0.41 Elective’s contribution to the students’ learning and understanding of oncologic pathology 4.8 0.51 a Calculated using a 5-point rating scale, with “1” representing the lowest possible score and “5” representing the highest possible score clinicopathological cases and virtual slides, since pathol- ogy teaching during the foundational courses at WCM- Q is grounded on these tools. Finally, we did not en- counter any barriers to this elective. All of these factors mentioned above are critical for the implementation and success of a new medical school curriculum. satisfaction and participation. Every student passed the elective. Assessment of the Elective and Students’ Feedback We surveyed our students to acquire some preliminary im- pressions and data about the elective’s performance and func- tioning (program assessment), and to make any necessary changes. We aimed to evaluate the elective’s overall quality and the quality of student and faculty online interactions, our students’ online and remote learning of pathology, any chal- lenges associated with their work and communications, and the most/least effective aspects of their remote learning expe- riences. We acknowledged that this appraisal would only be based on our learners’ input, assessment, and feedback. During the four iterations of the course, we encountered loss of Internet during 1 of 24 sessions (4.16%), which affect- ed the connectivity of a few students. This session was re- scheduled for the next day, resolving the problem. We also encountered loss of the virtual microscopy website connectiv- ity during 2 of 24 sessions (8.33%). This issue, though not ideal, was solved by sharing static images (JPGs) instead, prepared in advance from the same corresponding digital slides. We did not encounter any other barriers or challenges to our remote online program. A 2-part, anonymous survey, prepared by the WCM-Q Office of Educational Development (OED) for all virtual elec- tive courses, was distributed to the 29 students who had com- pleted our pathology elective, and 23 (79.31%) responded. Tables 1 and 2 summarize the survey results. While technical troubles, as described above, were rare, they underscored the critical importance of developing Internet and server back-up systems for institutions that offer online courses. In addition, although during our elective we used standard home-type Internet, networks that offer ade- quate bandwidth are crucial. In summary, efforts must be made to mitigate technical risks that can diminish student We additionally asked the students if they had or had not experienced any challenges due to their remote participation Fig. 1 WCM-Q faculty and students discussing a virtual microscopy pathology slide via Zoom videoconferencing Fig. 1 W Fig. 1 WCM-Q faculty and students discussing a virtual microscopy pathology slide via Zoom videoconferencing 553 Med.Sci.Educ. Table 2 Survey of students about the elective’s performance (program assessment)-Part B Discussion Third, our students have strong pa- t h o l o g y f o u n d a t io n s an d a r e f a m i l i a r wi t h Table 2 Survey of students about the elective’s performance (program assessment)-Part B Table 2 Survey of students about the elective’s performance (program assessment)-Part B y p (p g ) Meana Standard Deviation After completing this elective, my knowledge of some complex pathological entities and diseases has improved 4.6 0.49 After completing this elective, my recognition of the critical importance of robust clinicopathological correlations has improved 4.6 0.49 After completing this elective, my comprehension of the rationale supporting targeted therapies and preventive approaches in malignancies has improved 4.4 0.65 The elective’s Zoom sessions encouraged interactions between students 4.8 0.39 The elective’s Zoom sessions encouraged interactions between faculty and students 4.7 0.44 a Calculated using a 5-point Likert scale: [SD] Strongly Disagree = 1; [D] Disagree = 2; [N] Neutral = 3; [A] Agree = 4; [SA] Strongly Agree = 5 Meana Standard Deviation After completing this elective, my knowledge of some complex pathological entities and diseases has improved 4.6 0.49 After completing this elective, my recognition of the critical importance of robust clinicopathological correlations has improved 4.6 0.49 After completing this elective, my comprehension of the rationale supporting targeted therapies and preventive approaches in malignancies has improved 4.4 0.65 The elective’s Zoom sessions encouraged interactions between students 4.8 0.39 The elective’s Zoom sessions encouraged interactions between faculty and students 4.7 0.44 a Calculated using a 5-point Likert scale: [SD] Strongly Disagree = 1; [D] Disagree = 2; [N] Neutral = 3; [A] Agree = 4; [SA] Strongly Agree = 5 The elective’s Zoom sessions encouraged interactions between faculty and students 554 Med.Sci.Educ. (2021) 31:549–556 Centers for Medicare and Medicaid Services (CMS) [20–22]. In fact, as per CMS guidance, a temporary testing site may even include the pathologist’s home. [25–27]. Luckily, although the COVID-19 pandemic has harshly impacted education at many levels, we now have in- structional tools previously unavailable. Still, it is imperative to learn from recent events, since the possibility exists of fu- ture analogous challenges. The current pandemic has encour- aged instructional innovations that must be included in our medical education armamentarium. We were also interested in whether students carried on developing their SDL and self-discovery skills through this program. Discussion They were accordingly provided with cases and ac- cess to virtual slides, readings, and other activities 72 h in advance of Zoom meetings. During their preparatory time, the learners reviewed these items on their own and were ex- pected to create differential diagnoses, eventually formulating their own specific cancer diagnosis. Thus, this preparatory time was a great opportunity for SDL and self-discovery in line with LCME standards for accreditation of medical educa- tion programs [23] and World Federation for Medical Education (WFME) basic medical education global standards for quality improvement [24]. Nevertheless, we would need a considerably extended follow-up and further appraisals to de- termine if this approach enhances the SDL of our students in the long-term. The same applies to including USMLE-type questions in the cases, and correlating this strategy with the USMLE performance of our learners. A benefit of this remote curriculum is that it allows instruc- tion at any distance, for any reason, at any time, and from anywhere. This supports flexibility and globalization in med- ical education. As virtual teaching becomes more generalized, it will allow students to interact with peers and faculty at other institutions worldwide, supporting and increasing educational exchanges. Remote education, however, cannot replace face-to face instruction. We favor a blended system, including distanced and face-to-face teaching. Moreover, some key aspects of medical education (e.g., bedside teaching), can only occur in the context of direct contact with patients. It is also important that policymakers and governing bodies ensure that medical schools have all the necessary resources to face the next “emergency” in undergraduate medical educa- tion. This includes technology and faculty development. Similarly, it would be useful for appropriate national (e.g., LCME in the US) and supranational (e.g., WFME) organiza- tions to establish some common rules, standards, and guide- lines for medical education at a distance so that high-quality remote instruction is assured and maintained. During the Zoom meetings, as each student took their turn to discuss a case, they were first asked to share with the group the diagnoses they had generated during their preparatory time. This was followed by a group discussion and a final consensus diagnosis under faculty guidance. Students remotely controlled the virtual im- ages during the discussions, selecting and showing to others via Zoom, their fields of interest on the digital slides and important tumor features to support their di- agnoses. Compliance with Ethical Standards Conflict of Interest None. The authors have no relevant financial or non- financial interests to disclose. Funding Open access funding provided by the Qatar National Library. Funding Open access funding provided by the Qatar National Library. Data Availability all data and materials support the published claims and comply with field standards. All data generated or analyzed during this study are included in this article. Discussion This learning strategy can strengthen student autonomy and confidence, and eventually, self-efficacy and motivation for learning. Faculty also offered forma- tive feedback and reinforcements. In addition, sharing images, ideas, and questions and answers live through Zoom enhanced collaborative work and learning. In fact, sometimes students offered different perspectives on a difficult case, encouraging rich discussions during our meetings. Lastly, our work should be replicated by others and evalu- ated accordingly. Future research should look at hard evidence of learning outcomes associated with remote online instruction. Funding Open access funding provided by the Qatar National Library. References 1. Whelan A, Prescott J, Young G, Catanese VM. Guidance on Medical Students’ Clinical Participation: Effective Immediately. March 17, 2020. https://lcme.org/wp-content/uploads/filebase/ March-17-2020-Guidance-on-Mediical-Students-Clinical- Participation.pdf. 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https://openalex.org/W1997972137	https://pure.uva.nl/ws/files/15153899/Monte_Carlo_modeling_of_the_central_carbon_metabolism.pdf	English	null	Monte-Carlo Modeling of the Central Carbon Metabolism of Lactococcus lactis: Insights into Metabolic Regulation	PloS one	2,014	cc-by	14,641	Citation for published version (APA): Murabito, E., Verma, M., Bekker, M., Bellomo, D., Westerhoff, H. V., Teusink, B., & Steuer, R. (2014). Monte-Carlo modeling of the central carbon metabolism of Lactococcus lactis: insights into metabolic regulation. PLoS ONE, 9(9), Article e106453. https://doi.org/10.1371/journal.pone.0106453 UvA-DARE (Digital Academic Repository) Monte-Carlo modeling of the central carbon metabolism of Lactococcus lactis: insights into metabolic regulation Murabito, E.; Verma, M.; Bekker, M.; Bellomo, D.; Westerhoff, H.V.; Teusink, B.; Steuer, R. DOI 10.1371/journal.pone.0106453 Publication date 2014 Document Version Final published version Published in PLoS ONE License CC BY Link to publication Citation for published version (APA): Murabito, E., Verma, M., Bekker, M., Bellomo, D., Westerhoff, H. V., Teusink, B., & Steuer, R. (2014). Monte-Carlo modeling of the central carbon metabolism of Lactococcus lactis: insights into metabolic regulation. PLoS ONE, 9(9), Article e106453. https://doi.org/10.1371/journal.pone.0106453 Monte-Carlo modeling of the central carbon metabolism of Lactococcus lactis: insights into metabolic regulation Murabito, E.; Verma, M.; Bekker, M DOI 10.1371/journal.pone.0106453 Publication date 2014 Document Version Final published version Published in PLoS ONE License CC BY Link to publication Citation for published version (APA): Murabito, E., Verma, M., Bekker, M., Bellomo, D., Westerhoff, H. V., Teusink, B., & Steuer, R. (2014). Monte-Carlo modeling of the central carbon metabolism of Lactococcus lactis: insights into metabolic regulation. PLoS ONE, 9(9), Article e106453. https://doi.org/10.1371/journal.pone.0106453 Abstract Metabolic pathways are complex dynamic systems whose response to perturbations and environmental challenges are governed by multiple interdependencies between enzyme properties, reactions rates, and substrate levels. Understanding the dynamics arising from such a network can be greatly enhanced by the construction of a computational model that embodies the properties of the respective system. Such models aim to incorporate mechanistic details of cellular interactions to mimic the temporal behavior of the biochemical reaction system and usually require substantial knowledge of kinetic parameters to allow meaningful conclusions. Several approaches have been suggested to overcome the severe data requirements of kinetic modeling, including the use of approximative kinetics and Monte-Carlo sampling of reaction parameters. In this work, we employ a probabilistic approach to study the response of a complex metabolic system, the central metabolism of the lactic acid bacterium Lactococcus lactis, subject to perturbations and brief periods of starvation. Supplementing existing methodologies, we show that it is possible to acquire a detailed understanding of the control properties of a corresponding metabolic pathway model that is directly based on experimental observations. In particular, we delineate the role of enzymatic regulation to maintain metabolic stability and metabolic recovery after periods of starvation. It is shown that the feedforward activation of the pyruvate kinase by fructose-1,6-bisphosphate qualitatively alters the bifurcation structure of the corresponding pathway model, indicating a crucial role of enzymatic regulation to prevent metabolic collapse for low external concentrations of glucose. We argue that similar probabilistic methodologies will help our understanding of dynamic properties of small-, medium- and large-scale metabolic networks models. Citation: Murabito E, Verma M, Bekker M, Bellomo D, Westerhoff HV, et al. (2014) Monte-Carlo Modeling of the Central Carbon Metabolism of Lactococcus lactis : Insights into Metabolic Regulation. PLoS ONE 9(9): e106453. doi:10.1371/journal.pone.0106453 Editor: Ying Xu, University of Georgia, United States of America Received February 10, 2014; Accepted August 7, 2014; Published September 30, 2014 Received February 10, 2014; Accepted August 7, 2014; Published September 30, 2014 Copyright: 2014 Murabito 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. Ettore Murabito1, Malkhey Verma1, Martijn Bekker2, Domenico Bellomo3, Hans V. Westerhoff1,4,5, Bas Teusink3, Ralf Steuer6,7 1 Manchester Institute of Biotechnology, School of Chemical Engineering and Analytical Sciences (CEAS), Manchester Centre for Integrative Systems Biology (MCISB), The University of Manchester, Manchester, United Kingdom, 2 Molecular Microbial Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands, 3 Systems Bioinformatics IBIVU and Netherlands Institute for Systems Biology (NISB), VU University Amsterdam, Amsterdam, The Netherlands, 4 Synthetic Systems Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands, 5 Molecular Cell Physiology, FALW, VU University Amsterdam, Amsterdam, The Netherlands, 6 CzechGlobe - Global Change Research Center, Academy of Sciences of the Czech Republic, Brno, Czech Republic, 7 Humboldt-University Berlin, Institute for Theoretical Biology, Berlin, Germany Abstract Funding: HVW acknowledges support by the transnational program Systems Biology of MicroOrganisms (SysMO) and ERASysBio program and its funders EPSRC/ BBSRC for the following projects: R111828, MOSES project, ERASysBio, BB/F003528/1, BB/C008219/1, BB/F003544/1, BB/I004696/1, BB/I00470X/1, BB/I017186/1, EP/D508053/1, BB/I017186/1. RS acknowledges funding by the OPVK project "Local Team and International Consortium for Computational Modelling of a Cyanobacterial Cell, Reg. No. CZ.1.07/2.3.00/20.0256". As of 1.1.2013, RS is supported by the framework e:Bio of the BMBF (Fo¨rderkennzeichen 0316192). 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: ettore.murabito@manchester.ac.uk (EM); ralf.steuer@hu-berlin.de (RS) General rights I i i General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). 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Disclaimer/Complaints regulations UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:24 Oct 2024 September 2014 \| Volume 9 \| Issue 9 \| e106453 The Central Metabolism of Lactococcus lactis The starting point of our analysis is a stoichiometric represen- tation of the central metabolism of Lactococcus lactis, defined here as the carbon and energy metabolism of this organism that generates most of its free-energy and C3 carbon precursors during fermentative growth. Drawing upon earlier kinetic models [3,4,7,9,15] and several available genome-scale reconstructions [5,31], a set of enzymes involved in fermentative metabolism of L. lactis was selected. A graphical overview is shown in Figure 1. The metabolic network was chosen so as to describe the main glycolytic intermediates, the ATP regeneration cycle, and the dynamics of inorganic phosphate (Pi) and redox carriers (NAD/NADH). We neglect flux through the pentose phosphate pathway, since it accounts for less than 2% of glycolytic flux [32]. Main fermentation products are lactate (LAC), ethanol (EtOH), acetate and butanediol. Stoichiometric analysis reveals that the systems has three conserved moieties, ATP/ADP, NAD/NADH, as well as conservation of a phosphate group involving 11 metabolites. The concentrations of formate (FMT) and coenzyme A (CoA) are considered constant. The stoichiometry of the network allows for either LAC as the only fermentation product, or for equimolar amounts of butanediol and EtOH or acetate and EtOH as end products. The latter branch, fermentation to acetate and EtOH, results in the highest yield of ATP per glucose consumed. Building upon these methods, we aim to elucidate and understand the regulatory properties of the central metabolism of the lactic acid bacterium Lactococcus lactis. In particular, we seek to understand how allosteric regulation, together with its associated feedback and feedforward loops, influence the control exerted by the various enzymatic steps. Making only minimal assumptions about rate equations and kinetic parameters, and data from direct experimentation, we show that it is possible to study and elucidate the control properties of a metabolic pathway. In a second step, we investigate the dynamics of a corresponding kinetic pathway model in periods of starvation and show that allosteric control and regulatory interactions are crucial to maintaining metabolic viability in times of nutrient scarcity. Our probabilistic approach directly builds upon measured properties, such as the concentrations of metabolic intermediates and flux distributions, rather than enzyme-kinetic parameters, to constrain the possible dynamics of a metabolic pathway. Introduction but also via redox (NADH) and energy carriers (ATP). Under- standing the dynamic response arising from such a complex network of interactions is often not possible through heuristic reasoning alone, but requires the construction of detailed computational models. Such kinetic models of biochemical pathways are typically built in a bottom-up approach. First, the detailed enzyme-kinetic properties of each individual step are collected, having been sourced from either dedicated experiments or from literature research and databases. Subsequently, the model is constructed and revised until a faithful in-silico representation of the pathway is obtained. Despite considerable efforts, however, the construction of such models is still hampered by the extensive data requirements necessary to parametrize each enzymatic step. Several approaches have been suggested to alleviate this problem, including the use of approximative kinetics [18,19] and Monte-Carlo sampling of reaction parameters Lactic acid bacteria (LAB) are industrially important microor- ganisms used in the fermentation of milk, meat, and certain vegetables [1,2]. LAB produce lactic acid as their main catabolic byproduct and are frequently used as starter cultures in various food-fermentation processes. LAB have a long tradition of safe use and, because the central metabolism of LAB has high activity, is an interesting target for metabolic engineering. A variety of metabolic models, detailed kinetic models as well as large-scale stoichiometric reconstructions, have been proposed, in particular for the best- studied LAB Lactococcus lactis [3–10]. Despite a relative wealth of data, however, many aspects of the fermentative metabolism of LAB are still insufficiently understood [2,11–17]. Even compar- atively small metabolic pathways, such as the glycolytic pathway of LAB, are highly interconnected not only via carbon metabolites, September 2014 \| Volume 9 \| Issue 9 \| e106453 1 PLOS ONE \| www.plosone.org Monte-Carlo Modeling of L. lactis Central Metabolism Figure 1. A pathway map of L. lactis central metabolism. The pathway involves 21 metabolic interconversions between 24 metabolites and includes three conserved moieties and two internal metabolites whose concentrations are assumed to be constant (FMT, CoA). External metabolites are indicated by light blue boxes. The inset provides an overview of the regulatory interactions. Abbreviations are defined in Text S2. doi:10.1371/journal.pone.0106453.g001 Figure 1. A pathway map of L. lactis central metabolism. The pathway involves 21 metabolic interconversions between 24 metabolites and includes three conserved moieties and two internal metabolites whose concentrations are assumed to be constant (FMT, CoA). External metabolites are indicated by light blue boxes. Introduction The inset provides an overview of the regulatory interactions. Abbreviations are defined in Text S2. doi:10.1371/journal.pone.0106453.g001 regulatory properties that enable large-scale metabolic networks to function reliably in uncertain environments. [20,21]. As one of the first applications of a Monte-Carlo sampling procedure accounting for the uncertainty in kinetic parameters, Wang et al. [22] employed statistical tools for the identification of the rate-controlling steps in a model of yeast glycolysis. Shortly afterwards, the formalism of generalized modeling [23] was extended to study the dynamic properties of models of metabolic pathways by using a general parametric representation of their Jacobian matrix [24,25]. Later, these methods were adopted and modified by several other groups [26–30]. September 2014 \| Volume 9 \| Issue 9 \| e106453 Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism The resulting distribution of flux control coefficients allows us to assess, qualitative and fairly independent of the precise values of the enzyme kinetic parameters, the potential effects of perturba- tions in enzyme levels on the system flux at the metabolic state. Most importantly, the distributions of the control coefficients were not arbitrarily. Rather, the results shown in Figure 3 exhibit strong patterns and, in accordance with earlier studies [37], indicate that qualitative knowledge of model properties can already result in well-constrained predictions, even when individual parameters are only poorly constrained. With respect to the interpretation of the distributions of scaled flux control coefficients, three properties are of particular interest: (i) The width of the distribution. Narrow distributions indicate control coefficients that do not change appreciably due to parameter sampling, whereas broad distribu- tions indicate that the precise value of the coefficient is highly dependent on parameter values. (ii) The median of the distribu- tion. For several reactions, the respective distributions are appreciably shifted away from zero towards either negative or positive values. (iii) The dominant sign of the control coefficients. Often, it is only of interest whether an increase in enzyme amount will result in an increase or decrease of a specific flux in the network, irrespective of the exact magnitude. To this end, Figure 4 provides the probabilistic sign distribution of flux control coefficients, grey-scale coded to indicate the percentage of sampled control coefficients that lie on the positive semi-axis. therefore the concentration of free inorganic phosphate (Pi) is an (inverse) measure of the amount of the glycolytic intermediates plus ATP. Further reactions that are under regulatory control include the pyruvate dehydrogenase (PDH), as well as the alcohol dehydrogenases (ADH). A complete list of all regulatory interac- tions is summarized in Figure 1 and given in Text S1. Following the workflow outlined above, a kinetic rate equation was assigned to each reaction step. In good agreement with earlier models [3], reaction rates were assumed to follow conventional Michaelis- Menten kinetics. Regulatory interactions were implemented as multiplicative factors to the rate equations, using Hill-type kinetics [33]. Detailed reaction equations are provided in Text S2. Subsequently, equilibrium constants were assigned to all steps, using values obtained by the group contribution method [34]. Probabilistic Analysis of Control Properties To quantify the extent to which a system property, such as a specific flux or metabolite concentration, is influenced by a specific enzymatic step, we make use of the concepts of Metabolic Control Analysis (MCA). The control coefficient CP i is defined as the relative change of a steady-state property P (the response of the system) divided by the small relative modulation of the activity of an enzyme i that caused that change [35,36]. In the following, we mostly focus on the flux control coefficients CJ, where the system properties P represent the fluxes within central metabolism. These control coefficients can only be evaluated for a fully characterized system, that is, their evaluation requires knowledge of the stoichiometry, as well as of all kinetic parameters or elasticity coefficients. Because neither all kinetic parameters nor all elasticity coefficients were known, we employed a Monte-Carlo scheme to iteratively sample enzyme-kinetic parameters, in particular Michaelis-Menten constants, to obtain insight into what we shall call the probabilistic distribution of control coefficients. To determine the respective intervals for the Monte-Carlo sampling of parameters, we make use of the fact that the control coefficients are essentially determined by the scaled elasticities, the logarithmic partial derivatives of the rate equations with respect to metabolic compounds. In a first approximation, these values are determined by the ratio of the Michaelis-Menten constants to their respective concentration [24,28]. Motivated by the saturation properties of enzymatic rate equations, and making use of knowledge of the metabolic phenotype, we assume each Michaelis-Menten constant to be distributed around the concentration value of its associated metabolic compound. The width of the sampling interval is varied to test for robustness of the results; in the following all results are reported for a sampling interval spanning both, one order of magnitude above and below the respective concentration. After choosing all Michaelis constants, the forward velocity Vmax was adjusted so as to ensure that the reaction rate was not affected by the chosen constant. Applying this Monte-Carlo procedure, we obtain a distribution, rather than a single value, for each flux control coefficient. We emphasize that our approach ensures that each set of parameters is sampled such that it is consistent with the experimentally observed state. That is, each sampled set of paremeters indeed gives rise to the experimentally observed state. All subsequent evaluations are specific to this state. Probabilistic Analysis of Control Properties Prior to the calculation of control coefficients, the stability of the model is tested for each sampled set of parameters and only sets corresponding to a stable phenotype are retained (<92% of all sampled instances). Our workflow is outlined in Figure 2. Monte-Carlo Modeling of L. lactis Central Metabolism The metabolic state was based on values for extracellular fluxes and metabolites specifically measured for this study, supplemented with values from the literature. We consider a state with high external glucose (20 mM) and high-flux activity, see also Text S1. The Central Metabolism of Lactococcus lactis We demonstrate that (i) the control coefficients of biochemical network models show intelligible patterns and trends that are accessible without detailed knowledge of enzyme-kinetic parameters; (ii) the regula- tory structure of a biochemical network models has profound effects on the possible dynamics that are largely independent of specific kinetic parameters; and (iii) more specifically, that the topology of the regulation network is instrumental to ensure the stability of an observed state and to enable the patwhay to survive periods of starvation. We expect that our methodology will be of high utility to elucidate and understand the dynamic and Beyond the reaction stoichiometries, our model incorporates the currently known regulatory features found in L. lactis central metabolism. Fructose 1,6-bisphosphate (FBP) activates the forma- tion of pyruvate (PYR) by the pyruvate kinase (PYK), activates the conversion from PYR to LAC by the lactate dehydrogenase (LDH), and inhibits the phosphotransferase system (PTS). A potentially important regulator is the concentration of free phosphate. In our model, the total pool of phosphate is constant, September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 2 September 2014 \| Volume 9 \| Issue 9 \| e106453 The Control of Flux Figures 3 and Figure 4 show the resultant distributions of control coefficient based on the sampling procedure described above. All sampled sets of parameters share the common property that they are consistent with the experimentally observed metabolic state of the pathway. However, other than that, we have no knowledge as to whether an actual control coefficient is close to the median value of the respective distribution, or whether evolutionary pressure has selected for a value at the extreme fringe of this distribution. Clearly, for each control coefficient, both scenarios are possible. To provide a meaningful interpretion of these results, we therefore proceed in two steps. First, we asses to what extend the observed distributions correspond to our intuitive assumptions about control within the pathway. Overall, the pattern of flux control coefficients shown in Figures 3 and 4 are indeed in good agreement with conventional assumptions about the distribution of control within the pathway. For example, the glycolytic enzymes (PGI to ENO) predominantly show positive control over the glycolytic flux, with more than 80% of the sampled control coefficients having positive values. Reactions close to equilibrium, such as PGM or ENO, typically also have narrow distributions around zero, indicating that they typically exert little control on the fluxes through the system, irrespective of precise parameter values. Indeed, the average standard deviation of the scaled control coefficients correlates with the distance from equilibrium of the respective reaction, as shown in Figure 5, and in accordance with theoretical considerations [38]. Other straightforward results include the predominantly negative control the LDH exerts upon the competing branches involved in the production of ethanol, acetate and butanediol. However, other properties of the distributions shown in Figure 3 and Figure 4 are less straightforward to explain. For example, the predominantely negative control that the enzymes involved in acetate production (PTA and ACK) exert upon the glycolytic flux. Although increasing activity of PTA and ACK might be expected to pull more flux through the upper part of glycolysis, the flux control coefficients indicate that this is, for the vast majority of parameters, not the case. Rather, increasing the activity of the acetate branch September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 3 Monte-Carlo Modeling of L. lactis Central Metabolism Figure 2. Probabilistic Modeling of L. lactis. The topological properties of the pathway, including the stoichiometry and all known regulatory interactions, are assembled. The Control of Flux The analysis is then based on knowledge of a metabolic state of the system, as defined by a steady-state flux distribution and a set of associated thermodynamically consistent metabolite concentrations. Based on this information, the state-specific dynamic properties of the corresponding pathway model are evaluated. Of particular interest are control coefficients, the role of regulatory interactions, as well as the dynamic response to periods of starvation. doi:10.1371/journal.pone.0106453.g002 Figure 2. Probabilistic Modeling of L. lactis. The topological properties of the pathway, including the stoichiometry and all known regulatory interactions, are assembled. The analysis is then based on knowledge of a metabolic state of the system, as defined by a steady-state flux distribution and a set of associated thermodynamically consistent metabolite concentrations. Based on this information, the state-specific dynamic properties of the corresponding pathway model are evaluated. Of particular interest are control coefficients, the role of regulatory interactions, as well as the dynamic response to periods of starvation. doi:10.1371/journal.pone.0106453.g002 Figure 3. Probabilistic distribution of flux control coefficients. Shown is the distribution of the scaled flux control coefficients corresponding to the pathway model of L. lactis central metabolism given in Figure 1. Each plot corresponds to the interval [21,1] on the abscissa. The diagram in the i th column and on the j th row gives the distribution of the control coefficient quantifying the extent to which enzyme i controls the flux through the reaction j. Each distribution provides information about the magnitude and uncertainty of one control coefficient. Narrow distributions indicate control coefficients that do not change appreciably due to parameter sampling, whereas broad distributions indicate that the precise value of the coefficient is more strongly dependent on parameter values. The corresponding sign distribution is shown in Figure 4. doi:10.1371/journal.pone.0106453.g003 September 2014 \| Volume 9 \| Issue 9 \| e106453 September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 4 Monte-Carlo Modeling of L. lactis Central Metabolism Figure 4. Probabilistic sign distribution of flux control coefficients. Grey-scale representation of the sign distribution of the flux control coefficient shown in Figure 3. The shade of the entry represents the percentage of the calculated control coefficients that are positive. Dark colors correspond to a distribution of flux control coefficients that lies predominantly on the negative semiaxis, whereas light colors indicate that the sampled control coefficients are predominantely positive. For example, for this metabolic phenotype, an increase of the enzyme PYK will for almost all sampled parameter values result in decreased flux through the LDH reaction as indicated by the dark circle in the row for LDH and the column for PFK. doi:10 1371/journal pone 0106453 g004 Figure 4. Probabilistic sign distribution of flux control coefficients. Grey-scale representation of the sign distribution of the flux control coefficient shown in Figure 3. The shade of the entry represents the percentage of the calculated control coefficients that are positive. Dark colors correspond to a distribution of flux control coefficients that lies predominantly on the negative semiaxis, whereas light colors indicate that the sampled control coefficients are predominantely positive. For example, for this metabolic phenotype, an increase of the enzyme PYK will for almost all sampled parameter values result in decreased flux through the LDH reaction as indicated by the dark circle in the row for LDH and the column for PFK. doi:10.1371/journal.pone.0106453.g004 Figure 5. The width of the distribution of control coefficients correlates with distance from equilibrium. Shown is the average standard deviation of the sampled flux control distribution as a function of displacement C from equilibrium of the respective enzyme. Reactions close to equilibrium (C close to unity) typically have narrow distributions of flux control coefficients, centered at zero, indicating they can only exert little control over the flux through the system. Contrary, reactions far from equilibrium (C%1) exhibit broad distribu- tions, indicating a potential, but no necessity, for high control coefficients. For definitions see Materials and Methods. doi:10.1371/journal.pone.0106453.g005 prevents the regeneration of NAD required by the glyceraldehyde- 3-phosphate-dehydrogenase (GAPDH), consequently slowing down the glycolytic pathway. We emphasize that the probabilistic distributions shown in Figures 3 and 4 depict control properties of the pathway model for values of kinetic parameters that are consistent with the experimentally observed state, but are otherwise random. Clearly, the peaks of these distributions do not necessarily correspond to the actual control profile of the organism. Selection pressure during evolution might have easily led to parameters on the fringes or even outside of the range in which we sampled, precisely because the organism’s requirements were different from what arises as ‘typical’ from random sampling. We therefore must compare the distributions shown in Figures 3 and Figure 4 with known and empirically obtained control coefficients in L. lactis [39–42]. In particular, control coefficients that take values far from the median value of the respective distribution might point to additional evolutionary pressure, or errors in the topology of the pathway, and therefore provide valuable information for further analysis. Figure 5. The width of the distribution of control coefficients correlates with distance from equilibrium. Shown is the average standard deviation of the sampled flux control distribution as a function of displacement C from equilibrium of the respective enzyme. Reactions close to equilibrium (C close to unity) typically have narrow distributions of flux control coefficients, centered at zero, indicating they can only exert little control over the flux through the system. Contrary, reactions far from equilibrium (C%1) exhibit broad distribu- tions, indicating a potential, but no necessity, for high control coefficients. For definitions see Materials and Methods. doi:10.1371/journal.pone.0106453.g005 Using a series of mutants with altered GAPDH activity, Solem et al. (2003) [40] determined that changes in GAPDH activity had virtually no effect on glycolytic flux in growing as well as nongrowing cells. Control of Glycolytic Flux by PTS As a rather counter-intuitive finding, the distribution of control coefficients indicates that for most sets of parameters the PTS would exert a negative control on flux through the pathway. This finding is in contrast to our expectation that an increase in PTS activity results in higher glucose uptake, hence an increased flux through glycolysis. These results emphasize that the control coefficients are indeed systemic properties of the pathway and may exhibit non-intuitive patterns, depending on the specific metabolic phenotype and details of the pathway stoichiometry and regula- tion. To verify that the non-intuitive distribution of the influence of the glucose transport upon the glycolytic flux is indeed a true feature of the pathway model, we varied the maximal velocity (Vmax) of the PTS using an explicit kinetic model. The resulting flux is shown in Figure 6 for a specific set of reference parameters. Repeating the analysis for a large number of sampled parameters yielded similar results. At the specified metabolic state, the slope of the curve representing glucose uptake as a function of Vmax is almost always negative. The universality of the negative slope indicates that the control profile of the network model is indeed strongly dependent on the specific metabolic phenotype at which the probabilistic control profile is evaluated. That is, the experimentally observed metabolic state itself shapes the resultant distribution of control coefficients. To verify this assertion, we evaluated the control profile of a second metabolic state, corresponding to a situation with low external glucose. The results are provided and discussed in Text S2. In this case, the control of the PTS system on glycolytic flux is predominantly positive. Repeating the Monte-Carlo sampling of kinetic parameters in the absence of regulatory interactions, we first note that the percentage of stable models, as evaluated by an analysis of the largest real part of the eigenvalues of the Jacobian, drops to about 83% of sampled instances, as compared to 92% for the regulated system considered above. The analysis was repeated for the second metabolic state, characterised by low external glucose, in the Text S2, showing that a similar drop in average stability is again associated with the absence of regulatory interactions. Figure 7 shows the probabilistic distribution of the scaled flux control coefficients in the absence of metabolic regulation, the corre- sponding sign distribution is shown in Figure 8. Likewise, in a series of studies, the enzymes PFK, PYK, and LDH, encoded together on the las operon, were shown to have no significant control on the glycolytic flux in PLOS ONE \| www.plosone.org September 2014 \| Volume 9 \| Issue 9 \| e106453 5 Monte-Carlo Modeling of L. lactis Central Metabolism Figure 6. Dependency of the glycolytic flux on the maximal activity of the glucose transporter (PTS). Shown is the glucose uptake as a function of Vmax, evaluated using a kinetic model with a representative set of the sampled parameters. The black dot indicates the reference state. doi:10.1371/journal.pone.0106453.g006 exponentially growing cells [39,41]. These findings are in agreement with the rather narrow distributions, centered at zero, of the respective probabilistic control coefficients. Furthermore, Koebmann et al. (2005) [41] showed that the enzyme LDH has a strong negative control over the flux to mixed acids and formate formation, whereas PYK has a strong negative control over these fluxes – again in agreement with the respective distributions. PFK was found to have no control on either acetate or lactate flux, again corresponding to the respective distribution. An interesting case is the control of ATP consuming-processes outside of the pathway (ATPase) on the glycolytic flux. The respective probabi- listic distribution indicates a large potential for control, as manifested by the very broad distribution of the corresponding flux control coefficients. Such a high level of control by ATP consuming reactions outside of the pathway was indeed reported recently [17], and a similar high control of demand for ATP on glycolytic flux has been observed for E. coli [43]. From the introduction of an uncoupled ATPase activity, however, Koeb- mann et al. (2002) [39] concluded that the flux control by ATP demanding processes was close to zero over a range of ATP/ADP ratios. Both scenarios are consistent with the broad distribution shown in Figure 3. We emphasize that in the following, despite the good agreement with experimental data, we do not interpret the probabilistic distributions as a likelihood for the actual control coefficients. Rather the distributions indicate ranges of control coefficients that are typical for the metabolic state, given our sampling procedure, whereas deviations provide a highly valuable starting point for further analysis. We note, however, that as yet most empirically determined control coefficients available for L. lactis do seem to be consistent with the bulk of the respective distribution. Figure 6. Control of Glycolytic Flux by PTS The absence of regulation affects the control profile in terms of the dominant sign of the control coefficients, as well as the amplitude and width of their distribution. A comparison between Figures 3 and 7 shows, for example, that the control of PGI and PFK over the glycolytic flux is predominantly positive in the regulated system, but almost always negative in the unregulated systems. Differences are also Dependency of the glycolytic flux on the maximal activity of the glucose transporter (PTS). Shown is the glucose uptake as a function of Vmax, evaluated using a kinetic model with a representative set of the sampled parameters. The black dot indicates the reference state. doi:10.1371/journal.pone.0106453.g006 Each instance of sampled parameters included randomly selected values for all regulatory interactions. To delineate the impact of metabolic regulation, we subsequently removed all regulatory interactions and repeated the analysis to pinpoint specific differences between the regulated and unregulated network model. In particular, our re-scaling and sampling procedure allows us to analyze both systems at an identical steady state and therefore enables a direct comparison between both scenarios. We note that this approach is different to a simple change of parameters within a kinetic model, for example by decreasing the value of a certain feedback parameter. While such a change would indeed modify the feedback properties, it would also alter the metabolic state of the pathway and thereby complicate a direct comparison of systemic properties between both scenarios. In contrast, here, we assume that the metabolic phenotype, the set of concentrations and fluxes, has evolved to satisfy the particular functional requirements of the cell. By removing regulatory interactions while keeping the metabolic state unchanged, we ask the question how this evolved state would typically behave in the absence of metabolic regulation, whilst fulfilling the same metabolic function. As is demonstrated below, this approach allows us to perform a detailed assessment of the role of individual regulatory interac- tions. September 2014 \| Volume 9 \| Issue 9 \| e106453 Monte-Carlo Modeling of L. lactis Central Metabolism The probability of the system to have recovered from starvation also depends on the duration of the Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism recorded for the regulation of the glycolytic flux by AS, ACLACD and BDH, which is negative in the regulated case and evenly distributed between negative and positive values in the absence of regulation. A strong difference between the probabilistic control profiles is also observed for the ATPase, representing general ATP demand outside of the pathway. In the presence of regulation, the distribution of the control coefficients of the ATPase on the glycolytic flux is rather broad, indicating a high potential for control, and symmetrically distributed around zero. In contrast, in the absence of regulation, the distribution is shifted to higher values, with a median value close to unity. Consequently, in the absence of metabolic regulation, the pathway model exhibits a strong sensitivity with respect to ATP demand for almost all possible sets of parameters. Similar differences are observed for the possible control of PYK on the glycolytic flux. The respective distribution is significantly narrower in the presence of metabolic regulation, indicating less potential for control but an increased robustness of the flux with respect to perturbations in enzyme levels. Similar differences in widths and signs were observed for the second metabolic state as discussed in Text S2. numeric simulations for 103 instances of sampled parameters in the presence and absence of regulatory interactions. After a period of t~1 min, the external glucose was lowered from its original value of [GLCx] = 20 mM to a value of [GLCx] = 0:1 mM. The pathway model then adapted to the new conditions and converged to a new metabolic state. At a simulation time of t~10 min, the external glucose was restored to its original value [GLCx] = 20 mM. As expected, and shown in Figure 9, withdrawal of external glucose resulted in a quick drop of intracellular metabolites. Typical time-courses of selected model instances are provided in the Text S2. Due to the autocatalytic nature of the glycolytic pathway, the concentration of ATP typically first exhibits a slight increase, corresponding to the cessation of ATP utilization in the upper part of glycolysis. Subsequently, the concentration of ATP, and likewise of other metabolites, drops to a new steady-state, characterized by a significantly decreased ATP concentration. The average value of ATP concentration during starvation is 0:5+0:5 mM for regu- lated systems versus 0:11+0:07 mM in the absence of regulation. Monte-Carlo Modeling of L. lactis Central Metabolism Both scenarios exhibit drastically different behavior after external glucose is restored to its original value. For the regulated system, the distribution of ATP concentration after a suitable recovery time is clearly bimodal (Figure 9A). Approximately 54% of all models recover to the original metabolic state (542 of 1000 instances tested) and the ATP level reaches its original value (&4 mM). In the absence of regulation, the probability of the system to recover drops significantly. More than 95% of the simulations correspond to a failure in restoring the original concentration of ATP. The probability of the system to have recovered from starvation also depends on the duration of the numeric simulations for 103 instances of sampled parameters in the presence and absence of regulatory interactions. After a period of t~1 min, the external glucose was lowered from its original value of [GLCx] = 20 mM to a value of [GLCx] = 0:1 mM. The pathway model then adapted to the new conditions and converged to a new metabolic state. At a simulation time of t~10 min, the external glucose was restored to its original value [GLCx] = 20 mM. As expected, and shown in Figure 9, withdrawal of external glucose resulted in a quick drop of intracellular metabolites. Typical time-courses of selected model instances are provided in the Text S2. Due to the autocatalytic nature of the glycolytic pathway, the concentration of ATP typically first exhibits a slight increase, corresponding to the cessation of ATP utilization in the upper part of glycolysis. Subsequently, the concentration of ATP, and likewise of other metabolites, drops to a new steady-state, characterized by a significantly decreased ATP concentration. The average value of ATP concentration during starvation is 0:5+0:5 mM for regu- lated systems versus 0:11+0:07 mM in the absence of regulation. Both scenarios exhibit drastically different behavior after external glucose is restored to its original value. For the regulated system, the distribution of ATP concentration after a suitable recovery time is clearly bimodal (Figure 9A). Approximately 54% of all models recover to the original metabolic state (542 of 1000 instances tested) and the ATP level reaches its original value (&4 mM). In the absence of regulation, the probability of the system to recover drops significantly. More than 95% of the simulations correspond to a failure in restoring the original concentration of ATP. The Impact of Metabolic Regulation Up to this point, the system had been evaluated in the presence of metabolic regulation additional to substrate/product effects. September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 6 Starvation and Dynamic Recovery To investigate the impact of metabolic regulation on the dynamic properties of the system, we have to go beyond the steady-state control coefficients and consider explicit time-courses of the system. In particular, we are interested in the dynamic response to periods of starvation and in the subsequent recovery when external nutrients are replenished. To this end, we set up an ensemble of models, each at the steady-state defined by the metabolic state considered above. Subsequently, we ran explicit Figure 7. Probabilistic distribution of flux control coefficients in the absence of metabolic regulation. Same as Figure 3 except for the absence of metabolic regulation. Any diagram refers to the control of one flux (i.e. through the step indicated to the left of the row) by one enzyme (i.e. the enzyme indicated above each diagram corresponds to the interval [-1,1] on the abscissa. doi:10.1371/journal.pone.0106453.g007 Figure 7. Probabilistic distribution of flux control coefficients in the absence of metabolic regulation. Same as Figure 3 except for the absence of metabolic regulation. Any diagram refers to the control of one flux (i.e. through the step indicated to the left of the row) by one enzyme (i.e. the enzyme indicated above each diagram corresponds to the interval [-1,1] on the abscissa. doi:10.1371/journal.pone.0106453.g007 September 2014 \| Volume 9 \| Issue 9 \| e106453 September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 7 Monte-Carlo Modeling of L. lactis Central Metabolism Figure 8. Probabilistic sign distribution of flux control coefficients in the absence of regulation. The shade of the entry represents the percentage of the calculated control coefficients that are positive. Dark colors correspond to a distribution of flux control coefficients that lies predominantly on the negative semiaxis, whereas light colors indicate that the sampled control coefficients are predominantly positive. doi:10.1371/journal.pone.0106453.g008 Figure 8. Probabilistic sign distribution of flux control coefficients in the absence of regulation. The shade of the entry represents the percentage of the calculated control coefficients that are positive. Dark colors correspond to a distribution of flux control coefficients that lies predominantly on the negative semiaxis, whereas light colors indicate that the sampled control coefficients are predominantly positive. doi:10.1371/journal.pone.0106453.g008 starvation (Ts) and recovery time (Tr). Figure 10 shows the percentage of recovered systems as a function of Ts and Tr for both scenarios. Starvation and Dynamic Recovery In the presence of regulation, the probability to recover does not seem to depend appreciably on the duration of the starvation and recovery time. In the absence of regulation, however, a longer starvation time strongly decreases the proba- bility of recovery. In addition, unregulated systems tend to recover more slowly, as also observed in the individual time-courses provided in the Text S2. These results are in agreement with the previous observation that regulatory interaction can accelerate response times of simple biological network motifs [44,45]. FBP as an activator of PYK, followed by free inorganic phosphate (Pi) as an inhibitor of the same reaction. A slight, but significant, increase is also observed for FBP as an inhibitor of PTS. The results are in good agreement with previous work on metabolic stability using minimal pathway models [2], such as the study of Voit et al. [15] who investigate the feedforward activation system in L. lactis using a six variable model with generalized mass action kinetics. Likewise, recent work on regulation of PEP utilization in E. coli also emphasized the importance of FBP as a regulator allowing rapid adaptation to changing environmental conditions [46]. September 2014 \| Volume 9 \| Issue 9 \| e106453 FBP as a regulator of PYK and PTS The importance of allosteric regulation of PYK by FBP for metabolic functioning, in particular for the levels of ATP and glycolytic intermediates, is widely recognized and the regulatory mechanisms is present in almost all glycolytic pathways [2,46]. Our results suggest that for L. lactis at the metabolic state studied here, a major role of the activation of PYK by FBP is to prevent an irreversible metabolic collapse during brief times of starvation. Indeed, as argued previously [2,15], the mechanism introduces a safety valve for the utilization of PEP. In the absence of regulation, and after the withdrawal of glucose, PEP is continually converted to pyruvate and short-lived ATP until all resources are depleted. However, in the presence of regulation, a decrease of FBP concomitantly decreases the utilization of PEP by PYK. Since no external glucose is present, utilization of PEP by PTS is likewise diminished. Hence a substantial amount of PEP is retained in the To investigate the role of regulation in the response to periods of starvation and the subsequent metabolic recovery in more detail, we tested the response of the system in the presence of individual regulation mechanisms. To do so, we performed the aforemen- tioned parameter sampling on models of the pathway endowed with only a single regulatory interaction. The percentage of models that recover after a period of starvation strongly depends on the specific regulatory interaction. Table 1 lists the percentage of recovering models for each individual regulation mechanism, along with the percentage of stable Jacobians for the respective system. Most of the regulatory mechanisms do not have any appreciable effect on the ability of the system to recover from starvation. Three of the ten tested regulation mechanisms, however, result in a significantly increased probability to recover after a period of starvation. The strongest increase is observed for September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 8 Monte-Carlo Modeling of L. lactis Central Metabolism Figure 9. Metabolic recovery after periods of starvation. Starting at the defined metabolic state, external glucose is lowered from 20 mM to 0.1 mM at time t = 1 min, mimicking a brief period of starvation. At time t = 10 min external glucose is restored to its orginal value. The upper panel shows a histogram of intracellular ATP after a recovery period at time t = 100 min. FBP as a regulator of PYK and PTS For the regulated system (A), approximately 54% of all models recover to the initial metabolic state (542 of 1000 instances tested), whereas in the absence of regulation the probability of recovery is below 5% (39 of 1000 instances tested). The lower panel shows the median of the time-course, with .25 and 0.75 quantiles included as dashed lines. For the regulated system, the time-course is split between recovering and non-recovering instances. doi:10.1371/journal.pone.0106453.g009 Figure 9. Metabolic recovery after periods of starvation. Starting at the defined metabolic state, external glucose is lowered from 20 mM to 0.1 mM at time t = 1 min, mimicking a brief period of starvation. At time t = 10 min external glucose is restored to its orginal value. The upper panel shows a histogram of intracellular ATP after a recovery period at time t = 100 min. For the regulated system (A), approximately 54% of all models recover to the initial metabolic state (542 of 1000 instances tested), whereas in the absence of regulation the probability of recovery is below 5% (39 of 1000 instances tested). The lower panel shows the median of the time-course, with .25 and 0.75 quantiles included as dashed lines. For the regulated system, the time-course is split between recovering and non-recovering instances. doi:10.1371/journal.pone.0106453.g009 system, which allows glucose uptake and regeneration of ATP as soon as external glucose is restored to its pre-starvation levels. Figure 11 shows the time-course of the median of the concentra- tions of FBP, ATP, and PEP following the withdrawal of external glucose at t~1 min for models that include the activation of PYK by FBP. As expected, after withdrawal of glucose, the concentra- tion of PEP quickly rises and attains a new steady state – system, which allows glucose uptake and regeneration of ATP as soon as external glucose is restored to its pre-starvation levels. Figure 11 shows the time-course of the median of the concentra- tions of FBP, ATP, and PEP following the withdrawal of external glucose at t~1 min for models that include the activation of PYK by FBP. As expected, after withdrawal of glucose, the concentra- tion of PEP quickly rises and attains a new steady state – interestingly with a median concentration similar to the value before starvation. With the restoration of external glucose at t~10 min, PEP undergoes a quick drop, fuelling the uptake of glucose, and the subsequent production of ATP. FBP as a regulator of PYK and PTS The percentage of the recovering systems is always relative to the instances of stable systems tested. doi:10 1371/journal pone 0106453 t001 The table gives the percentage of stable instances of the Jacobian in the presence of a single regulation mechanisms (83% in the absence of regulation) and the probability of metabolic recovery (4% in the absence of any regulation). The percentage of the recovering systems is always relative to the instances of stable systems tested. d i 10 1371/j l 0106453 t001 stable instances of the Jacobian in the presence of a single regulation mechanisms (83% in the absence of regulation) and the 4% in the absence of any regulation). The percentage of the recovering systems is always relative to the instances of stable systems systems that fail to recover after the external glucose has been restored to its original value must at least possess one additional stable state for the respective concentration of external glucose, that is, the systems must be at least bistable. To test for this hypothesis, we varied the concentration of external glucose and progressively decreased the concentration from the initial 20 mM to 0:1 mM. For each level of external glucose, the respective systems were allowed to relax to a new steady state. Subsequently, the external glucose was progressively increased again, back to the original value of 20 mM. Two typical examples are shown in Figure 12. Indeed, bistability and hysteresis was observed for all non-recovering systems. For recovering systems, in almost all cases (approximately 99%) no hysteresis was observed. A small subset of recovering systems (approximately 1%), however, also exhibited hysteresis. In this case the hysteresis loop is usually fully contained within the considered interval of external glucose and the system is monostable for an external glucose concentration of 20 mM. In corresponding plot and its discussion is provided the Text S2. We note that a similar reasoning might also hold in the absence of a PTS. In this case, the substrate ATP is required for the hexokinase and PFK in upper glycolysis, which is provided by the PYK reaction. The functional role of the FBP-mediated negative feedback on the PTS is more difficult to delineate and has received only little attention so far [2]. While the feedback also contributes to metabolic stability, its quantitative effect is rather small. Functionally, the regulation constitutes a traditional negative feedback loop, providing a mechanism for pathway homeostasis. FBP as a regulator of PYK and PTS The time-course shown in Figure 11 is in strong contrast to the situation in the absence of feedforward regulation, even when selecting for parameters that likewise allow for metabolic recovery. The Figure 10. Recovery as a function of starvation time. Shown is the percentage of model instances that recovered to the original metabolic state after a starvation time Ts and a recovery time Tr in the presence (left panel) and absence (right panel) of regulation. We emphasise the different scales on the z-axis on both panels. In the presence of regulation, the probability to recover does not appreciably depend on length of starvation and recovery time. In the absence of regulation a longer starvation time decreases the probability to recover. doi:10.1371/journal.pone.0106453.g010 Figure 10. Recovery as a function of starvation time. Shown is the percentage of model instances that recovered to the original metabolic state after a starvation time Ts and a recovery time Tr in the presence (left panel) and absence (right panel) of regulation. We emphasise the different scales on the z-axis on both panels. In the presence of regulation, the probability to recover does not appreciably depend on length of starvation and recovery time. In the absence of regulation a longer starvation time decreases the probability to recover. doi:10.1371/journal.pone.0106453.g010 September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 9 Monte-Carlo Modeling of L. lactis Central Metabolism Table 1. Percentage of recovering systems in the presence of individual regulation mechanisms. REACTION REGULATOR TYPE PERCENTAGE STABLE STATES PERCENTAGE RECOVERING SYSTEMS PTS FBP Inhibitor 87% 11% GAPDH NADH Inhibitor 81% 4% PYK Pi Inhibitor 97% 26% PYK FBP Activator 87% 51% PDH DHAP Inhibitor 83% 4% PDH GAP Inhibitor 83% 3% ADH ATP Inhibitor 82% 3% LDH Pi Inhibitor 84% 2% LDH FBP Activator 83% 2% LDH NADH/NAD Inhibitor 83% 3% The table gives the percentage of stable instances of the Jacobian in the presence of a single regulation mechanisms (83% in the absence of regulation) and the probability of metabolic recovery (4% in the absence of any regulation). The percentage of the recovering systems is always relative to the instances of stable systems tested. doi:10.1371/journal.pone.0106453.t001 The table gives the percentage of stable instances of the Jacobian in the presence of a single regulation mechanisms (83% in the absence of regulation) and the probability of metabolic recovery (4% in the absence of any regulation). Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism very rare cases, the system also returned to its original state, despite hysteresis and the existence of a second stable state with low metabolic activity. In these cases, the sudden withdrawal and reestablishment of external glucose may induce dynamic transients that allow the system to leave the lower state. Figure 12 shows a non-recovering system in the absence of regulation (panels A and B), as well as a recovering system in the presence of regulation (panels C and D). We note that hysteresis was tested numerically and therefore the existence of further metabolic states cannot be excluded. However, the numeric results obtained from the tested 103 instances clearly showed that recovery was predominantely due to the monostability of the steady state for the respective concentration of the external glucose. Furthermore, the absence of bistability was clearly linked to the regulatory structure of the system, almost irrespective of the precise paramater values. Our results therefore imply that the regulatory structure indeed has a structural influence on the possible bifurcations of the pathway independent of a specific fine-tuned set of parameters. dynamic properties of a corresponding pathway model. To this end, our first aim was to demonstrate that the distributions of control coefficients obtained from conditional Monte-Carlo sampling is highly structured. Based on a sampling scheme, such that all considered sets of parameters are in accordance with the experimentally observed state, the resulting patterns of control coefficients were consistent with many well-known control control principles of the pathway. Given these findings, two aspects of our study must be emphasized: First, we did not conduct a straightforward Monte- Carlo study such that the kinetic parameters were drawn from a random distribution. Such an approach would likely result in a plethora of different metabolic states – most of which are totally irrelevant for the functioning of the system under any condition. Rather, our approach is based on the assertion that evolution and selective pressure can easily restrict the metabolic state, and hence the parameters, to values that are beneficial for the organism’s survivial, even if these parameter sets represent only an exceedingly small portion within parameters. Therefore, we restrict the sampling to an evolved metabolic state, such that all sampled parameters are consistent with this experimentally observed state. Monte-Carlo Modeling of L. lactis Central Metabolism The underlying assumption is that the primary function of a metabolic pathway is embodied in its flux and, to a lesser extend, in the set of metabolite concentrations. It is these values that are the targets of selective pressure, not the kinetic parameters per se. We therefore seek to study how the dynamic properties of the pathway differ for different potential sets of kinetic parameters that are equally suited to support the observed steady-state flux. Such an approach allows us to straightforwardly Metabolic Collapse and Hysteresis As demonstrated, the regulatory structure of the glycolytic pathway is crucial to allow for metabolic recovery after periods of starvation. This raises the question about the detailed dynamic mechanisms through which recovery is achieved. By definition, Figure 11. Time-courses of intermediate metabolites. The median of the concentration of FBP, ATP, and PEP following a withdrawal of external glucose at t~1 min in models that include activation of PYK by FBP is shown. After withdrawal of glucose, the concentration of PEP quickly rises and attains a new steady state. With the restoration of external glucose at t~10 min, PEP undergoes a quick drop, fuelling glucose uptake and subsequent production of ATP. The corresponding figure for systems that lack regulatory interactions but are nonetheless able to recover from periods of starvation is discussed in the Text S2. doi:10.1371/journal.pone.0106453.g011 Figure 11. Time-courses of intermediate metabolites. The median of the concentration of FBP, ATP, and PEP following a withdrawal of external glucose at t~1 min in models that include activation of PYK by FBP is shown. After withdrawal of glucose, the concentration of PEP quickly rises and attains a new steady state. With the restoration of external glucose at t~10 min, PEP undergoes a quick drop, fuelling glucose uptake and subsequent production of ATP. The corresponding figure for systems that lack regulatory interactions but are nonetheless able to recover from periods of starvation is discussed in the Text S2. doi:10.1371/journal.pone.0106453.g011 September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 10 From Stoichiometry to Dynamics y y Our approach consists of a series of well-defined steps and is based upon related strategies that utilize Monte-Carlo sampling in the study of metabolic networks [22,24,26,28,30,48]. The starting point of our analysis is a stoichiometric representation of a metabolic system, as obtained either from textbook knowledge or extracted from genome-scale models of the respective organism. The stoichiometric representation is tested to allow for meaningful flux patterns, for example by an analysis of metabolic flux modes [49] or flux-balance analysis [50]. In addition to stoichiometric dependencies, we assume that the basic regulatory interactions are known. That is, for each enzymatic interconversion there may be a set of metabolites that either inhibits or activates the respective step, albeit with unknown strength. Differing from conventional bottom-up modelling, and following the definitions given in Grimbs et al. [51] and Murabito et al. [28], the subsequent analysis is then based on knowledge of a specific metabolic phenotype of the system. That phenotype is defined by a steady- state flux value for each metabolic reaction, as well as by a set of concentration values for all metabolic intermediates, and has to fulfill two prerequisites. First, its flux distribution must be consistent with the mass-balance constraint. Second, the set of concentration values must be thermodynamically consistent with the directions of the fluxes, namely that given a set of concentration values and the set of equilibrium constants, the Gibbs free energy of all reactions must be negative in the direction indicated by the phenotype’s flux distribution. To obtain insights into kinetic properties, each reaction rate is then assigned a rate equation that specifies the dependence of the reaction rate with respect to its substrates and products, as well as with respect to possible allosteric or competitive effectors. In case the actual rate equation of the respective step is unknown, a generic Michaelis- Menten equation is employed [33,52]. Once these data are assembled, the system of differential equations that determines the dynamic properties of the network has been fully specified except for lacking numerical values for most enzyme-kinetic parameters. As shown previously [28], however, with these definitions and the known metabolic phenotype, it is possible to systematically sample the parametric space, such that the resulting set of enzyme-kinetic parameters is consistent with the known metabolic state. Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism compare the distributions of dynamic properties in the presence and absence of a specific metabolic feedback. In summary, sampling unknown kinetic parameters for a kinetic model of a metabolic pathway, such that all sampled parameters give rise to the same metabolic state, can lead to fundamental insights into the control properties of the underlying system. In particular, the emergent control profile exhibits a structure that helps us to draw conclusions about the possible behavior of the system. The probabilistic control profile represents what dynamics we should expect as typical, given no further knowledge about specific constraints the system is subject to – a tremendously useful information when interpreting experimentally observed data. In this respect, some of the probabilistic distributions are in line with what we expect given the topology of the network, while other properties are counter-intuitive and therefore point the direction for further investigation. Our approach is particularly suited to investigate the qualitative effects that result from changes in pathway topology, in particular from the presence or absence of regulatory interactions. Our computational approach is straight- forward to implement and numerically efficient even for large systems. We therefore expect it to be of high utility also in other studies of metabolic, gene-expression and signal transduction systems. p The second aspect of our study relates to the question: How relevant are such distributions of control properties, given that any actual system only attains a single set of parameters. To what extent is, therefore, average behavior an indicator for actual behavior, given that the actual set of parameters may as well be located on the extreme fringes of the distribution. While we reject any interpretation of average behavior as a likelihood for actual behavior, we claim that the distributions obtained using our computational procedure indeed have functional, as well as explanatory, relevance. The explanatory relevance lies in the fact that probabilistic control profiles allow us to evaluate to what extent experimental findings are rooted in the topological structure of the pathway, rather than in fine-tuning of Michaelis-Menten parameters. Monte-Carlo Modeling of L. lactis Central Metabolism Specifically, if almost all sampled parameters attain a value within a certain narrow range, and the empirically observed value is in good agreement with these values, then we face no further explanatory challenge: The empirical value simply corresponds to what we expect as typical behavior, given no further knowledge about additional contraints the system is subject to. On the other hand, an empirically obtained control coefficient that is located at the extreme fringe of the respective distribution provides valuable information for further analysis and points to additional constraints or requirements the pathway is subject to. The functional relevance of our probabilistic evaluation is then given by the fact that control properties that are already inherent in the topological structure allow for an evolutionary adaptation of parameters with respect to other objectives. Specifically, it seems favorable, within the course of evolution, to adopt a network topology that exhibits a certain desired behavior, such as recovery after periods of starvation, for a broad range of parameter values. These parameters can then be further fine-tuned according to other objectives, without impeding the core functionality of the pathway. September 2014 \| Volume 9 \| Issue 9 \| e106453 Conclusions Metabolic regulation is a crucial aspect of cellular function. While significant progress has been made on the stoichiometric properties of large-scale metabolic systems, the organizing principles of metabolic regulation that enable stability and rapid adaptation are still insufficiently understood [15,29,38,47]. In this work, we have presented a Monte-Carlo approach to study the regulatory feedback topology of the homo-fermentative LAB strain Lactococcus lactis. In particular, we have used knowledge of an experimentally acquired metabolic state to evaluate typical Figure 12. Bistability and hysteresis with respect to external glucose. Shown is a non-recovering system in the absence of regulation (A,B) and a recovering system in the presence of regulation (C,D). In the upper panels (A,C), the concentration of external glucose was varied between the initial level of 20 mM to a lower value of 0:1 mM and back. The lower panels (B,D) show a corresponding time-course of the rate of glucose uptake. The original level of external glucose, 20 mM, was lowered to 0:1 mM within the time interval t = 1 min to 10 min. doi:10.1371/journal.pone.0106453.g012 Figure 12. Bistability and hysteresis with respect to external glucose. Shown is a non-recovering system in the absence of regulation (A,B) and a recovering system in the presence of regulation (C,D). In the upper panels (A,C), the concentration of external glucose was varied between the initial level of 20 mM to a lower value of 0:1 mM and back. The lower panels (B,D) show a corresponding time-course of the rate of glucose uptake. The original level of external glucose, 20 mM, was lowered to 0:1 mM within the time interval t = 1 min to 10 min. doi:10.1371/journal.pone.0106453.g012 September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 11 Monte-Carlo Modeling of L. lactis Central Metabolism Defining the system and its evaluation All code is provided in File S1. J’ : ~N’:Lv LSDS0 :L: ð4Þ ð4Þ The control coefficients are only evaluated for stable metabolic states, characterized by an invertible Jacobian with all real parts of the eigenvalues below zero. The information required to evaluate the control coefficients therefore consists of: (i) The stoichiometry of the system, as encoded by N’ and L; (ii) the metabolic state, as encoded in the matrices Dv 0 and DS0; and (iii) the kinetic properties of the reactions, as encoded in the partial derivatives. The partial derivatives are also known as the unscaled elasticity coefficients. Our probabilistic evaluation of the system is then based on the fact that the metabolic state is often directly experimentally accessible, whereas information about kinetic parameters, and hence the elasticities, is generally lacking. We therefore evaluate the possible values of the unscaled elasticities by drawing random instances of parameters and evaluating the equations for the control coefficients. In particular, we proceed along the following steps: First, each reaction is associated with a kinetic reaction equation. We adopt general Michaelis-Menten kinetics of the form From Stoichiometry to Dynamics In this In this sense, we argue that our approach has led to increased understanding of principles of metabolic regulation in Lactococcus lactis: We compared the typical response of the pathway with respect to short periods of starvation in the presence and absence of metabolic regulation. It was shown that the regulatory interactions, irrespective of the particular parameter values, result in qualitative differences in the dynamics. Our results therefore shows that the topology of the regulation alone is, to a large extent, already sufficient to ensure dynamic stability and recovery of the pathway. While fine-tuning of parameters may achieve a similar increase in stability, an appropriate regulatory structure dramat- ically increases the set of accessible parameters space and therefore opens the possibility to optimize Michaelis-Menten parameters for other secondary objectives, such as the trade-off between affinity and catalytic rate which are not considered here. The main result presented in this study is therefore that the regulatory architecture of the Lactococcus lactis central metabolism induces qualitative changes in the probabilistic control profile, as well as in the dynamic behavior after a brief period of starvation. Further, we were able to delineate the role of individual regulation terms: Three of the known regulatory mechanisms play a major role in the recovery ability of the system. These are (i) the inhibition of PYK by inorganic phosphate, (ii) the activation of PYK by FBP and (iii) the inhibition of PTS by FBP. We showed that in cases of non-recovery the zero-flux metabolic state is an attractor for the system. In the presence of regulation the ability of the system to escape metabolic death is, in the overwhelming number of cases, mirrored by the absence of such attractor. September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 12 Monte-Carlo Modeling of L. lactis Central Metabolism way, insight into the typical dynamic properties of a specific metabolic phenotype can be obtained. way, insight into the typical dynamic properties of a specific metabolic phenotype can be obtained. u~Vmax:f (S,K,Keq), ð5Þ ð5Þ Defining the system and its evaluation where K denotes a vector of unknown Michaelis-Menten parameters and Keq denotes an equilibrium constant. The function f includes terms for possible inhibition and activation. For example, for an unregulated uni-uni reaction A?B, the equation reads The probabilistic approach used within this work has been described previously [28]. In the following, we briefly outline our workflow. We assume that a metabolic system of interest consisting of m metabolites and r reactions is described by a system of ordinary differential equations of the form, u~Vmax: A{ B Keq K1 1z A K1 z B K2 ð6Þ dS dt ~N:v(S), ð1Þ ð1Þ ð6Þ where N denotes the m\|r stoichiometric matrix and S the m{dimensional vector of metabolite concentrations. The r{dimensional vector v specifies the nonlinear dependencies of the reaction rates as a function of the associated metabolite concentrations and kinetic parameters. To evaluate the dynamics, we assume the existence of a feasible metabolic state, defined by a concentration vector S0 and its associated flux values v(S0), such that Nv(S0)~0. We note that the metabolic state does not necessarily has to be asymptotically stable. The matrices of flux control coefficients CJ and concentration control coefficients CS can be expressed as, The number of unknown reaction parameters typically equals the number of associated substrates, products and modifiers (plus Vmax and the equlibrium constant). A full list of rate equations is provided in the Text S2. Second, the kinetic parameters K are sampled from intervals chosen according to the associated metabolite concentration, such that Ki[ 10{a1S ,10za2S : ð7Þ i0 i0 Ki[ 10{a1S ,10za2S : ð7Þ i0 i0 ð7Þ CS~{D{1 S0 :L:J’{1:N’:Dv0 ð2Þ For each simulation, the set of Michaelis-Menten parameters is chosen at random. All results are reported for a1~a2~1, but the results are highly robust for different choices of a and b. Sampling was linear in log space, i.e., the logarithm of Ki=S i0 is equidistributed in the interval. Once the parameters are specified, the values of Vmax are adjusted so as to deliver the known steady- state flux, ð2Þ and and CJ~1zD{1 v0 :Lv LSDS0 :DS0:CS ð3Þ ð3Þ where Dv0 and DS0 denote diagonal matrices with elements v0 and S0 on the diagonal, respectively, N’ denotes the reduced stoichiometric matrix and L the link matrix. See [28] for details. Defining the system and its evaluation The Jacobian J’ accounts for possible mass conservation and is defined as Vmax~ u0 f (S0,K,Keq) , ð8Þ ð8Þ and the derivatives of the reaction equation can be computed. Parameter sampling and the evaluation of control coefficients were repeated 2:104 times for each regulatory scenario. The values obtained for the control coefficients were largely robust with respect to different choices of the rate equations. Specifically, the evaluation of the control coefficient only depends on the values attained by the partial derivative, which are primarily determined by the ratio of substrate concentrations with respect to their corresponding Michaelis-Menten constants, as well as by param- eter-independent thermodynamic contributions. Using a similar sampling scheme with slightly modified reaction equations is therefore unlikely to result in major deviations with respect to overall control properties [38]. We note that our approach is straightforward to implement and its robustness with respect to different sampling schemes has been tested previously [28]. In particular, the evaluation of the control coefficients does not require explicit kinetic simulation of nonlinear differential equations, making it applicable also for medium- and large-scale systems. The key requirements are knowledge of the network topology, including its regulatory interactions, the metabolic state, as well as the respective thermodynamic equilibrium constants. All code is provided in File S1. and the derivatives of the reaction equation can be computed. Parameter sampling and the evaluation of control coefficients were repeated 2:104 times for each regulatory scenario. The values obtained for the control coefficients were largely robust with respect to different choices of the rate equations. Specifically, the evaluation of the control coefficient only depends on the values attained by the partial derivative, which are primarily determined by the ratio of substrate concentrations with respect to their corresponding Michaelis-Menten constants, as well as by param- eter-independent thermodynamic contributions. Using a similar sampling scheme with slightly modified reaction equations is therefore unlikely to result in major deviations with respect to overall control properties [38]. We note that our approach is straightforward to implement and its robustness with respect to different sampling schemes has been tested previously [28]. In particular, the evaluation of the control coefficients does not require explicit kinetic simulation of nonlinear differential equations, making it applicable also for medium- and large-scale systems. The key requirements are knowledge of the network topology, including its regulatory interactions, the metabolic state, as well as the respective thermodynamic equilibrium constants. Fermentation experiments KH2PO4, 0.6 g ammonium citrate, 1 g acetate, 0.25 g tyrosine, 0.24 g alanine, 0.125 g arginine, 0.42 g aspartic acid, 0.13 g cysteine, 0.5 g glutamic acid, 0.15 g histidine, 0.21 g isoleucine, 0.475 g leucine, 0.44 g lysine, 0.275 phenylalanine, 0.675 g proline, 0.34 g serine, 0.225 g threonine, 0.05 g tryptophan, 0.325 g valine, 0.175 g glycine, 0.125 g methionine, 0.1 g asparagine, 0.2 g glutamine, 10 g glucose, 0.5 g L-ascorbic acid, 35 mg adenine sulfate, 27 mg guanine, 22 mg uracil, 50 mg cystine, 50 mg xanthine, 2.5 mg D-biotin, 1 mg vitamin B12, 1 mg riboflavin, 5 mg pyridoxamine-HCl, 10 mg p-aminobenzoe¨ic acid, 1 mg pantothenate, 5 mg inosine, 1 mg nicotinic acid, 5 mg orotic acid, 2 mg pyridoxine, 1 mg thiamine, 2.5 mg lipoic acid, 5 mg thymidine, 200 mg MgCl2, 50 mg CaCl2, 16 mg MnCl2, 3 mg FeCl3, 5 mg FeCl2, 5 mg ZnSO4, 2.5 mg CoSO4, 2.5 mg CuSO4, (NH4)6Mo7O24. Mid-exponentially grown cells were harvested by centrifugation at 5000 RPM for 10 minutes at room temperature, washed twice with 50 mM MES buffer (pH = 6.5), and finally suspended in the indicated buffer solution. Anaerobic conditions were established by flushing with nitrogen for 10 min. Glucose (20 mM for L. lactis) was added and samples were taken at regular time intervals. 400 ml samples were taken and mixed immediately with 200 ml of a cold perchloric acid (3.5 M) solution. The extracts were kept on ice for maximal 60 minutes. The pH was neutralized with 160 ml 2 M KOH. The pH-adjusted samples were centrifuged and the supernatants were stored at 280uC for subsequent analysis. All metabolites were quantified by enzymatic methods coupled to the spectrophotometric determination of NAD(P)H. The strain NZ9000 was used [54]. where DRG0 denotes the change in standard Gibbs free energy occurring in the corresponding reaction and RT the gas constant multiplied by the absolute temperature. The value of DRG0 was estimated using, DRG0~ X products Df G0{ X substrates Df G0, ð10Þ ð10Þ where Df G0 denotes the Gibbs free energy of formation of the different metabolites. The values were adopted from Feist et al. [34], who use the group contribution method developed by Mavrovouniotis [56,57]. The full list of equilibrium constants is provided in the Text S1. The metabolic state was checked for thermodynamics feasibility. Acknowledgments We would like to thank Adam Wilkins for his kind help and contribution to revise linguistic aspects of the manuscript. Determination of the metabolic state Fluxes for lactate, acetate, formate, pyruvate and ethanol were calculated using their fermentation broth concentration, dilution rate (0:5h{1) and steady state bacterial cell dry weight. Fluxes are given in Text S1. Steady state intracellular metabolites concen- trations were gathered from previously published articles in various journals. The steady state data and its sources are provided in Text S1. Fermentation experiments In particular for any reaction S1zS2?P1zP2 with positive flux, the following relationship must hold at steady state, S 1 0S 2 0{P 1 0P 2 0=Keq w0: ð11Þ 2 0{P 1 0P 2 0=Keq w0: ð11Þ ð11Þ In Figure 5, we consider the distance C from equilibrium, defined as the ratio C : ~ 1 Keq Pj P j0 Pi S i0 , ð12Þ ð12Þ for a reaction that converts a set of substrates S into a set of products P. Only considering the positive direction of flux, the value of C is zero for irreversible reactions and approaches unity for reactions close to equilibrium. Determination of the equilibrium constants Determination of the equilibrium constants The equilibrium constants Keq were derived using the following equation, Conceived and designed the experiments: EM MV MB BT HVW RS. Performed the experiments: EM MV MB. Analyzed the data: EM MV MB DB HVW RS. Contributed reagents/materials/analysis tools: MB DB BT. Wrote the paper: RS MV EM. DRG0~{RT ln Keq, ð9Þ ð9Þ Analysis of carbon fluxes Bacterial dry weight was measured as described previously [55]. External glucose, pyruvate, lactate, formate, acetate, succinate, and ethanol were determined by high-pressure liquid chromatog- raphy (HPLC; LKB) with a Rezex organic acid analysis column (Phenomenex) at a temperature of 45uC with 7.2 mM H2SO4 as the eluent, using a RI 1530 refractive index detector ( Jasco) and AZUR chromatography software for data integration. Discrimi- nation between d- and l-lactate was performed using a d-/l-lactate assay kit (Megazyme). Fermentation experiments L. lactis cells were grown anaerobically at 37uC in CDM-LAB medium [53]. The medium contained per liter: 1 g K2HPO4, 5 g September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 13 Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism KH2PO4, 0.6 g ammonium citrate, 1 g acetate, 0.25 g tyrosine, 0.24 g alanine, 0.125 g arginine, 0.42 g aspartic acid, 0.13 g cysteine, 0.5 g glutamic acid, 0.15 g histidine, 0.21 g isoleucine, 0.475 g leucine, 0.44 g lysine, 0.275 phenylalanine, 0.675 g proline, 0.34 g serine, 0.225 g threonine, 0.05 g tryptophan, 0.325 g valine, 0.175 g glycine, 0.125 g methionine, 0.1 g asparagine, 0.2 g glutamine, 10 g glucose, 0.5 g L-ascorbic acid, 35 mg adenine sulfate, 27 mg guanine, 22 mg uracil, 50 mg cystine, 50 mg xanthine, 2.5 mg D-biotin, 1 mg vitamin B12, 1 mg riboflavin, 5 mg pyridoxamine-HCl, 10 mg p-aminobenzoe¨ic acid, 1 mg pantothenate, 5 mg inosine, 1 mg nicotinic acid, 5 mg orotic acid, 2 mg pyridoxine, 1 mg thiamine, 2.5 mg lipoic acid, 5 mg thymidine, 200 mg MgCl2, 50 mg CaCl2, 16 mg MnCl2, 3 mg FeCl3, 5 mg FeCl2, 5 mg ZnSO4, 2.5 mg CoSO4, 2.5 mg CuSO4, (NH4)6Mo7O24. Mid-exponentially grown cells were harvested by centrifugation at 5000 RPM for 10 minutes at room temperature, washed twice with 50 mM MES buffer (pH = 6.5), and finally suspended in the indicated buffer solution. Anaerobic conditions were established by flushing with nitrogen for 10 min. Glucose (20 mM for L. lactis) was added and samples were taken at regular time intervals. 400 ml samples were taken and mixed immediately with 200 ml of a cold perchloric acid (3.5 M) solution. The extracts were kept on ice for maximal 60 minutes. The pH was neutralized with 160 ml 2 M KOH. The pH-adjusted samples were centrifuged and the supernatants were stored at 280uC for subsequent analysis. All metabolites were quantified by enzymatic methods coupled to the spectrophotometric determination of NAD(P)H. The strain NZ9000 was used [54]. Supporting Information Text S1 A pdf document detailing the experimental procedure and the metabolic state. 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Solem C, Koebmann B, Yang F, Jensen PR (2007) The las enzymes control pyruvate metabolism in Lactococcus lactis during growth on maltose. J Bacteriol 189: 6727–6730. 16. Voit EO, Almeida J, Marino S, Lall R, Goel G, et al. (2006) Regulation of glycolysis in Lactococcus lactis: an unfinished systems biological case study. Syst Biol (Stevenage) 153: 286–298. 43. Koebmann BJ, Westerhoff HV, Snoep JL, Nilsson D, Jensen PR (2002) The glycolytic flux in Escherichia coli is controlled by the demand for ATP. J Bacteriol 184: 3909–3916. ( g ) 17. Papagianni M, Avramidis N, Filiousis G (2007) Glycolysis and the regulation of glucose transport in Lactococcus lactis spp. lactis in batch and fed-batch culture. Microb Cell Fact 6: 16. J 44. Rosenfeld N, Elowitz MB, Alon U (2002) Negative autoregulation speeds the response times of transcription networks. J Mol Biol 323: 785–793. 18. Heijnen JJ (2005) Approximative kinetic formats used in metabolic network modeling. 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September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 14 Monte-Carlo Modeling of L. lactis Central Metabolism Murabito E, Smallbone K, Swinton J, Westerhoff HV, Steuer R (2011) A probabilistic approach to identify putative drug targets in biochemical networks. J R Soc Interface 8: 880–895. 54. Wouters JA, Frenkiel H, de Vos WM, Kuipers OP, Abee T (2011) Cold shock proteins of lactococcus lactis MG1363 are involved in cryoprotection and in the production of cold-induced proteins. Appl Environ Microbiol 67: 5171–5178. 29. Girbig D, Grimbs S, Selbig J (2012) Systematic analysis of stability patterns in plant primary metabolism. PLoS One 7: e34686. p p y 30. Murabito E (2013) Targeting breast cancer metabolism: A metabolic control analysis approach. Curr Synthetic Sys Biol 1: 104. 55. Alexeeva S, de Kort B, Sawers G, Hellingwerf KJ, de Mattos MJ (2000) Effects of limited aeration and of the arcAB system on intermediary pyruvate catabolism in escherichia coli. 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https://openalex.org/W2165794817	https://dash.harvard.edu/bitstream/1/3693471/2/Min_ImagingChromophores.pdf	English	null	Imaging chromophores with undetectable fluorescence by stimulated emission microscopy	Nature	2,009	cc-by	6,011	Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:3693471 Published Version doi:10.1038/nature08438 Published Version doi:10.1038/nature08438 Citation Min, Wei, Sijia Lu, Shasha Chong, Rahul Roy, Gary R. Holtom, and Xiaoliang Sunney Xie. 2009. Imaging chromophores with undetectable fluorescence by stimulated emission microscopy. Nature: 461(7267): 1105-1109. Published Version doi:10.1038/nature08438 Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Imaging Chromophores with Undetectable Fluorescence by Stimulated Emission Microscopy ei Min, Sijia Lu, Shasha Chong, Rahul Roy, Gary R. Holtom, X. Sunney Xie† Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138 (USA) Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138 (USA) † To whom correspondence should be addressed. E-mail: xie@chemistry.harvard.edu (X.S.X.) † To whom correspondence should be addressed. E-mail: xie@chemistry.harvard.edu (X.S.X.) 1 1 Fluorescence, i.e., spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging1, 2. However, many chromophores, such as hemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non- radiative decay3 and hence overwhelmed by background signals. By allowing stimulated emission to effectively compete with the nonradiative decay, here we make the chromphores detectable and report a new contrast mechanism for optical microscopy. In a pump-probe experiment, upon photoexcitation by a pump pulse, the sample is stimulated down to the ground state by a time-delayed probe pulse, whose intensity is concurrently increased. We extract the miniscule intensity increase with shot-noise limited sensitivity by using a lock-in amplifier and intensity modulation of the pump beam at a high megahertz-frequency. The signal is generated only at the laser foci due to the nonlinear dependence on the input intensities,which offers intrinsic three-dimensional optical sectioning capability. In contrast, conventional one beam absorption measurement suffers from poor sensitivity, lack of 3D sectioning capability, and complication by sample scattering. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distributions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of hemoglobin. In all these cases, sensitivity has been orders of magnitude higher than those of spontaneous emission or absorption contrast, allowing more reporters for molecular imaging. Fluorescence, i.e., spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging1, 2. However, many chromophores, such as hemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non- radiative decay3 and hence overwhelmed by background signals. By allowing stimulated emission to effectively compete with the nonradiative decay, here we make the chromphores detectable and report a new contrast mechanism for optical microscopy. Imaging Chromophores with Undetectable Fluorescence by Stimulated Emission Microscopy In a pump-probe experiment, upon photoexcitation by a pump pulse, the sample is stimulated down to the ground state by a time-delayed probe pulse, whose intensity is concurrently increased. We extract the miniscule intensity increase with shot-noise limited sensitivity by using a lock-in amplifier and intensity modulation of the pump beam at a high megahertz-frequency. The signal is generated only at the laser foci due to the nonlinear dependence on the input intensities,which offers intrinsic three-dimensional optical sectioning capability. In contrast, conventional one beam absorption measurement suffers from poor sensitivity, lack of 3D sectioning capability, and complication by sample scattering. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distributions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of hemoglobin. In all these cases, sensitivity has been orders of magnitude higher than those of spontaneous emission or absorption contrast, allowing more reporters for molecular imaging. 2 The phenomenon of stimulated emission was first described by Einstein in 19174. An atom or molecule in its excited state can be stimulated down to the ground state by an incident light field, resulting in the creation of a new coherent photon identical to those in the original incident field. This process only occurs when the frequency of the incident field matches the transition energy. Stimulated emission was later used as a fundamental principle for light amplification in the laser5. The depopulation aspect of stimulated emission has been used for population dumping from excited states6, super-resolution fluorescence (STED) microscopy7, and fluorescence lifetime imaging8. Here we utilize the light amplification aspect of stimulated emission as a contrast mechanism for highly sensitive imaging of chromophores with undetectable fluorescence. Those chromophores have very short lived excited states with much faster non- radiative decay rates than their spontaneous emission rates. As a result, their feeble fluorescence is overwhelmed by backgrounds, such as stray light, solvent Raman scattering, and detector dark counts. Our solution to this problem is to conduct a dual beam experiment in order to interrogate the short lived excited state by stimulated emission, which can compete with the non-radiative decay under strong enough stimulating field strength (Fig. 1a). The resulting “amplification” of the stimulation beam can then be detected in the presence of the background signals. Imaging Chromophores with Undetectable Fluorescence by Stimulated Emission Microscopy Considering the optical excitation at frequency 01 ω (Fig. 1a), the absorption cross section [ ] 1 0→ abs σ is ~ 10-16 cm2 for a single chromophore at room temperature2, 9. As shown in Fig. 1b, under a tightly focused laser beam with a beam waist area of S (~ 10-9 3 3 cm2), the integrated intensity attenuation of the excitation beam, E E I I Δ , is proportional cm2), the integrated intensity attenuation of the excitation beam, E E I I Δ , is proportional to the ratio between [ ] 1 0→ abs σ and S: e ratio between [ ] 1 0→ abs σ and S: to the ratio between [ ] 1 0→ abs σ and S: [ ] S N I I abs E E 1 0 0 → ⋅ − = σ Δ (1) (1) where N0 is the number of molecules in the ground state. For a single chromophore, E E I I Δ is on the order of 10-7. Such small attenuation cannot be detected by conventional absorption microscopy. We note that single molecule absorption was previously achieved in cryogenic temperatures using a frequency modulation method10, which is however difficult to implement at room temperature because of the broad molecular absorption spectrum. Moreover, absorption measurement is often complicated by scattering when imaging biological specimen. Instead of detecting direct absorption, here we detect the stimulated emission followed by the excitation of the molecule. where N0 is the number of molecules in the ground state. For a single chromophore, E E I I Δ is on the order of 10-7. Such small attenuation cannot be detected by conventional absorption microscopy. We note that single molecule absorption was previously achieved in cryogenic temperatures using a frequency modulation method10, which is however difficult to implement at room temperature because of the broad molecular absorption spectrum. Moreover, absorption measurement is often complicated by scattering when imaging biological specimen. Instead of detecting direct absorption, here we detect the stimulated emission followed by the excitation of the molecule. where N0 is the number of molecules in the ground state. For a single chromophore, E E I I Δ is on the order of 10-7. Such small attenuation cannot be detected by conventional absorption microscopy. Imaging Chromophores with Undetectable Fluorescence by Stimulated Emission Microscopy We note that single molecule absorption was previously achieved in cryogenic temperatures using a frequency modulation method10, which is however difficult to implement at room temperature because of the broad According to Einstein4, the molecular cross section . .em sti σ for stimulated emission is comparable to abs σ , because of microscopic reversibility. Unlike the absorption which results in attenuation, the stimulation beam will experience an intensity gain after interacting with the molecules [ ] S N I I em sti S S 3 2 . . 2 → ⋅ + = Δ σ (2) (2) (2) where N2 is the number of excited molecules transiently probed by the stimulation pulses (Fig. 1b). For a single chromophore, S S I I Δ is also ~10-7. Without special techniques, such a small signal would be again buried in the noise (~1%) of the stimulation beam. 4 To overcome this noise problem and promote the advantage of performing stimulated emission, we implemented a high-frequency (> 1 MHz) phase-sensitive detection technique. In so doing, the laser intensity fluctuation, which occurs primarily at low frequency (kHz to DC), can be circumvented, as has been previously applied in other spectroscopic11 and recently stimulated Raman scattering (SRS) microscopy12 and two photon absorption microscopy13. In the scheme shown in Fig. 1b and c, the intensity of the excitation beam is modulated at 5 MHz, and this creates a modulation of the To overcome this noise problem and promote the advantage of performing stimulated emission, we implemented a high-frequency (> 1 MHz) phase-sensitive detection technique. In so doing, the laser intensity fluctuation, which occurs primarily at low frequency (kHz to DC), can be circumvented, as has been previously applied in other spectroscopic11 and recently stimulated Raman scattering (SRS) microscopy12 and two photon absorption microscopy13. In the scheme shown in Fig. 1b and c, the intensity of the excitation beam is modulated at 5 MHz, and this creates a modulation of the stimulated emission signal at the same frequency, because only when the excitation beam is present can the gain of the stimulation beam occur. Such an induced modulation signal can then be sensitively extracted by a lock-in amplifier referenced to this high frequency. In this way, the stimulated emission signal can be detected against the vanishing laser noise with a superior sensitivity. Imaging Chromophores with Undetectable Fluorescence by Stimulated Emission Microscopy In contrast, it is difficult to implement such modulation scheme for high sensitivity one-beam absorption measurement11. Specifically, we use a ~200 fs (FWHM) pulse train for excitation, and another ~200 fs pulse train for stimulation. The time delay between these two pulse trains is chosen to be ~300 fs (Fig. 1c), which is shorter than the excited state lifetime (sub-ps) of the chromophores. This delay also eliminates contributions from other instantaneous optical processes, such as two photon absorption13, cross phase modulation and SRS12. We conduct the experiment under a non-saturating condition of the four-level system (Fig. 1a). Under this condition, N2 in equation (2) originates from a linear excitation: [ ] S I N N abs E 1 0 0 2 → ⋅ ⋅ ∝ σ . This relation, together with equation (2), indicates that the final signal SI Δ is linearly dependent on both E I and SI , 5 [ ] ( ) [ ] ( ) S S I I N I em sti abs S E S 3 2 . . 1 0 0 → → ⋅ ⋅ ⋅ ⋅ ∝ Δ σ σ (3) (3) The overall quadratic power dependence, as experimentally demonstrated (Fig.2a), would allow three-dimensional (3D) optical sectioning, as in many other multi-photon techniques14, 15. Such dependence also confirms the non-saturation condition within the intensity range used. Moreover, it offers, in principle, a spatial resolution of twice as high (in spatial frequency) as in conventional fluorescence microscopy. The overall quadratic power dependence, as experimentally demonstrated (Fig.2a), would allow three-dimensional (3D) optical sectioning, as in many other multi-photon techniques14, 15. Such dependence also confirms the non-saturation condition within the intensity range used. Moreover, it offers, in principle, a spatial resolution of twice as high (in spatial frequency) as in conventional fluorescence microscopy. Fig. 2b shows the stimulated emission signal as a function of the time-delay between the excitation and stimulation pulses. In pump-probe experiment, the signal could arise from ground state recovery, excited state absorption or stimulated emission. Ground state recovery is avoided here by the large Stokes shift of stimulation beam from molecular absorption band. Excited state absorption is negligible compared to stimulated emission based on the observed intensity increase of the stimulation beam, consistent with a study of fluorescence quenching in STED microscopy16. The initial rise in Fig. 2b is due to vibrational relaxation from level 1 to level 2 (Fig. Imaging Chromophores with Undetectable Fluorescence by Stimulated Emission Microscopy 1a), while the subsequent decay indicates the short excited state lifetime (~0.6 ps) of level 2, which underlies the undetectable fluorescence. Such a short lifetime also reduces the probability of going into the triplet state, effectively protecting the molecule from photo-bleaching. The stimulated emission spectrum (Fig. 2c), recorded by tuning the wavelength of the stimulated beam, is also in agreement with the reported emission spectrum of crystal violet in glycerol17, where the high viscosity increases the fluorescence quantum yield. Fig. 2d shows that the stimulated emission signal depends linearly on analyte concentration, as predicted by equation (3). This allows straightforward quantitative 6 analysis. The limit of detection is ~60 nM for crystal violet with 1 sec integration time. This sensitivity effectively corresponds to a few (<5) molecules in focus, which has surpassed the detection limit of recently reported for SRS microscopy12 by orders of magnitude because of the strong electronic transition. The ultimate sensitivity would be achieved under the condition of saturated excitation but near-saturated stimulation, and is fundamentally determined by the shot noise of the stimulation beam ( S S I I Δ ~10-7.5 for ~1mW averaged over 1 sec). As the first biological application, we imaged distributions of chromoproteins in live E. coli cells. Genetically encodable chromoproteins, such as gtCP18 and cjBlue19, are naturally existing variants of green fluorescent proteins20, and only absorb light but do not fluoresce. When the gene encoding for gtCP is expressed in E. coli cells, tetrameric gtCP can be clearly visualized to reside homogeneously inside cytoplasm by stimulated emission microscopy (Fig. 3 a&b). Similarly, the distribution of another chromoprotein, cjBlue, can be imaged (Fig. 3 c&d). Unlike gtCP which is expressed in most cells, cjBlue is expressed in a fraction of them, as we see from the corresponding transmission image. Therefore, stimulated emission microscopy opens the possibility of visualizing chromoproteins that are not accessible by fluorescence microscopy. Next, we show the stimulated emission imaging in live E. coli cells of a non- fluorescent indigo reaction product commonly used to assay gene expression. β- galactosidase catalyzes the cleavage of the glycosidic linkage of X-gal, a popular chromogenic substrate, eventually forming an indigo product after oxidation21. The gene encoding β-galactosidase, lacZ, is a classic reporter gene. Traditionally, the indigo product has to accumulate in sufficient concentration for its blue color to be visible21. Imaging Chromophores with Undetectable Fluorescence by Stimulated Emission Microscopy 7 With stimulated emission, the accumulation of the indigo product can now be monitored in wild-type cells without induction of lacZ (Fig. 3 e-g). The inhomogeneous distribution of indigo product inside individual cells (Fig. 3 e&f) is consistent with the fact that this product is insoluble and tends to form localized precipitates. In contrast, the corresponding transmission image (Fig. 3 g) shows no signs of any color. We note that an assay using a fluorogenic substrate has been recently developed22, but it requires a microfluidics to enclose individual cells because the hydrolysis product there is quickly pumped out by the cell. Hence, stimulated emission microscopy allows monitoring of a product presumed to report basal lacZ gene activity with superb sensitivity. Monitoring transdermal delivery of non-fluorescent drug with intrinsic 3D optical sectioning is another application. In particular, we show mapping of a cationic thiazine dye toluidine blue O (TBO) at both the cellular and tissue levels. Having a selective affinity for cancer cells in vivo, TBO is a photosensitizer in photodynamic therapy23, 24. Subcellular localization of TBO is crucial since it influences both the level and the kinetics of apoptosis induction. However, it is difficult to image the true TBO distribution, because its fluorescence is quenched when bound to tissue substrates and only the non- specific stain residue in the tissue retains its native fluorescence24. Independent of the complication from fluorescence contrast, stimulated emission microscopy is suitable for this problem. The stimulated emission image of TBO inside the cancer cells after incubation shows its local accumulation (Supplementary Fig. 1). When topically applied to skin tissue, being hydrophilic and water soluble, TBO is enriched in the center of the protein phase of the polygonal stratum corneum cells rather than in the intercellular space which is in lipid phase (Fig. 4a). At a 20 μm deeper depth, TBO displays a rich Monitoring transdermal delivery of non-fluorescent drug with intrinsic 3D optical sectioning is another application. In particular, we show mapping of a cationic thiazine dye toluidine blue O (TBO) at both the cellular and tissue levels. Having a selective affinity for cancer cells in vivo, TBO is a photosensitizer in photodynamic therapy23, 24. 8 subcellular distribution in the cytoplasm of epidermis where cellular proliferation actively takes place (Fig. 4b). These imaging results support the hydrophilic delivery pathway as well as the recent hypothesis of TBO binding to cytoplasmic RNA to initiate apoptosis23. Imaging Chromophores with Undetectable Fluorescence by Stimulated Emission Microscopy Finally, we demonstrate label-free imaging of microvascular based on endogenous contrast from non-fluorescent hemoglobin. Structure and hemodynamics of blood vessels play a major role in many biomedical processes such as angiogenesis in tumors25 and cerebral oxygen delivery in brain26, 27. However, established techniques such as MRI, CT, PET, ultrasound, confocal and two-photon fluorescence microscopy either lack the spatial resolution needed for resolve individual capillaries or require exogenous contrast agents. Here we perform ex vivo stimulated emission imaging of the well-developed vascular network from a nude mouse ear, by exciting the Soret band of hemoglobin through efficient two-photon absorption28 and subsequently stimulating the emission from its Q band which has longer excited state lifetime than Soret band29. As shown in Fig. 4c, blood vessels (in red) branch and loop around sebaceous glands (in green). In addition, single red blood cells can be clearly seen to reside within individual capillaries (~5 μm in diameter). Two photon absorption has been recently developed to image hemoglobin13. We note that stimulated emission is more sensitive because it involves direct one-photon transition. Furthermore, our new technique offers the prospect of 3D mapping of blood oxygenation level for addressing broad physiological and pathological problems25-27. We note that the complexity and cost of the instrument can be much reduced as fiber laser sources are adapted. Stimulated emission microscopy allows imaging of non- fluorescent chromophores with 3D optical sectioning and high sensitivity, and extends the repertoire of reporters for biological imaging beyond fluorophores. 9 9 Figure Captions Fig. 1. Principle of stimulated emission microscopy. a, Energy diagram of spontaneous emission, non-radiative decay and stimulated emission processes for a typical four-level energy system. Non-radiative decay dominates spontaneous emission for chromophores with undetectable fluorescence. However, when the stimulation field is designed to have the correct energy and timing, the stimulated emission can compete with non-radiative decay and become the dominating decay pathway. b, Stimulated emission microscopy. The incident excitation and delayed stimulation pulse trains are spatially overlapped and focused onto the common focal spot in the sample. A modulator switches the intensity of the excitation beam on-and-off at 5 MHz. The spectrally filtered stimulation beam is detected by a large-area photodiode (PD), and demodulated by a lock-in amplifier to create the image contrast while raster scanning the collinear exciting and stimulating beams. The relative energy gain or loss of the stimulation and excitation beam, respectively, for a single chromophore at the laser focus (area of S), is given by the equations (inset). c, Principle of high-frequency modulation. As the intensity of the excitation beam is modulated on-and-off in time, the corresponding stimulated emission signal is also modulated at the same frequency. The stimulation pulses is made to lag behind the excitation pulses by an appropriate amount t Δ . 10 Fig. 2 Characterizations of stimulated emission microscopy. a, Stimulated emission signal from 30μM crystal violet/water solution is proportional to the product of excitation beam power, IE, and stimulation beam power, IS. b, Stimulated emission signal as a function of time delay between the excitation (590nm) and the stimulation (660nm) pulses. The decay of the signal (~ 0.6 ps) reflects the short excited state lifetime. The FWHM of the pulses is ~200fs. c, Measured stimulated emission spectrum is in agreement with the reported fluorescence spectrum of crystal violet in glycerol solution16. 590nm excitation is fixed while the stimulation wavelength is scanned by tuning the other OPO wavelength. A time delay of 0.3 ps is used. Inset is the absorption spectrum of crystal violet in water. d, Stimulated emission signal scales linearly with crystal violet concentration in aqueous solution. Continuous flow of the sample is used to replenish molecules. 590nm and 660nm for excitation and stimulation beam, respectively. 0.3 ps time delay is used. Error bars show 1 s.d. of the signals from a 30 sec recording. Figure Captions The detection limit was determined to be 60nM with a signal-to-noise ratio of 1:1 (Supplementary Fig. 3). The excitation and stimulation beam intensities at the focus are 0.2 mW (~0.14 MW/cm2) and 1 mW (~0.70 MW/cm2), respectively. For 1 sec time constant at the lock-in amplifier, a relative signal of 10-7 for S S I I Δ can be detected. Fig. 2 Characterizations of stimulated emission microscopy. a, Stimulated emission signal from 30μM crystal violet/water solution is proportional to the product of excitation beam power, IE, and stimulation beam power, IS. b, Stimulated emission signal as a function of time delay between the excitation (590nm) and the stimulation (660nm) pulses. The decay of the signal (~ 0.6 ps) reflects the short excited state lifetime. The FWHM of the pulses is ~200fs. c, Measured stimulated emission spectrum is in agreement with the reported fluorescence spectrum of crystal violet in glycerol solution16. 590nm excitation is fixed while the stimulation wavelength is scanned by tuning the other OPO wavelength. A time delay of 0.3 ps is used. Inset is the absorption spectrum of crystal violet in water. d, Stimulated emission signal scales linearly with crystal violet concentration in aqueous solution. Continuous flow of the sample is used to replenish molecules. 590nm and 660nm for excitation and stimulation beam, respectively. 0.3 ps time delay is used. Error bars show 1 s.d. of the signals from a 30 sec recording. The detection limit was determined to be 60nM with a signal-to-noise ratio of 1:1 (Supplementary Fig. 3). The excitation and stimulation beam intensities at the focus are 0.2 mW (~0.14 MW/cm2) and 1 mW (~0.70 MW/cm2), respectively. For 1 sec time constant at the lock-in amplifier, a relative signal of 10-7 for S S I I Δ can be detected. Fig. 3 Imaging non-fluorescent chromoproteins and chromogenic reporter for gene expression. Imaging distributions of cytoplasmic chromoproteins gtCP (a) and cjBlue (c) in live E. coli cells, respectively, by stimulated emission microscopy. b and d are the corresponding wide-field transmission images. Plasmids containing the genes encoded for gtCP and cjBLue are transformed into E. coli, respectively. Compared to gtCP, cjBlue 11 is expressed less abundantly inside cells. e, Stimulated emission imaging of the oxidation product (4,4'-dichloro-5,5'-dibromoindigo) resulting from X-gal hydrolysis catalyzed by β-galactosidase, the protein encoded by lacZ reporter gene. Indigo accumulation in E. coli cells is detected without any lacZ induction. The signal is detected only when both the cells and X-gal are present. Unlike the homogeneous protein images in a & c, indigo product shows inhomogeneous localized distribution inside cells due to its insolubility, as shown in the magnified image f. The direct transmission image g shows the morphology of the same region. gtCP and cjBlue absorb around 580nm18 and 610nm19, respectively; indigo product has a broad absorption around 600~650nm. In a & c, 590nm and 660nm for excitation and stimulation, respectively; in e & f, 600nm and 680nm for excitation and stimulation, respectively. The excitation and stimulation beams of 0.1 and 0.3 mW, respectively, and ~0.3 ps of time delay are used for all the above images. All of the images were taken between 5 sec and 45 sec, depending on the image size. Fig. 4 Transdermal drug distribution in 3D and microvascular imaging. a& Fig. 4 Transdermal drug distribution in 3D and microvascular imaging. a&b, Drug delivery of toluidine blue O (TBO), a drug used as photosensitizer in photodynamic therapy, in a freshly cut mouse ear skin the same area at two different depths, 3 and 25 μm, respectively, 30 min after topical application of 10 μM TBO/PBS solution. At the surface layer (3 μm) of stratum corneum, (a) shows TBO is accumulated in the protein phase of the polygonal cells rather than in the lipid-rich intercellular space. At the layer of epidermis (25 μm), (b) shows a rich TBO distribution following the subcellular cytoplasm of nucleated basal keratinocytes. These images support the 'hydrophilic path' as a main pathway for transdermal drug delivery of TBO. 600nm and 680nm for 12 excitation and stimulation, respectively. The excitation and stimulation beams have 0.1 and 0.3 mW, respectively, at the focus. ~0.3 ps time delay is used. The color table (cyan hot) shows the magnitude of S S I I Δ . c, Ex vivo imaging of microvasculature network of a mouse ear based on endogenous hemoglobin contrast. Stimulated emission image (red channel, maximum intensity projection) shows the blood vessel network surrounding sebaceous glands (green channel, simultaneously recorded by confocal reflectance). Shown by the zoomed-in image, individual red blood cells are lined up within a single capillary (~5 μm in diameter). In c, 830nm (~20mW) and 600nm (~3mW) are used for two-photon excitation of Soret band and one-photon stimulated emission of Q band of hemoglobin, respectively. Pulse widths of both excitation and stimulation beams are about 0.2 ps with ~0.2 ps time delay between them. METHODS SUMMARY In the scheme depicted in Fig. 1, two fs optical parametric oscillators (OPO) In the scheme depicted in Fig. 1, two fs optical parametric oscillators (OPO) (Coherent/APE) are synchronously pumped by a fs mode-locked 76 MHz Ti:Sapphire laser (Coherent). Two independent frequency-doubled outputs from these two OPO signal waves, in the wavelength range of 560 to 700 nm with pulse widths around 200 fs, serve as the either excitation or stimulation pulse trains. A pulse compressor consisting of a pair of SF11 prisms is built to control the pulse width. Collinear excitation and stimulation beams are combined and focused with a high numerical aperture (NA=1.2) objective onto a common focal spot. The temporal delay between the synchronized excitation and stimulation inter-pulse is adjusted to between 0.2 and 0.3 ps. The intensity (Coherent/APE) are synchronously pumped by a fs mode-locked 76 MHz Ti:Sapphire laser (Coherent). Two independent frequency-doubled outputs from these two OPO 13 of the excitation beam is modulated by an acousto-optical modulator (Crystal technology) at 5 MHz. A condenser with a N.A.=0.9 is used to collect the forward propagating stimulation beam, which is spectrally filtered before detected by a photodiode. To acquire images with a laser scanning microscope (FV300, Olympus), we used a 100 μs time constant for a lock-in amplifier (SR844, Stanford Research) and pixel dwell time of 190 μs. μs. μs. (1) Pawley, J. B. (ed.) Handbook of Biological Confocal Microscopy. Springer, 3rd edition. (2) Lakowicz, J. R. 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Acknowledgements We thank K. Lukyanov and A. Miyawaki for providing the generous gifts of chromoprotein gtCP and cjBlue plasmid DNA, respectively; Coherent Inc. for providing a loan for one fs-OPO; P. Choi for preparing X-gal E. coli cells; and B. G. Saar, C. W. Freudiger, S. Basu, J. W. Lichtman and C. B. Schaffer for helpful discussions. METHODS SUMMARY This work was supported by National Science Foundation (grant CHE- 0634788) and U.S Department of Energy’s Basic Energy Sciences Program (DE-FG02- 07ER15875). Harvard University has filed a patent application based on this work. Author Contributions W. M., S. L. and S. C. performed experiments and analyzed data. R. R. constructed E. coli cells expressing chromoproteins. G. R. H. and S. C. helped to construct the laser systems. W. M., S. L. and X. S. X. conceived the concept, designed the experiments and wrote the paper. 17 Author Information Reprints and permissions information is available at www.nature.com/reprints. Correspondence and requests for materials should be addressed to X. S. X.(xie@chemistry.harvard.edu). 18 on ay n [2 3] sti em SI σ → Δ E eous emissi diative deca ted emissio . .[2 3] sti em S SI S → = S ω23 Spontane Non-rad Stimulat Modulator ω01 ω23 I I σ Stimulation beam Δt Excitation beam Vibrational relaxation IE IS I IS IE ΔIS t t Figure 1 Input pulse train Output pulse train t t [2 3] sti em SI σ → Δ . .[2 3] sti em S SI S → = S ulator ω01 ω23 I I σ m tion beam IE IS ΔIS t Output pulse train t ΔIS t Output pulse train t [2 3] sti em SI σ → Δ . METHODS SUMMARY .[2 3] sti em S SI S → = S or ω01 ω23 I I σ n beam IE IS I I IE IS l utp ay n diative deca ted emissio ω23 Non-rad Stimulat Modul Stimulation beam Δt Excitati ibrational relaxation I IS IE t Figure 1 Input pulse train t IS [10 -0.4 0.0 0.4 0.8 1.2 0 ΔIS / I 0.0 0.5 1.0 1.5 4 d 0 2 3 0 5] 0.4 0.0 0.4 0.8 1.2 Time delay (ps) IE IS (10 -6 W 2) 2 3 IS / IS [10 -6] 500 600 700 0 1 2 ε [1 λ (nm) 0.0 0.5 1.0 1.5 0 1 ΔI Crystal violet concentration (μM) 25 650 675 700 5 Stimulation wavelength (nm) Figure 2 y (μ ) g ( ) 0.4 0.8 1.2 0.4 0.8 1.2 e delay (ps) 0.5 1.0 1.5 let concentration (μM) (μ ) IS -0.4 0.0 0 ΔIS / I 1.0 1.5 4 d 2 3 0 5] 0.4 0.0 Tim (10 -6 W 2) 2 3 / IS [10 -6] 500 600 700 0 1 2 ε [1 λ (nm) IS / 0.0 0 0 1 ΔI Crystal viol 675 700 wavelength (nm) Figure 2 y g ( ) 5 c d 2μm 1μm f e 4μm g 1μm c d 2μm f e 4μm g d 2μm f g e 15μm 15μm c 10-5 c 50 μm Figure 4 10 50 μm Figure 4
https://openalex.org/W4384155735	https://dl.acm.org/doi/pdf/10.1145/3597926.3598107	English	null	Guiding Greybox Fuzzing with Mutation Testing	null	2,023	cc-by	14,012	1 INTRODUCTION Greybox fuzzing [9, 49, 52, 81] and coverage-guided property test- ing [44, 58] have become increasingly popular for automated testing. Their key idea is to evolve a corpus of test inputs via an evolutionary search that maximizes code coverage: in each iteration, a new input is synthesized by randomly mutating an existing input from the cor- pus. The mutated input is added to the corpus if the corresponding execution of the test program increases code coverage. In this paper, we develop and evaluate Mu2, a Java-based frame- work for incorporating mutation analysis in the greybox fuzzing loop, with the goal of producing a test-input corpus with a high mu- tation score. Mu2 makes use of a diﬀerential oracle for identifying inputs that exercise interesting program behavior without causing crashes. This paper describes several dynamic optimizations imple- mented in Mu2 to overcome the high cost of performing mutation analysis with every fuzzer-generated input. These optimizations introduce trade-oﬀs in fuzzing throughput and mutation killing ability, which we evaluate empirically on ﬁve real-world Java bench- marks. Overall, variants of Mu2 are able to synthesize test-input corpora with a higher mutation score than state-of-the-art Java fuzzer Zest. Fuzzing is traditionally used to discover inputs that crash pro- grams and reveal security vulnerabilities [5, 11, 14, 20, 25, 42, 50, 54, 59, 68]. In the absence of new bugs, fuzzers are evaluated based on code coverage achieved during the fuzzing campaign [10, 48]. However, in the vast majority of fuzzing research, the end goal is to ﬁnd bugs in the moment [42]; not much attention is paid to the inputs saved along the way. In this paper, we explicitly focus on the quality of the test-input corpus produced at the end of a fuzzing campaign. Such a corpus can be used for continuous regression testing during subsequent program development. This practice is recommended by Google’s OSS-Fuzz [28], and is already adopted by some mature projects. For example, in SQLite, “Historical test cases from AFL, OSS Fuzz, and dbsqlfuzz are collected [...] and then rerun by the fuzzcheck utility program whenever one runs make test” [71]. Similarly, OpenSSL uses several distinct fuzzer-generated corpora and their correspond- ing fuzz drivers for continuous testing [72]. Even though these test corpora are used for regression testing, the only metric being tar- geted by conventional greybox fuzzers is code coverage. ACM Reference Format: Vasudev Vikram, Isabella Laybourn, Ao Li, Nicole Nair, Kelton OBrien, Rafaello Sanna, and Rohan Padhye. 2023. Guiding Greybox Fuzzing with Mutation Testing. In Proceedings of the 32nd ACM SIGSOFT International Symposium on Software Testing and Analysis (ISSTA ’23), July 17–21, 2023, Seattle, WA, USA. ACM, New York, NY, USA, 13 pages. https://doi.org/10. 1145/3597926.3598107 Greybox fuzzing and mutation testing are two popular but mostly independent ﬁelds of software testing research that have so far had limited overlap. Greybox fuzzing, generally geared towards searching for new bugs, predominantly uses code coverage for selecting inputs to save. Mutation testing is primarily used as a stronger alternative to code coverage in assessing the quality of regression tests; the idea is to evaluate tests for their ability to identify artiﬁcially injected faults in the target program. But what if we wanted to use greybox fuzzing to synthesize high-quality regression tests? CCS CONCEPTS • Software and its engineering →Software testing and debug- ging. Guiding Greybox Fuzzing with Mutation Testing Vasudev Vikram Carnegie Mellon University Pittsburgh, PA, USA vasumv@cmu.edu Ao Li Carnegie Mellon University Pittsburgh, PA, USA aoli@cmu.edu Isabella Laybourn Carnegie Mellon University Pittsburgh, PA, USA ilaybour@andrew.cmu.edu Nicole Nair Swarthmore College Swarthmore, PA, USA nnair1@swarthmore.edu Kelton OBrien University of Minnesota Minneapolis, MN, USA obri0707@umn.edu Rafaello Sanna University of Rochester Rochester, NY, USA rsanna@u.rochester.edu Rohan Padhye Carnegie Mellon University Pittsburgh, PA, USA rohanpadhye@cmu.edu 1 INTRODUCTION However, coverage alone is not the necessarily the strongest predictor of fault detection ability [15, 36]. 2.1 Greybox Fuzzing and Corpus Generation Coverage-guided greybox fuzzing (CGF) is a technique for auto- matic test-input generation using lightweight program instrumenta- tion. It was ﬁrst popularized by open-source tools such as AFL [81] and libFuzzer [49], but has since been heavily studied and variously extended in academic research [5, 9, 14, 20, 25, 44, 50, 52, 58, 59]. [ ] Algorithm 1 describes the basic greybox fuzzing algorithm, with many details elided. First, a corpus of test inputs is initialized with a set of one or more seed inputs (Line 2), which could be user-provided or randomly generated. Then, in each iteration of the fuzzing loop (Line 3), a new input is synthesized by ﬁrst picking an existing input 푥from the corpus (Line 4) and then performing random mu- tations to produce 푥′ (Line 5). The heuristics to sample an input (PickInput) vary, and often use some sort of energy schedule [9]. Some inputs may also be marked as favored, and receive higher energy than other inputs. The random mutations performed on 푥to get 푥′ (MutateInput) also vary depending on the known format of inputs (e.g., bitﬂips for binary data or random keyword insertion for text ﬁles). Structure-aware fuzzing tools [4, 44, 59, 65, 76] perform mutations that preserve the syntax or type safety of inputs, e.g. by mutating parse trees using a grammar or by mutating pseudoran- dom choices backing a Quickcheck-like [16] generator function. The program under test 푃is then executed with the new input 푥′, using lightweight instrumentation to collect code coverage during execution. The function coverage referenced in Algorithm 1 re- turns a set of program locations executed when processing an input. If the run of 푥′ causes new code to be covered (Line 8), then 푥′ is saved to the corpus (Line 9); thus, 푥′ may be used as the basis for further input mutation in subsequent iterations of the fuzzing loop. This paper details two main aspects of Mu2’s design. First, with a conventional fuzzing oracle that only identiﬁes program crashes or aborts, many inputs will be discarded for not killing any mu- tant even though they exercise interesting program functionality. For mutation testing to be useful, we need a stronger test oracle. Mu2 incorporates the idea of diﬀerential mutation testing, which validates the output of program execution. Second, evaluating each fuzzer-generated input on the set of all program mutants is pro- hibitively expensive, thereby reducing fuzzing throughput. KEYWORDS fuzz testing, mutation testing, test generation This work is licensed under a Creative Commons Attribution 4.0 Interna- tional License. ISSTA ’23, July 17–21, 2023, Seattle, WA, USA © 2023 Copyright held by the owner/author(s). ACM ISBN 979-8-4007-0221-1/23/07. https://doi.org/10.1145/3597926.3598107 Now, the technique of mutation testing [19], which evaluates the ability of tests to catch artiﬁcially injected bugs (a.k.a. mutation 929 ISSTA ’23, July 17–21, 2023, Seatle, WA, USA Vasudev Vikram, Isabella Laybourn, Ao Li, Nicole Nair, Kelton OBrien, Rafaello Sanna, and Rohan Padhye Execute Save? Execution feedback No Yes Add Input’ Input Input Input Input Seeds 𝑚𝑢𝑡𝑎𝑡𝑖𝑜𝑛_𝑠𝑐𝑜𝑟𝑒 New mutants killed? Input Pick <foo></foo> Input’ Random Mutation <woo>?</oo> Program’ Program’ Program’ (Program Mutants) Figure 1: A mutation-analysis-guided fuzzing loop. Each fuzzer-generated input is run through a set of program mu- tants to compute a mutation score. Inputs are saved to the corpus if they improve mutation score. Execute Save? Execution feedback No Yes Add Input’ Input Input Input Input Seeds 𝑚𝑢𝑡𝑎𝑡𝑖𝑜𝑛_𝑠𝑐𝑜𝑟𝑒 New mutants killed? Input Pick <foo></foo> Input’ Random Mutation <woo>?</oo> Program’ Program’ Program’ (Program Mutants) (Program Mutants) Mu2 employing diﬀerent strategies for improving performance. Our combined evaluation represents 21,600 CPU-hours (2.5 CPU-years) of fuzzing campaigns. Random Mutation Our results indicate: (1) an optimized version of Mu2 has an overall improvement of up to 20% in mutation scores across ﬁve benchmarks (5% increase on average); (2) mutation-analysis feed- back generates test-input corpora with higher reliability of killing nontrivial mutants compared to coverage-only feedback; (3) the diﬀerential testing oracle is signiﬁcantly valuable to Mu2, detecting 30% more mutants on average than a conventional fuzzing oracle; To summarize, this paper makes the following contributions: (1) We investigate the various challenges of combining muta- tion testing and greybox fuzzing, and propose solution ap- proaches to include in our framework. Figure 1: A mutation-analysis-guided fuzzing loop. Each fuzzer-generated input is run through a set of program mu- tants to compute a mutation score. Inputs are saved to the corpus if they improve mutation score. (2) We incorporate diﬀerential testing as an oracle for mutation testing in the fuzzing loop and ﬁnd that it signiﬁcantly im- proves the strength of the fuzzing oracle. (3) We employ multiple sound performance optimizations that enable mutation analysis to run in the fuzzing loop, and propose aggressive optimizations that are able to scale Mu2 to larger programs. KEYWORDS analysis), has shown promise as an adequacy criteria for improving test-suite eﬀectiveness [15, 39, 64]. A test is said to kill a program mutant if it fails when executed on the mutant, whereas mutants that fail no tests are said to survive. A goal of mutation testing is to produce a test corpus that has a high mutation score, deﬁned as the fraction of all mutants that are killed by the test suite. A natural question thus arises: can we use mutation scores to guide the fuzzer? (4) We present an empirical evaluation of Mu2 on 5 real-world Java benchmarks, with Zest [59] as a baseline. (5) We open-source Mu2 for use by practitioners and to enable reproduction and extension by researchers. q g f In this paper, we develop and evaluate a framework for incor- porating mutation analysis in the fuzzing loop, building on our previous work which ﬁrst proposed the approach [46]. The idea is as follows (see Fig. 1): after a new input is synthesized by a fuzzer via random mutation of a previously saved input, it is evaluated by executing a set of mutants of the program under test. If the new in- put kills any previously surviving program mutant, then it is added to the corpus. In this process, we distinguish between input muta- tions (e.g., randomly setting input bits or ﬁelds to zero) and program mutations (e.g., replacing the expression a+b with a-b in the tar- get’s source code). Our Java-based implementation, called Mu2—for Mutation-Based Greybox Fuzzing + Mutation Testing—incorporates program mutations from the popular PIT toolkit [17] into a custom guidance in the JQF [58] greybox fuzzing framework. Mu2 is open source and available at: https://github.com/cmu-pasta/mu2. 2 BACKGROUND 2.1 Greybox Fuzzing and Corpus Generation 2.1 Greybox Fuzzing and Corpus Generation Mu2 prunes the set of mutants to run at each fuzzing iteration using dynamic analysis of the original program’s execution in two ways: (a) sound optimizations that prune mutants which cannot be killed by a given input, and (b) aggressive optimizations that select only a bounded subset of candidate mutants to run in each iteration. We evaluate Mu2 on ﬁve real-world Java targets using state-of- the-art greybox fuzzer Zest [59], which is also built on top of the JQF framework, as a baseline. We also empirically evaluate 7 variants of 930 ISSTA ’23, July 17–21, 2023, Seatle, WA, USA Guiding Greybox Fuzzing with Mutation Testing Algorithm 1 Coverage-guided greybox fuzzing 1: procedure CGF(Program 푃, Set of inputs seeds, Budget 푇) 2: corpus ←seeds ⊲Initialize saved inputs 3: repeat ⊲Fuzzing loop 4: 푥←PickInput(corpus) ⊲Sample using heuristics 5: 푥′ ←MutateInput(푥) ⊲Synthesize new input 6: if running 푃(푥′) leads to a crash then 7: raise 푥′ ⊲Bug found! 8: if coverage(푃,푥′) ⊈Ð 푥∈corpus coverage(푃,푥) then 9: corpus ←corpus ∪푥′ 10: until budget 푇 11: return corpus ⊲Final corpus Algorithm 1 Coverage-guided greybox fuzzing 푃(푥) = 푃′(푥). Similar to code coverage—where 100% may not be achievable due to unreachable code—the best use of the adequacy score is as a relative measurement rather than an absolute one. One of the most mature and actively developed mutation testing frameworks, PIT [17], targets Java programs by mutating JVM bytecode. PIT’s default mutation operators include: • Conditional boundary mutator (e.g., a<b to a<=b) • Increments mutator (e.g., a++ to a--) • Invert negatives (e.g., -a to a) • Negate conditionals (e.g., a==b to a!=b) ⊲Final corpus ⊲Final corpus • Return values mutator (e.g., replacing operands in return statements with a constant such as null, 0, 1, false, etc. depending on type). If the execution of any synthesized input 푥′ causes the program to crash, then a bug is reported (Line 7). The fuzzing loop continues until a user-provided resource budget 푇runs out (Line 10), where this budget may be in terms of the number of fuzzing trials (i.e., iterations of the fuzzing loop) or in terms of wall-clock time. The corpus of fuzzer-synthesized test inputs is ﬁnally returned (Line 11) and may be used either as a regression test suite, for seeding future fuzzing campaigns, or for other applications [28, 55, 71, 72, 75]. 2.2 Mutation Testing Mutation testing (also known as a mutation analysis) is a method- ology for assessing the adequacy of a set of tests using artiﬁcially injected “bugs”, or program mutants [19, 37]. In assessing test ade- quacy [27], we are given a program 푃and a suite of passing tests 푋. The goal is to evaluate the quality of 푋by computing a score that grows monotonically [79] with additions to the set 푋. Code coverage is an example of a test adequacy criteria. In mutation testing, a set of program mutants, say Mutants(푃), is ﬁrst generated. Each mutant 푃′ ∈Mutants(푃) is a program that diﬀers from 푃in a very small way. Most commonly, mutations are replacements of program expressions. For example, an expression a+b at line 42 in 푃may be replaced with the expression a-b. We can use the notation ⟨푃, a+b, a-b, 42⟩to refer to this mutation. For purposes of this paper, we use the notation: Scope. Since there is a vast amount of literature on the many variables involved in mutation analysis, as surveyed by Papadakis et al. [63], we restrict ourselves in this paper to investigating only the aspects of combining mutation analysis with greybox fuzzing. In particular, we (1) work with the assumption that a high muta- tion score is a desirable property of a test-input corpus used for regression testing, referring the reader to several empirical stud- ies examining the relationship between mutation scores and real faults [2, 12, 15, 32, 39, 41, 64], and (2) directly use the default set of mutation operators provided by PIT (ref. Section 2.2), which have been chosen based on several empirical studies of eﬀectiveness, suﬃciency, and to align with developer expectations [1, 17, 45, 57]. 푃′ = ⟨푃,푒,푒′,푛⟩ to refer to a program mutant 푃′ as a modiﬁcation of program 푃 where expression 푒is replaced with 푒′ at program location 푛. The main idea is that a program mutation simulates a simple program- mer error or an artiﬁcially injected “bug”. The test suite 푋is then run on each mutant 푃′. If some test 푥∈푋 fails when run on mutant 푃′, then the mutant 푃′ is said to be killed, which we denote as Kills(푃′,푥). If the test suite 푋still passes, then the mutant 푃′ is said to survive. 3 MUTATION-ANALYSIS-GUIDED FUZZING 3 MUTATION-ANALYSIS-GUIDED FUZZING 3.1 Problem Statement and Scope 3.1 Problem Statement and Scope In this paper, we focus on the following problem: In this paper, we focus on the following problem: Can we use mutation analysis to guide greybox fuzzing in order to synthesize a test-input corpus with high mutation score? Recently, Gopinath et al. [31] have identiﬁed and discussed sev- eral challenges of combining mutation analysis with fuzzing, in- cluding (1) the strength of oracles used by the fuzzer, (2) the compu- tational expense of performing mutation analysis, (3) dealing with equivalent mutants, and (4) the lack of mutation testing frameworks that focus on fuzzers. We directly address such challenges in this paper. Oracles are discussed in Section 3.3 and performance con- cerns in Section 3.4. Our evaluation is not dependent on identifying equivalent mutants, since we only care about relative mutation scores (higher=better) rather than the exact number of mutants killed by a test-input corpus. Section 3.4.2 deals with reducing the performance impact of equivalent mutations. 2.1 Greybox Fuzzing and Corpus Generation The quality of the ﬁnal test-input corpus is often evaluated using code coverage [10, 42], though mutation scores—which we describe in the next section—have also been used [75]. deﬁned in Figure 2 and the following test method, which is written in the property-testing style using JQF’s @Fuzz annotation: deﬁned in Figure 2 and the following test method, which is written in the property-testing style using JQF’s @Fuzz annotation: 1 @Fuzz // Inputs generated using greybox fuzzing 2 void fuzzInsertionSort(int[] input) { 3 assert(isSorted(Sort.insertionSort(input))); 4 } 1 @Fuzz // Inputs generated using greybox fuzzing 2 void fuzzInsertionSort(int[] input) { 3 assert(isSorted(Sort.insertionSort(input))); } We then determine whether the input 푥′ is the ﬁrst input to kill some mutant 푃′. If 푃′ is killed by 푥′ and 푃′ has not previously been killed by any input in the corpus (Lines 9 and 14), then we add 푥′ to the corpus (Line 10). Broadly, this algorithm saves fuzzer-generated inputs if they increase either code coverage or mutation score. Addi- tionally, inputs that increase mutation score are marked as favored, giving them more energy to be picked for fuzzing (Line 4). As be- fore, the ﬁnal corpus of fuzzer-generated inputs is returned as the result (Line 12). For Mu2, we could use this property test as an oracle. Con- sider the following examples, using the notation introduced in Section 2.2: executing mutant 푃′ 1 = ⟨Sort, i+1, i, 10⟩with input array 푥 = [3, 2, 1] would result in an uncaught IndexOutOfBoundsException (-1) on line 10, triggering a failure via the implicit oracle. Additionally, executing 푃′ 2 = ⟨Sort, i>=0, i>0, 5⟩with 푥would result in an assertion failure in the property test because the result of 푃′ 2(푥) would be the array [3, 1, 2], which is not sorted. So, both mutants 푃′ 1 and 푃′ 2 would get killed by the fuzzer if it discovers such an input. We have implemented Algorithm 2 for fuzzing Java programs by integrating PIT [17] into JQF [58]. We call this system Mu2, since it combines Mutation-based Greybox Fuzzing with Mutation Testing. We chose PIT and JQF because of their maturity, extensibility, and their common target platform. As described in Section 2.2, PIT is an actively developed mutation testing framework that operates on JVM bytecode. The JQF framework [58] was originally designed for coverage-guided property-based testing, which is a structure- aware variant of greybox fuzzing (ref. Section 2.1) and instruments JVM bytecode for collecting code coverage. JQF also has a highly extensible design for creating pluggable guidances, which supports rapid prototyping of new fuzzing algorithms [43, 55, 56, 59, 69, 75, 83]. deﬁned in Figure 2 and the following test method, which is written in the property-testing style using JQF’s @Fuzz annotation: Unfortunately, the property test is not a complete oracle in that it does not fully specify the expected behavior of the sort function. Consider a third mutant 푃′ 3 = ⟨Sort, arr[i], 1, 7⟩, which assigns a constant to every array element at line 7. This is clearly a bug in insertion sort, yet the output is always sorted. For example, when 푥 = [3, 2, 1], the result of 푃′ 3(푥) is [1, 1, 1]. Such a mutant would incorrectly survive on any input the fuzzer generates. Writing a complete oracle for testing insertion sort is possible, but quite cumbersome. In general, this is a hard problem [6]. For many applications, a complete oracle would need to be as complex (or in some cases exactly the same) as the original program itself. In Mu2, Mutants(푃) includes all of PIT’s default expression mutation operators (ref. Sections 2.2 and 3.1). For heuristics such as PickInput and MutateInput, Mu2 reuses the logic and code from Zest [59], which we also use as a baseline for evaluation (Section 4). In Mu2, we use the well-known concept of diﬀerential testing to deﬁne our oracle. In diﬀerential testing [21, 53], diﬀerent im- plementations of a program that are expected to satisfy the same speciﬁcation are executed on a single input, and their results are compared to identify discrepancies. In Mu2, our diﬀerent "imple- mentations" are the original program and program mutants; any discrepancy between the original program output and a mutant’s output leads to that mutant being killed. 3.2 The Mu2 Framework To address our problem statement, we present the mutation- analysis-guided greybox fuzzing technique in Algorithm 2. This is an extension of Alg. 1, with changes highlighted in grey. The key additions of this algorithm are in evaluating whether a fuzzer- generated input 푥′ should be saved to the corpus. The function ProgMuts2Run (Line 8) returns a set of program mutants to eval- uate with input 푥′. For now, assume it to return Mutants(P) as deﬁned in Section 2.2, though we will reﬁne this in Section 3.4.2. Ideally, we want our tests to be able to identify “bugs” and so we hope to have tests that fail on each mutant 푃′. So, the adequacy of test suite 푋is deﬁned by the mutation score, which is computed as the fraction of mutants killed: \|{푃′∈Mutants(푃) \|∃푥∈푋:kills(푃′,푥)}\| \|Mutants(푃) \| . \| ( ) \| In general, a mutation score of 100% is rarely achievable because some mutants 푃′ may actually be equivalent to 푃—that is, ∀푥: 931 Vasudev Vikram, Isabella Laybourn, Ao Li, Nicole Nair, Kelton OBrien, Rafaello Sanna, and Rohan Padhye Vasudev Vikram, Isabella Laybourn, Ao Li, Nicole Nair, Kelton OBrien, Rafaello Sanna, and Rohan Padhye ISSTA ’23, July 17–21, 2023, Seatle, WA, USA Algorithm 2 Mutation-analysis-guided fuzzing. Changes to Alg. 1 are highlighted. 1: procedure Mu2 (Program 푃, Set of inputs seeds, Budget 푇) 2: corpus ←seeds 3: repeat 4: 푥←PickInput(corpus) 5: 푥′ ←MutateInput(푥) 6: if coverage(푃,푥′) ⊈Ð 푥∈corpus coverage(푃,푥) then 7: corpus ←corpus ∪푥′ 8: for all 푃′ ∈ProgMuts2Run(푃, corpus,푥′) do 9: if kills(푃′,푥′) ∧푃′ ∉killed(푃, corpus) then 10: corpus ←corpus ∪푥′ 11: until budget 푇 12: return corpus 13: function killed(Program 푃, Set of inputs X) 14: return {푃′ \| 푃′ ∈Mutants(푃) ∧∃푥∈X : kills(푃′,푥)} 1 class Sort { 2 static int[] insertionSort(int[] arr) { 3 for (int j = 1; j < arr.length; j++) { 4 int key = arr[j], i = j-1; 5 while (i >= 0 && // P'2 changes `>=` to `>` 6 key < arr[i]) { 7 arr[i+1] = arr[i]; // P'3 sets RHS to `1` 8 i = i-1; // P'4 removes `-1` 9 } 10 arr[i+1] = key; // P'1 removes `+1` 11 } return arr; 12 }} Algorithm 2 Mutation-analysis-guided fuzzing. Changes to Alg. 1 are highlighted. Figure 2: Java program that implements insertion sort, anno- tated with four sample program mutants. 3.2 The Mu2 Framework Figure 2: Java program that implements insertion sort, anno- tated with four sample program mutants. deﬁned in Figure 2 and the following test method, which is written in the property-testing style using JQF’s @Fuzz annotation: 1The Closure Compiler benchmark was too large to run without the execution and infection optimizations, so we did not include the speedups in this table. Figure 3: A Mu2 diﬀerential mutation test driver and com- parison method for the insertionSort method (Fig. 2). Algorithm 3 Logic for determining which mutants to run in a given iteration of the fuzzing loop (Alg. 2) Algorithm 3 Logic for determining which mutants to run in a given iteration of the fuzzing loop (Alg. 2) 1: function ProgMuts2Run(Program 푃, Old inputs corpus, New input 푥) 2: surviving ←mutants(푃) \ killed(푃, corpus) 3: killable ←{푃′ = ⟨푃,푒,푒′,푛⟩\| (푃′ ∈surviving) ∧ 4: (푛∈coverage(푃,푥)) ∧(infect(푃,푒,푒′,푥))} 5: if AGGRESSIVE_OPT is conﬁgured then 6: return filter(killable, AGGRESSIVE_OPT ) 7: return killable is shown in Figure 3. The @Diff method runInsertionSort returns an output value of type int[]. The user-deﬁned com- parison method checkEq simply determines if the output ar- rays are equal. If unspeciﬁed, the @Compare function defaults to the java.lang.Objects.equals() method. Our interface is general enough to support complex diﬀerential testing oracles such as the ones used in CSmith [80]. With diﬀerential mutation testing, we are able to kill mutants such as 푃′ 3 described above with an input like [3, 2, 1], where the output of insertionSort on the original program—[1, 2, 3]—is not equal to the output of the mutant—[1, 1, 1]. We can now precisely deﬁne Kills(푃′,푥) which was referenced in Algorithm 2. Given a mutant 푃′ = ⟨푃,푒,푒′,푛⟩and an input 푥, With diﬀerential mutation testing, we are able to kill mutants such as 푃′ 3 described above with an input like [3, 2, 1], where the output of insertionSort on the original program—[1, 2, 3]—is not equal to the output of the mutant—[1, 1, 1]. methods are expected to be self-contained and not depend on global state. Like JQF and Zest, Mu2 is designed to work in a single JVM. We can now precisely deﬁne Kills(푃′,푥) which was referenced in Algorithm 2. Given a mutant 푃′ = ⟨푃,푒,푒′,푛⟩and an input 푥, Kills(푃′,푥) returns true iﬀ: state. Like JQF and Zest, Mu2 is designed to work in a single JVM. Mu2 thus adopts a diﬀerent strategy than PIT and takes advan- tage of the Java class-loader mechanism to load and run program mutants within the same JVM, essentially by having copies of the entire class hierarchy (one per mutant) in memory at the same time. First, a CoverageClassLoader (CCL) is responsible for loading the original target program 푃and collecting code coverage using on-the-ﬂy instrumentation. For diﬀerential testing, the CCL-loaded classes compute the ground-truth outcome 푃(푥). 3.3 Oracle: Diﬀerential Mutation Testing One challenge of mutation-analysis-guided fuzzing is determining whether a program mutant is killed by a particular input. This corresponds to the kills function invoked in line 9 of Algorithm 2. One challenge of mutation-analysis-guided fuzzing is determining whether a program mutant is killed by a particular input. This corresponds to the kills function invoked in line 9 of Algorithm 2. In mutation testing, a program mutant 푃′ is considered killed if any test in the test suite fails. The logic that determines whether a test passes or fails is known as the test oracle. In mutation testing, a program mutant 푃′ is considered killed if any test in the test suite fails. The logic that determines whether a test passes or fails is known as the test oracle. To support the comparison of outputs, we create a diﬀerential mutation testing framework. This allows for (1) output values to be returned from a fuzzing driver (as opposed to the void returns used by conventional property testing methods) and (2) a user- deﬁned comparison function for specifying how outputs from the original program and a program mutant should be compared. An example of diﬀerential mutation testing methods in our framework Greybox fuzzing generally relies on implicit oracles, which aim to detect anomalous behavior such as crashes or uncaught excep- tions, or property tests, which assert a predicate over the output of some computation. For example, consider the insertion sort method 932 ISSTA ’23, July 17–21, 2023, Seatle, WA, USA Guiding Greybox Fuzzing with Mutation Testing Guiding Greybox Fuzzing with Mutation Testing Table 1: Geometric mean of speedups achieved by the execu- tion and infection based optimizations (Alg. 3, Line 4) from the PIE model [38] across 10 repetitions of 3 hours each1. 1 @Diff // inputs generated by Mu2 2 int[] runInsertionSort(int[] input) { 3 return Sort.insertionSort(input); 4 } 5 @Compare // outputs compared with mutant 6 boolean checkEq(int[] outOrig, int[] outMut) { 7 return Arrays.equals(outOrig, outMut); 8 } 1 @Diff // inputs generated by Mu2 2 int[] runInsertionSort(int[] input) { 3 return Sort.insertionSort(input); 4 } 5 @Compare // outputs compared with mutant 6 boolean checkEq(int[] outOrig, int[] outMut) { 7 return Arrays.equals(outOrig, outMut); 8 } Figure 3: A Mu2 diﬀerential mutation test driver an Mean Speedup From: Execution Opt. Infection Opt. ChocoPy 3.6× 7.4× Gson 18.2× 23.2× Jackson 60.5× 77.4× Tomcat 13.6× 23.8× Figure 3: A Mu2 diﬀerential mutation test driver and com- parison method for the insertionSort method (Fig. 2). Second, a family of MutationClassLoaders (MCL) are used to load program mutants; one MCL per mutant 푃′ = ⟨푃,푒,푒′,푛⟩. When a mutant test program is loaded by the MCL, it performs on-the-ﬂy bytecode instrumentation exactly at location 푛, replacing expression 푒with 푒′ and loading the rest of the program without changing semantics. The MCL adds instrumentation at backward jumps (i.e., loops) in order to detect timeouts and exit test execution cleanly if necessary. (1) 푃(푥) = 푦∧푃′(푥) = 푦′ ∧¬Compare(푦,푦′), where Compare is the user-deﬁned @Compare method (e.g., checkEq in Fig- ure 3) or Object.equals() if one is not deﬁned; or (2) 푃(푥) = 푦but executing 푃′(푥) results in an uncaught run-time exception being thrown; or (3) Executing 푃′(푥) takes longer than a predeﬁned TIMEOUT. The timeout is required for killing mutants such as 푃′ 4 = ⟨Sort, i-1, i, 8⟩, which eﬀectively removes the decrement of i, leading to an inﬁnite loop on the input [3, 1, 2]. We evaluate the improvement in completeness using the diﬀer- ential oracle over the greybox fuzzing implicit oracle in Section 4.4. 4 EVALUATION Table 1 shows the results of prelim- inary experiments on 4 benchmarks included in our evaluations in Section 4 to validate these optimizations; clearly, they improve performance signiﬁcantly. (2) Gson [29] JSON Parser (~26K LoC): The test driver parses a input JSON string and returns a Java object output. (3) Jackson [22] JSON Parser (~49K LoC): The test driver acts similar to that of Gson. (4) Apache Tomcat [3] WebXML Parser (~10K LoC): The test driver parses a string input and returns the WebXML repre- sentation of the parsed output. (5) Google Closure Compiler [30] (~250K LoC): The test driver (reused from [59] and [75]) takes in a JavaScript program and performs source-to-source optimizations. It then returns the optimized JavaScript code. We note that all the pruning methods mentioned above are sound optimizations: a mutant is pruned only if it is guaranteed to sur- vive when executed. Eﬀectively, we are pruning mutants that are equivalent modulo inputs [47]. Mutation selection. Following previous work on semantic fuzzing [59, 75], we ﬁlter on package names to identify classes relating to the core logic of the program under test. The mutation operators are then applied on these classes. We use the same gen- erators, oracles, and ﬁlters for both Zest and Mu2. All of the test drivers return objects that override Object.equals, and were thus properly compared by the diﬀerential oracle. 3.4.3 Aggressive Mutant Selection Optimizations. While the execu- tion and infection optimizations signiﬁcantly improve the overall throughput of Mu2, the 푀factor in Equation 1 still grows linearly with the size of the program (more code = more mutants). We can be aggressive about reducing 푀by attempting to bound it by a constant 푘, at the risk of potentially missing out on analyzing some mutants that could have been killed by a given input. We call these aggressive optimizations. We use the function filter in Algorithm 3 (Line 6) to optionally apply a selection strategy [66, 73] that returns a bounded subset of the killable mutants. We have implemented two types of ﬁlters in Mu2: Duration. Following best practices [42], we use a time bound of 24 hours for each experiment. Repetitions. To account for the randomness in fuzzing, we run each experiment 20 times and report statistics. Metrics. For our evaluations, we compute the branch coverage and mutation scores across each fuzzer-generated test-input corpus. 4 EVALUATION We evaluate Mu2 on 5 diﬀerent Java program benchmarks, using state-of-the-art coverage-guided fuzzer Zest [59] as the baseline. We structure our evaluation around four research questions: RQ1: Does mutation-analysis guidance produce a higher quality test-input corpus than coverage-only feedback in greybox fuzzing? RQ2: How do the performance optimizations impact the quality of the test-input corpus produced by mutation-analysis guidance? RQ3: How does the reliability of killing nontrivial mutants diﬀer between mutation-analysis guidance and coverage guidance? RQ4: How much stronger is the diﬀerential mutation testing oracle than the implicit oracle? We evaluate Mu2 on 5 diﬀerent Java program benchmarks, using state-of-the-art coverage-guided fuzzer Zest [59] as the baseline. We structure our evaluation around four research questions: So, we begin by applying the following conditions for a given 푃′ = ⟨푃,푒,푒′,푛⟩, which are shown in Algorithm 3, lines 2–4: (1) If 푃′ ∈killed(푃, corpus), then 푃′ does not need to be exe- cuted for any future inputs. (2) If the program mutant 푃′ applies a mutation to a program location 푛, but 푛is not covered when executing the original program on푥, then 푃′ cannot be killed by푥. This corresponds to execution-based pruning in the PIE model [38]. (3) If we can guarantee that all dynamic evaluations of 푒during the execution of 푃on 푥are equivalent to the corresponding evaluations of mutated expression 푒′, then 푃′ cannot be killed by 푥. This corresponds to infection-based pruning in the PIE model [38], which we implemented as a dynamic analysis of the execution of the original program 푃(푥). Benchmarks. We consider ﬁve real-world Java programs:2 Benchmarks. We consider ﬁve real-world Java programs:2 (1) ChocoPy [7, 61] reference compiler (~6K LoC): The test driver (reused from [75]) reads in a program in ChocoPy (a statically typed dialect of Python) and runs the semantic analysis stage of the ChocoPy reference compiler to return a type-checked AST object. The last two strategies from the PIE model require additional over- head when executing 푥: (1) the execution-based pruning depends on coverage instrumentation, and (2) infection-based pruning requires evaluating and comparing the mutation expression 푒each time that it is executed by 푥. Referring to Equation 1, the optimization results in a trade-oﬀfor trialTime due to the increase in timeorig and decrease in the number of mutants to run 푀. However, we ﬁnd this is quite beneﬁcial overall. 3.4 Performance Further, assuming that fuzz tests do not aﬀect global state, Mu2 loads only one copy of each library class (deﬁned as classes outside a speciﬁed package identifying the target application as long as they and their transitive dependencies do not reference any application class) using a common SharedClassLoader—this dramatically reduces memory pressure when mutating large programs. The biggest challenge with incorporating mutation testing inside a fuzzing loop is performance. Given its need to execute many mutants on each iteration, mutation testing is in general a very expensive technique [63], so scaling Mu2 to real-world software is a non-trivial task. Two aspects of improving scalability are: (1) reducing the average time required to execute each program mutant, and (2) reducing the number of program mutants that must be evaluated at each iteration of the fuzzing loop. To validate our design, we ran an informal preliminary exper- iment of performing mutation analysis with PIT and Mu2’s in- memory set-up on a ﬁxed corpus of seed inputs for the Google Closure Compiler [30]. In the steady state (after the ﬁrst 8 inputs), Mu2’s in-memory analysis runs with a 9.6× speed-up over PIT. 3.4.1 Improving Performance of Mutant Execution. When running a mutation testing tool such as PIT [17], each mutant and test is run in a diﬀerent JVM. For general mutation testing, this is ideal because it simpliﬁes managing multiple copies of the same program (sans mutations), and prevents global state changes from one program mutant aﬀecting the state of another program mutant. However, this is not necessary for Mu2. For in-process fuzzing, test driver 3.4.2 Reducing the Number of Mutants to Run in the Fuzzing Loop. For each trial—i.e., iteration of the fuzzing loop—(1) the input must be executed once by the original program and (2) the input must be executed by each mutant. Thus, we can model the time required to execute each trial as the following: (1) trialTime = timeorig + 푀∗avgTimemut (1) where 푀= \|ProgMuts2Run(푃, corpus,푥)\| as per Algorithm 2. 933 ISSTA ’23, July 17–21, 2023, Seatle, WA, USA Vasudev Vikram, Isabella Laybourn, Ao Li, Nicole Nair, Kelton OBrien, Rafaello Sanna, and Rohan Padhye Observe that the time per trial scales linearly with 푀. We can improve the fuzzing throughput (i.e., the number of trials executed per unit time) directly by reducing 푀. 2While we note lines of code (LoC) for completeness, only a fraction of this code is reachable from fuzz drivers. Fig. 5 indicates actual code coverage. 3.4 Performance From Algorithm 2 (Lines 9– 10), we can see that we only care about executing a program mutant if it will help us determine if a given input is the ﬁrst input to kill it. We can therefore reduce 푀by dynamically pruning mutants whose execution will necessarily lead to Line 9 evaluating to false. fuzzing campaign. This is a novel reduction strategy designed speciﬁcally for the fuzzing loop. Section 4.2 evaluates the impact of these aggressive optimizations. p ) y y g g ( 10), we can see that we only care about executing a program mutant if it will help us determine if a given input is the ﬁrst input to kill it. We can therefore reduce 푀by dynamically pruning mutants whose execution will necessarily lead to Line 9 evaluating to false. 4 EVALUATION The number of branches covered by Zest (used to normalize) is listed below each target. Error bars represent 95% conﬁdence intervals. signiﬁcant increases in all three. Additionally, Figure 5 shows equiv- alent branch coverage between Zest and Mu2 for these benchmarks. For the Tomcat WebXML parser, the number of killed mutants saturated at 239 in almost all of the repetitions of the fuzzing cam- paigns. For the Closure Compiler, our largest benchmark, the Mu2- Default corpora achieve, on average, approximately 17% less branch coverage than Zest (shown in Figure 5). This is likely due to the performance overhead of running mutation analysis for a large benchmark, and also likely accounts for the Zest corpora on aver- age killing 12 more mutants than Mu2-Default, as covering code is a necessary condition for killing mutants in that part of the code. This suggests Mu2-Default may not scale well to very large programs. One way to mitigate this slowdown is to add mutation-analysis feedback to coverage-guided fuzzing later in the campaign. The Mu2-Split variant utilizes coverage-only feedback for the ﬁrst half of the campaign (which is very eﬃcient) and then introduces ex- pensive mutation-analysis feedback for the second half. This is based on an idea by Gopinath et al. [31], who suggested saturating coverage before adding mutation analysis to the fuzzing loop. The Mu2-Split-generated corpora show statistically signiﬁcant increases in mutation score over Zest for the ﬁrst 4 benchmarks (Fig. 4), al- though the eﬀect for Tomcat is very small. There is also a major improvement over Mu2-Default in the Closure benchmark; Mu2- Split is able to bridge the gap in coverage (Fig. 5) and mutation scores (Fig. 4) that Mu2-Default had with the Zest baseline. Figure 5: Branch coverage across all benchmarks normal- ized to the mean coverage achieved by Zest. The number of branches covered by Zest (used to normalize) is listed below each target. Error bars represent 95% conﬁdence intervals. nontrivial mutants across the repetitions. When reporting statistical signiﬁcance, a Mann-Whitney-U test was performed with 훼= 0.05. Default variant. Unless explicitly qualiﬁed with an aggressive optimization strategy, the default variant of Mu2 used in our evalu- ation only uses sound optimizations described in Section 3.4.2. Reproducibility and Data Availability. We have published a repli- cation package and evaluation data at: https://doi.org/10.5281/ zenodo.7647828. The evaluation data contains logs of fuzzing cam- paigns used to generate all evaluation ﬁgures and tables [74]. 4 EVALUATION We report mutation scores as the absolute number of mutants killed instead of as a fraction (ref. Section 2.2), since we only care about comparing these numbers across fuzzing variants, and since the denominator is meaningless when considering a single test entry point. We additionally compute the kill frequency of each of the (1) 푘-Random Mutant Filter: For each generated input,푘mutants are randomly sampled from the killable set in Alg. 3. (2) 푘-Least-Executed Mutant Filter: For each generated input, the killable mutants are sorted by the number of times they have been executed on previous inputs. The ﬁrst 푘mutants are then selected. The goal is to prioritize executing mutants that have not been tested as frequently during the fuzzing 934 ISSTA ’23, July 17–21, 2023, Seatle, WA, USA Guiding Greybox Fuzzing with Mutation Testing Zest Mu2- Default Mu2- Split Mu2- OPT 270 275 280 285 290 Killed Mutants ChocoPy Zest Mu2- Default Mu2- Split Mu2- OPT 280 290 300 310 Gson Zest Mu2- Default Mu2- Split Mu2- OPT 340 360 380 400 Jackson Zest Mu2- Default Mu2- Split Mu2- OPT 225 230 235 Tomcat Zest Mu2- Default Mu2- Split Mu2- OPT 230 240 250 260 270 Closure Figure 4: Box plots showing the number of killed mutants by Zest and Mu2-generated test corpora across 20 repetitions of 24-hour fuzzing campaigns (higher is better). Mu2-Split and Mu2-OPT are two variants of Mu2 detailed in Section 4.1. Zest Mu2- Default Mu2- Split Mu2- OPT 5 0 5 Tomcat Zest Mu2- Default Mu2- Split Mu2- OPT 230 240 250 260 270 Closure Zest Mu2- Default Mu2- Split Mu2- OPT 340 360 380 400 Jackson Zest Mu2- Default Mu2- Split Mu2- OPT 0 0 0 0 0 Closure Figure 4: Box plots showing the number of killed mutants by Zest and Mu2-generated test corpora across 20 repetitions of 24-hour fuzzing campaigns (higher is better). Mu2-Split and Mu2-OPT are two variants of Mu2 detailed in Section 4.1. ChocoPy (4856) Gson (645) Jackson (2217) Tomcat (990) Closure (30792) Benchmark 0.00 0.25 0.50 0.75 1.00 Coverage Relative to Zest Branch Coverage Zest Mu2-Default Mu2-Split Mu2-OPT ChocoPy (4856) Gson (645) Jackson (2217) Tomcat (990) Closure (30792) Benchmark 0.00 0.25 0.50 0.75 1.00 Coverage Relative to Zest Branch Coverage Zest Mu2-Default Mu2-Split Mu2-OPT Figure 5: Branch coverage across all benchmarks normal- ized to the mean coverage achieved by Zest. 4 EVALUATION Another method of scaling Mu2 is to apply the aggressive opti- mizations detailed in Section 3.4.3. Mu2-OPT is a particular vari- ant we chose that applies the k-Least-Executed ﬁlter with 푘= 10 mutants. The Mu2-OPT generated corpus similarly achieves sta- tistically signiﬁcant increases in mutation scores across the ﬁrst four benchmarks over Zest, with up to 20% increase in the Jackson JSON parser (Fig. 4). There is no signiﬁcant diﬀerence between the mutation scores of Mu2-OPT and Zest on the Closure Compiler. Mu2-OPT achieves slightly less coverage than Zest on two bench- marks (ChocoPy and Closure) and more on one (Jackson)—however, the diﬀerences are fairly small (below 2%). 4.1 RQ1: Test-Input Corpus Quality 280 290 300 Gson 225 230 235 Tomcat 280 285 290 Killed Mutants ChocoPy Zest Mu2- Default Mu2- Split Mu2- OPT ChocoPy 864 ± 34 711 ± 47 725 ± 36 746 ± 40 (18.6 s ± 2.1 s) (9.8 s ± 0.8 s) (11.7 s ± 1.3 s) (10.4 s ± 1.0 s) Gson 467 ± 18 461 ± 17 469 ± 15 489 ± 21 (1.7 s ± 0.1 s) (1.7 s ± 0.1 s) (1.7 s ± 0.0 s) (1.7 s ± 0.0 s) Jackson 598 ± 19 655 ± 16 641 ± 19 673 ± 15 (2.4 s ± 0.1 s) (2.4 s ± 0.1 s) (2.4 s ± 0.0 s) (2.4 s ± 0.1 s) Tomcat 138 ± 7 122 ± 6 136 ± 6 171 ± 5 (2.6 s ± 0.1 s) (2.5 s ± 0.1 s) (2.6 s ± 0.1 s) (2.7 s ± 0.1 s) Closure 4885 ± 205 1075 ± 219 4044 ± 146 4037 ± 192 (554 s ± 82 s) (58 s ± 13 s) (360 s ± 27 s) (353 s ± 30 s) 392.5 395.0 397.5 400.0 402.5 405.0 407.5 Jackson 230 240 250 260 270 280 Closure 280 285 290 killed Table 3: Geometric mean of speedups achieved by each ag- gressively ﬁltered variant of Mu2 over Mu2-Default. 20/10/5 refer to the sizes of the ﬁltered subset of mutants. Random (20/10/5) LeastExecuted (20/10/5) ChocoPy 1.1/1.7/2.8× 1.1/1.6/2.5× Gson 0.9/1.1/1.3× 1.0/1.2/1.3× Jackson 1.0/1.1/1.3× 1.1/1.0/1.2× Tomcat 3.4/3.5/8.2× 2.3/6.0/7.9× Closure 10.4/13.8/21.3× 13.8/19.2/24.9× Figure 6: Box plots showing number of killed mutants by each aggressively optimized variant of Mu2 and the default, across 20 repetitions of 24 hour campaigns (higher is better). produced by Zest. Thus, mutation-analysis-guided fuzzing is able to produce a higher quality test-input corpus and can be feasibly used for regression testing. Closure benchmark. This makes sense, as the main purpose of aggressive optimizations is to enable scaling to large programs. We believe that an aggressively optimized version of mutation- analysis-guided fuzzing can be used as a replacement for coverage- guided fuzzing if the goal is to produce a test input corpus with high mutation score. Mu2-OPT provides an improvement for 4 bench- marks and scales to the largest target without paying a performance penalty. 4.1 RQ1: Test-Input Corpus Quality Due to the aggressive nature of the mutant ﬁltering, it is possible that input candidates that do kill mutants are not saved simply because those killable mutants were ﬁltered. To determine whether the speedup actually results in a test-input corpus with higher muta- tion score, we must also measure the impact of these optimizations on the mutation score of the generated corpus. Figure 6 displays the mutation scores of all of the variants for each of the 5 benchmarks. At least one optimized variant was bet- ter than the default in all benchmarks. Somewhat surprisingly, we observe similar mutation scores between the Mu2-LeastExecuted-푘 and Mu2-Random-푘variants for the same value of 푘in the ﬁrst four benchmarks. The one exception is Closure Compiler, where Mu2-LeastExecuted-10 achieves a statistically signiﬁcantly higher mutation score than Mu2-Random-10. Again, the eﬀect of aggres- sive optimizations is most pronounced in the largest target. 4.1 RQ1: Test-Input Corpus Quality Does mutation-analysis guidance produce a higher quality test- input corpus than coverage-only feedback in greybox fuzzing? RQ1 focuses on evaluating mutation-analysis-guided fuzzing with a ﬁxed time budget. Higher mutation score from the Mu2- produced corpus and comparable coverage results would demon- strate that mutation-analysis can be used as an oﬀ-the-shelf re- placement for coverage-only guidance. We ﬁrst discuss results for Mu2-Default, then evaluate two variants against the Zest baseline. Figure 4 visualizes the mutation scores for each fuzzer-generated corpus. The default mutation-analysis guidance (Mu2-Default) is able to produce a corpus with higher mutation scores than coverage- only feedback in the ﬁrst three benchmarks, achieving statistically RQ1 focuses on evaluating mutation-analysis-guided fuzzing with a ﬁxed time budget. Higher mutation score from the Mu2- produced corpus and comparable coverage results would demon- strate that mutation-analysis can be used as an oﬀ-the-shelf re- placement for coverage-only guidance. We ﬁrst discuss results for Mu2-Default, then evaluate two variants against the Zest baseline. We are also curious about whether the additional saving of mutant-killing inputs in Mu2 may bloat the size of the generated test-input corpus, impacting its use in regression testing. Table 2 displays the average sizes and runtimes for each fuzzer-generated corpus and show that no such bloat occurs in Mu2. While there are some diﬀerences in the number of test inputs, the runtime of the Mu2-produced corpora are not signiﬁcantly higher than those Figure 4 visualizes the mutation scores for each fuzzer-generated corpus. The default mutation-analysis guidance (Mu2-Default) is able to produce a corpus with higher mutation scores than coverage- only feedback in the ﬁrst three benchmarks, achieving statistically 935 ISSTA ’23, July 17–21, 2023, Seatle, WA, USA Vasudev Vikram, Isabella Laybourn, Ao Li, Nicole Nair, Kelton OBrien, Rafaello Sanna, and Rohan Padhye 280 285 290 Killed Mutants ChocoPy 280 290 300 Gson 392.5 395.0 397.5 400.0 402.5 405.0 407.5 Jackson 225 230 235 Tomcat 230 240 250 260 270 280 Closure 280 285 290 killed Variant (in order) Mu2-Default Mu2-Random-20 Mu2-LeastExecuted-20 Mu2-Random-10 Mu2-LeastExecuted-10 Mu2-Random-5 Mu2-LeastExecuted-5 Figure 6: Box plots showing number of killed mutants by each aggressively optimized variant of Mu2 and the default, across 20 repetitions of 24 hour campaigns (higher is better). Table 2: Average number of test inputs (and average runtime, in parentheses below) of fuzzer-generated corpora. Corre- sponding standard deviations also listed. 4.4 RQ4: Diﬀerential Mutation Testing How much stronger is the diﬀerential mutation testing oracle than the implicit oracle? Described in Section 3.3, the diﬀerential mutation testing oracle is responsible for determining whether an input kills a mutant by comparing the outputs of the executions. We contrast it with the incomplete greybox fuzzing implicit oracle, which only detects un- caught exceptions or failed property checks. To study the strength of the diﬀerential oracle, we evaluate the improvement in the num- ber of killed mutants over the implicit oracle. Mu2-OPT is able to achieve a signiﬁcantly higher kill frequency of nontrivial mutants in ChocoPy and Jackson. In fact, there are 29 mutants in Jackson that are killed during all repetitions of Mu2-OPT and zero repetitions of Zest. This is a strong indication that mutation-analysis feedback can consistently discover mutant- killing inputs that coverage-only feedback is incapable of ﬁnding. For the Gson parser, there are 22 vs. 24 nontrivial mutants killed more reliably by Zest and Mu2-OPT respectively, though the X- axis values are generally higher for Mu2-OPT. For Closure, there are over 60 mutants killed by at least one more repetition of Mu2- OPT compared to the 4 by Zest. Overall, Mu2-OPT is able to kill nontrivial mutants more reliably than Zest. Figure 8 shows the diﬀerence in mutant kills across the bench- marks with the two types of oracles. The diﬀerential oracle is able to kill a signiﬁcantly higher number of mutants across all 5 bench- marks, with an average increase of 25%. In the ChocoPy benchmark, over 85 more mutants are caught! This is because certain mutants are unkillable by the implicit oracle due to their eﬀect on program behavior. We describe one of these mutants below. For brevity, we describe the code functionality, omitting the actual code snippet. We also note that Figure 7 provides some insight into the di- versity of mutants, particularly redundant mutants. By deﬁnition, redundant mutants are grouped together in the same bars since they are always killed at the same frequency. Flattening the size of each bar to 1 removes at least all redundant mutants and acts as a lower bound on the number of nonredundant mutants. The ChocoPy type-checker has a function to check that the left and right operand types of an expression match when using the “+" operator. 4.2 RQ2: Aggressive Optimizations -20 -15 -10 -5 0 5 10 15 20 0 5 10 15 20 Gson -20 -15 -10 -5 0 5 10 15 20 0 2 4 6 8 10 ChocoPy Mu2-Random-5, and Mu2-LeastExecuted-5 kill ∼15 more mutants on average than Mu2-Default. We found that Mu2-LeastExecuted-10 and Mu2-LeastExecuted-5 were the strongest variants, as they had a statistically signiﬁcant increase in mutation score over Mu2-Default in the most bench- marks (3 out of 5) out of all variants. There was no signiﬁcant diﬀerence in mutation scores between these two variants in any benchmarks, so we arbitrarily picked Mu2-LeastExecuted-10 as the optimized version of mutation-analysis-guided fuzzing (Mu2-OPT) in our evaluation of RQ1 and RQ4. We do however note for future practitioners that the best aggressively optimized variant of Mu2 may change depending on the target program. -20 -15 -10 -5 0 5 10 15 20 0 10 20 30 40 Jackson -20 -15 -10 -5 0 5 10 15 20 0 1 2 3 4 5 6 Tomcat 4.3 RQ3: Nontrivial Mutants -20 -15 -10 -5 0 5 10 15 20 Difference in Kill Frequency (bars further right indicate higher reliability of Mu2) 0 10 20 30 40 50 Number of Mutants Closure Mu2-OPT Zest How does the reliability of killing nontrivial mutants diﬀer between mutation-analysis guidance and coverage guidance? How does the reliability of killing nontrivial mutants diﬀer between mutation-analysis guidance and coverage guidance? Not all mutants are equal—some mutants are easier to kill than others. We deﬁne a mutant 푃′ = ⟨푃,푒,푒′,푛⟩as trivial if it is killed by the ﬁrst input that executes 푛in every experiment (this is the dynamic version of Kaufman et al.’s deﬁnition [40]). Since trivial mutants are killed as soon as the corresponding code is covered, conventional coverage-guided fuzzing like Zest suﬃces to capture them. On the other hand, since nontrivial mutants may or may not be killed even after the mutated expression is covered, we are interested to know whether these get killed based on pure luck or whether these get killed reliably across repetitions potentially due to the guidance in the fuzzing algorithm. We measure reliability by counting the number of repetitions in which each mutant is killed. In particular, we study the diﬀerence in reliability of killing nontrivial mutants between Zest and the best variant of Mu2. Figure 7: Histogram of diﬀerence in kill-rate of nontrivial mutants between Zest and Mu2-OPT over 20 experiments. X-axis is the diﬀerence in repetitions (ranging from -20 to 20), and Y-axis is the number of mutants. Larger positive dif- ferences (right) are better for Mu2-OPT, and larger negative diﬀerences (left) are better for Zest. Figure 7 is a histogram showing the diﬀerence in kill rate of nontrivial mutants between Mu2-OPT and Zest. The values on the right side (green) correspond to mutants killed more reliably by Mu2-OPT than Zest. For the sake of visualization, the mutants with no diﬀerence in kill rate (X-axis value 0) are excluded from the charts. 4.2 RQ2: Aggressive Optimizations How do the performance optimizations impact the quality of the test-input corpus produced by mutation-analysis guidance? This RQ focuses on understanding the beneﬁt of the aggressive optimizations in mitigating the scalability concerns of Mu2-Default. We created variants Mu2-LeastExecuted-푘and Mu2-Random-푘, each applying the corresponding ﬁlter described in Section 3.4.3, and chose three diﬀerent values of 푘∈{5, 10, 20}. Another interesting observation is that we can visualize the trade-oﬀbetween execution speed and mutation score in the Jack- son benchmark: although the Mu2-Random-5 variant has a faster execution speed than Mu2-Random-10 (Tab. 3) due to the smaller number of mutants, the mutation score slightly decreases (Fig. 6) since the optimization might skip some mutants at the wrong time. Nonetheless, the speedup displayed by the variants for the Closure Compiler results in better test-input corpus quality. All of the Mu2 variants are able to achieve statistically signiﬁcantly higher muta- tion scores than Mu2-Default. Speciﬁcally, Mu2-LeastExecuted-10, First, we measure just the performance beneﬁt. Table 3 shows the speedups achieved—in terms of number of inputs evaluated over a 24-hour period—by each variant over Mu2-Default. The improvement for the benchmarks Gson and Jackson is relatively minor due to the already small number of mutants executed for each input after applying the execution and infection optimizations (ref. Section 3.4.2 and Table 1). However, the aggressive optimizations provide signiﬁcant improvement for the larger benchmarks, with almost 25× speedup for the Mu2-LeastExecuted-5 variant on the 936 ISSTA ’23, July 17–21, 2023, Seatle, WA, USA Guiding Greybox Fuzzing with Mutation Testing -20 -15 -10 -5 0 5 10 15 20 0 2 4 6 8 10 ChocoPy -20 -15 -10 -5 0 5 10 15 20 0 5 10 15 20 Gson -20 -15 -10 -5 0 5 10 15 20 0 10 20 30 40 Jackson -20 -15 -10 -5 0 5 10 15 20 0 1 2 3 4 5 6 Tomcat -20 -15 -10 -5 0 5 10 15 20 Difference in Kill Frequency (bars further right indicate higher reliability of Mu2) 0 10 20 30 40 50 Number of Mutants Closure Mu2-OPT Zest Figure 7: Histogram of diﬀerence in kill-rate of nontrivial mutants between Zest and Mu2-OPT over 20 experiments. X-axis is the diﬀerence in repetitions (ranging from -20 to 20), and Y-axis is the number of mutants. Larger positive dif- ferences (right) are better for Mu2-OPT, and larger negative diﬀerences (left) are better for Zest. 4.4 RQ4: Diﬀerential Mutation Testing If so, the type is returned and assigned to the corresponding expression node in the output AST; otherwise, an 937 ISSTA ’23, July 17–21, 2023, Seatle, WA, USA Vasudev Vikram, Isabella Laybourn, Ao Li, Nicole Nair, Kelton OBrien, Rafaello Sanna, and Rohan Padhye ChocoPy Gson Jackson Tomcat Closure Benchmark 0 100 200 300 400 Killed Mutants Strength of Oracle Implicit Differential Figure 8: Number of killed mutants detected by the diﬀeren- tial oracle vs. the implicit oracle across 20 repetitions of 24 hour campaigns with Mu2-Default (higher is better). Error bars represent 95% conﬁdence intervals. of mutation-analysis-guided fuzzing on mutation score and code coverage. Our implementation simply reused all the fuzzing hyperparame- ters (e.g., PickInput and MutateInput in Algorithms 1 and 2) that were set by the baseline Zest fuzzer. Tuning these heuristics could aﬀect our results, but the size of this search space is too large for us to explore systematically. We stick with the baseline-provided defaults for simplicity and make sure to use the same hyperparam- eters for both Zest and Mu2 so that our conclusions are exclusively based on the inclusion of mutation-analysis guidance in Mu2 only. Threats to external validity. Since our implementation is based on JQF [58] and PIT [17], which both target JVM bytecode, we used Zest as the baseline. We do not know if our conclusions will generalize to other programming languages or fuzzing platforms, such as the family of tools based on AFL [81] and libFuzzer [49]. The available mutation testing infrastructure for C/C++ appears to be less mature than that for Java/JVM. Another threat to exter- nal validity arises from our selection bias in choice of benchmark programs. Our targets have input and output formats which make them amenable to diﬀerential mutation testing. This is not always true for all applications that can be fuzzed—e.g., PDF viewers and other programs whose output is graphical. The study of the general test oracle problem [6] is outside the scope of this paper. Figure 8: Number of killed mutants detected by the diﬀeren- tial oracle vs. the implicit oracle across 20 repetitions of 24 hour campaigns with Mu2-Default (higher is better). Error bars represent 95% conﬁdence intervals. error message for the expression is added to the output AST error list. Consider a mutant 푃′ that modiﬁes this function to return null instead of the correct type. 5 THREATS TO VALIDITY Threats to construct validity. First, the measurement of mutation score is of course dependent on the set of mutation operators be- ing applied to generate program mutants [62]. We aim to mitigate this threat by using the default set of operators in the widely used PIT framework, as justiﬁed in Section 3.1. Second, our test oracles (ref. Section 3.3) report an outcome of TIMEOUT if a mutant execu- tion does not terminate within a predeﬁned limit. Such a bound is necessary to catch inﬁnite loops (e.g., for mutants that negate loop conditions). However, if this bound is too small, then it is possible in theory that some mutants could be marked as “killed” by a fuzzer- generated input even if their execution would eventually produce a correct output. To mitigate this threat, we compute the mutation scores for the ﬁnal test-input corpus by re-running saved inputs on all program mutants using a larger timeout. We also manually analyzed a sample of reported timeouts to conﬁrm correspondence to inﬁnite loops—we found no false kills. Greybox fuzzing for regression testing. A family of techniques have been developed for directing fuzz testing towards speciﬁc code locations [8, 13, 77] or code commits [84], which can be used for identifying regressions. However, this still requires running a full fuzzing campaign, which can take hours or days. In contrast, we focus on synthesizing a high-quality test-input corpus which can be quickly executed in CI—usually taking a few seconds or minutes—as is often already practiced (ref. Section 1). Guiding fuzzing with mutation testing. We ﬁrst proposed the idea of using mutation testing to augment greybox fuzzing in a student research competition [46]; independently, Qian et al. [67] published a similar idea at a regional symposium. However, we believe the current paper is the ﬁrst to thoroughly evaluate the performance and scalability of incorporating mutation testing in the fuzzing loop. In particular, we identiﬁed that the evaluation in Qian et al.’s paper [67] uses an unsound comparison to the baseline Zest; they use mutation analysis with multiple threads but run Zest only single threaded for the same time bound, hence giving higher CPU time to their technique and obscuring the eﬀects of the increased overhead of performing mutation testing. Additionally, they use a selection strategy to choose 10 mutants at random, but Threats to internal validity. 6 RELATED WORK Greybox fuzzing. The ﬁeld of coverage-guided greybox fuzzing has a vast literature, as surveyed by Manès et al. [52]; a more recent and evolving publication list is maintained by Wen [78]. The ma- jority of fuzzing research focuses on improving heuristics such as seed-picking power schedules [9], input mutations [5, 48, 50], and coverage feedback [14, 25]. FuzzFactory [60] generalizes the feed- back of greybox fuzzing beyond code coverage to domain-speciﬁc metrics that satisfy certain conditions. Our proposed mutation- analysis guidance ﬁts into this framework. We conclude that the diﬀerential oracle is substantially stronger than a traditional implicit oracle and is valuable for capturing a larger set of mutant program execution behaviors. 4.4 RQ4: Diﬀerential Mutation Testing Executing 푃′ on the well-typed ChocoPy program [1]+([2]+[3]) results in a type-checking error, since the [int] type of [1] does not match the mutated null return type of ([2]+[3]). The diﬀerential oracle kills 푃′ since the output AST produced by 푃′ contains a type error, whereas 푃does not. The implicit oracle fails to kill this mutant since no exceptions are triggered. ACKNOWLEDGMENTS This research was funded in part by NSF grant CCF-2120955, a seed grant from CMU’s CyLab, and an Amazon Research Award. This research was funded in part by NSF grant CCF-2120955, a seed grant from CMU’s CyLab, and an Amazon Research Award. This research was funded in part by NSF grant CCF-2120955, a seed grant from CMU’s CyLab, and an Amazon Research Award. 5 THREATS TO VALIDITY Our evaluation uses mutation score when comparing the quality of the generated test-input corpora since our goal was to synthesize a test-input corpus with high mutation score (ref. Section 3.1). We assume that a high mutation score is a valuable objective for fuzzers. However, there is a potential bias from using mutation score as an evaluation metric, as Mu2 beneﬁts from incorporating mutation testing in the fuzzing loop. Our results nevertheless capture the performance overhead impact 938 Guiding Greybox Fuzzing with Mutation Testing ISSTA ’23, July 17–21, 2023, Seatle, WA, USA 7 CONCLUSION do not measure the impact on the overall mutation score, since they never run all killable mutants. We were unable to perform a head-to-head evaluation between Mu2 and their technique since their implementation is not open source. We investigated the challenges of incorporating mutation analysis to guide greybox fuzzing. Our implementation, Mu2, integrates PIT mutation testing into the JQF framework, and is aimed at producing a test-input corpus with high mutation score. In our design, we in- corporated a diﬀerential testing as an oracle for killing mutants and proposed optimizations to improve fuzzing throughput by dynami- cally pruning the number of mutants to be executed. We applied both sound and aggressive optimizations for Mu2 to help scale it to larger programs. After conducting a thorough evaluation on Mu2 and several variants, we found that mutation-analysis feedback can improve the mutation score of a test-input corpus and more reliably kill nontrivial mutants than coverage-guided fuzzing. Using mutation testing in automated test generation. In a regis- tered report, Groce et al. [33] propose fuzzing specially mutated targets to ﬁnd inputs triggering interesting control ﬂow not in the original program, and then use those inputs as seeds for coverage- guided fuzzing. However, they do not target maximizing mutant kills—for example, a mutant which only changes return codes gets low fuzzing priority in their approach because it won’t aﬀect con- trol ﬂow [33]. In contrast, Mu2 aims to ﬁnd inputs that diﬀerentiate program output on potentially semantics-altering mutants, which often change data values but not necessarily control ﬂow. Our ap- proach is therefore orthogonal to Groce et al.’s and could potentially even be combined. 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ChocoPy: A Program- ming Language for Compilers Courses. In Proceedings of the 2019 ACM SIGPLAN Symposium on SPLASH-E (Athens, Greece) (SPLASH-E 2019). Association for Computing Machinery, 41–45. https://doi.org/10.1145/3358711.3361627 [ ] k d k h h d k d g [76] Junjie Wang, Bihuan Chen, Lei Wei, and Yang Liu. 2019. Superion: Grammar- Aware Greybox Fuzzing. In 41st International Conference on Software Engineering (ICSE ’19). https://doi.org/10.1109/ICSE.2019.00081 [62] Mike Papadakis, Christopher Henard, Mark Harman, Yue Jia, and Yves Le Traon. 2016. Threats to the validity of mutation-based test assessment. In Proceedings of the 25th International Symposium on Software Testing and Analysis. 354–365. https://doi.org/10.1145/2931037.2931040 p g [77] Pengfei Wang, Xu Zhou, Kai Lu, Tai Yue, and Yingying Liu. 2022. 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https://openalex.org/W2920899512	https://interactive-plus.ru/e-articles/602/Action602-485967.pdf	Russian	null	Training of cadets of educational institutions of the Ministry of internal Affairs of Russia competence and learning the moral side of communication for better adaptation in society	Upbringing and education: theory, methods and practice	2,019	cc-by	4,527	Scientific Cooperation Center "Interactive plus" Scientific Cooperation Center Interactive plus Човдырова Гульшат Сулеймановна д-р психол. наук, д-р мед. наук, профессор, профессор ФГКОУ ВО «Московский университет МВД России им. В.Я. Кикотя» г. Москва Клименко Тимур Станиславович канд. психол. наук, соискатель г. Москва Пяткина Ольга Андреевна слушательница Институт психологии служебной деятельности органов внутренних дел ФГКОУ ВО «Московский университет МВД России им. В.Я. Кикотя» г. Москва DOI 10.21661/r-485967 ОБУЧЕНИЕ КУРСАНТОВ ОБРАЗОВАТЕЛЬНЫХ УЧРЕЖДЕНИЙ МВД РОССИИ КОМПЕТЕНТНОСТИ И ПОЗНАВАНИЮ МОРАЛЬНО- НРАВСТВЕННОЙ СТОРОНЫ ОБЩЕНИЯ ДЛЯ ЛУЧШЕЙ АДАПТАЦИИ В СОЦИУМЕ Аннотация: цель работы – развитие у курсантов личностных коммуника- тивных способностей и компетентности. Научная новизна заключается во включении в процесс обучения психологического познавания морально-нрав- ственной стороны общения. Для выявления негативных коммуникативных установок авторами использована тестовая методика В.В. Бойко, для обуче- ния – дидактический метод. В работе даются разработанные этапы проведе- ния – общения полицейских с гражданами для развития у курсантов универси- тета навыков общения и профессиональной коммуникативной компетентно- сти. Делается вывод о том, что знание и соблюдение морально-нравственных DOI 10.21661/r-485967 Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) ОБУЧЕНИЕ КУРСАНТОВ ОБРАЗОВАТЕЛЬНЫХ УЧРЕЖДЕНИЙ МВД РОССИИ КОМПЕТЕНТНОСТИ И ПОЗНАВАНИЮ МОРАЛЬНО- НРАВСТВЕННОЙ СТОРОНЫ ОБЩЕНИЯ ДЛЯ ЛУЧШЕЙ АДАПТАЦИИ В СОЦИУМЕ Аннотация: цель работы – развитие у курсантов личностных коммуника- тивных способностей и компетентности. Научная новизна заключается во включении в процесс обучения психологического познавания морально-нрав- ственной стороны общения. Для выявления негативных коммуникативных установок авторами использована тестовая методика В.В. Бойко, для обуче- ния – дидактический метод. В работе даются разработанные этапы проведе- ния – общения полицейских с гражданами для развития у курсантов универси- тета навыков общения и профессиональной коммуникативной компетентно- сти. Делается вывод о том, что знание и соблюдение морально-нравственных принципов общения поможет повышению имиджа полицейских в популяции и 1 Центр научного сотрудничества «Интерактив плюс» успешной адаптации будущих сотрудников полиции к требованиям социальной среды. успешной адаптации будущих сотрудников полиции к требованиям социальной среды. Ключевые слова: морально-нравственная сторона общения, вербальное об- щение, невербальное общение, тренинг, полицейские, курсанты, развитие, ком- петентность. 2 https://interactive-plus.ru Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) 2 https://interactive-plus.ru 2 https://interactive plus.ru Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) 2 https://interactive-plus.ru Введение Компетентность – (лат. competens – подходящий, соответствующий, надле- жащий, способный, знающий) – качество человека, обладающего всесторонними знаниями в какой-либо области. Компетентность – наличие знаний, умений навыков, опыта, необходимых для эффективной деятельности в заданной пред- метной области. Много и других определений компетентности. Наша задача сде- лать выпускников профессионально компетентными специалистами, дав им зна- ния, умения, навыки, тем самым подготовив базу для лучшей адаптации к соци- альной среде, в которой им придется вступать в общение с широкими кругами населения из разных социальных слоев и этносов. Многие отечественные и зарубежные педагоги и психологи занимались про- блемой коммуникативной компетентности. Л. Хъел и Д. Зинглер рассматривали коммуникативную компетентность как психосоциальное качество, которое спо- собствует эффективному взаимодействию и общению с окружением. Направле- ние – «компетентностный подход» в образовании получило широкое распро- странение сравнительно недавно, в 80-е годы в России (Н. Хомский, Н.В. Кузь- мина, А.К. Маркова, В.Н. Куницина, Г.Э.Белицкая и др.) и стало какое-то время популярным среди психологов, хотя имеет изначально чисто педагогическое зна- чение. Л.А. Петровская рассматривает коммуникативную компетентность как совокупность знаний, умений навыков, опыта т.е. как психологическое образо- вание [1, с. 5]. Р. Вердебер, К. Вердеб считают, что коммуникативная компетентность – это умение общаться так, что другие воспринимают эту коммуникацию как соответ- ствующую ситуации с определенными целями и навыками [2, с. 27]. Scientific Cooperation Center "Interactive plus" Компонентами этой системы требований могут быть владение составляю- щими сторон общения: перцептивными, интерактивными, коммуникативным [1, c. 25], и с нашей точки зрения-морально-нравственными [4, с. 53–60]. Эти со- ставляющие общения рассматриваются в единой системе требований к личности коммуникаторов, а именно: – владение грамотной устной речью и той или иной лексикой, в зависимости от социального статуса общающихся людей, – вербальными признаками обще- ния; – знание внешних сигналов общения: мимика, пантомимика, жесты, прок- семика – невербальных сигналов общения. – знание внешних сигналов общения: мимика, пантомимика, жесты, прок- семика – невербальных сигналов общения. Как курсанту, так и действующему сотруднику полиции, при общении с гражданами, необходимо владеть двумя большими группами инструментов про- стой психодиагностики: – вербальными (речевыми) и невербальными [4, с. 56]. К вербальным – относится все то, что может определить отношение к чело- веку, в зависимости от словесного речевого общения с ним и выяснения: цели общения, направленности, установок, мотивов, и т. д., т.е., то, что узнается о че- ловеке, в основном, при речевой беседе, и при официальных допросах подозре- ваемых, свидетелей, потерпевших. Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) 4 https://interactive-plus.ru Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) 4 https://interactive-plus.ru https://interactive-plus.ru Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4. ttps://interactive-plus.ru Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) Введение К невербальным – относится все то, что воспринимается в партнере, до сло- весного контакта с ним: внешность – физические данные, одежда, особенности походки, мимика, общие манеры, топомимика – (движения частями тела) и т. п. То, что могут наблюдать полицейские при скрытом наблюдении, при рассмот- рении фото и видео данных. Актуальность данной темы вытекает из того, что в целом, наличие всех этих знаний, умений, навыков и виртуозное владение ими можно назвать комму- никативной культурой – общей коммуникативной компетентностью, которую необходимо развивать и постоянно совершенствовать у наших курсантов и слу- шателей – будущих полицейских, для повышения их имиджа в популяции, сле- довательно лучшей адаптации к учебной среде и дальнейшей к деятельности. Очень часто в деятельности полицейских общение происходит в конфликтных 3 Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) Центр научного сотрудничества «Интерактив плюс» ситуациях и в рамках часто встречающихся на практике критических инциден- тов, которые описаны в работах R.A. Blak, W.A. Foreman, W.W. Lippert [7–9]. ситуациях и в рамках часто встречающихся на практике критических инциден- тов, которые описаны в работах R.A. Blak, W.A. Foreman, W.W. Lippert [7–9]. Научная новизна заключается во включении в процесс обучения психологи- ческого познавания морально-нравственной стороны профессионального обще- ния, являющейся одновременно повышением специальной компетентности по- лицейских. Цель работы – развитие у курсантов личностных коммуникативных способ- ностей и компетентности, являющихся профессионально важными качествами, способствующими познанию морально-нравственной стороны общения и – успешной адаптации к учебе и будущей деятельности. Методы: проведение игровых методов обучения на занятиях ( тренинг меж- личностной вербальной и невербальной коммуникации), диагностический ин- струментарий: определение психологических негативных коммуникативных установок оценивались по методике В. В. Бойко и методом бальной оценки зна- ний на семинарах и практических занятиях. Результаты исследования и анализа полученных данных При проведении предварительного ранжированного экспертного опроса наиболее опытных полицейских, оперативные сотрудники на первое по важно- сти место вынесли коммуникативные качества личности, которые особенно важны при расследовании и раскрытии преступления для добывания скрываемой информации. Поэтому в данной работе уделено внимание именно компетентно- сти и развитию у курсантов этих качеств. Нами разработана структура установ- ления психологического контакта сотрудников полиции с населением, которая включает несколько этапов и эти этапы усваиваются курсантами в ходе проведе- ния дискуссионных, игровых, тренинговых методов обучения на занятиях (тре- нинг органов чувств, тренинг межличностной чувствительности или сензитив- ный и др.) [4, с. 70–80]. В данной статье представлена общая начальная часть преподаваемого на лекциях, семинарах и, уточняемых на практических занятиях материала. Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) Введение Разработанные нами этапы профессионального общения Scientific Cooperation Center "Interactive plus" разбираются теоретически на семинарах и разыгрываются (деловые игры) в по- следующем на практических занятиях. 1. Начальный этап общения-перцептивный, восприятие, построение пред- ставления. Прогнозирование результатов общения и представление образа граж- данина (гражданина, подозреваемого, свидетеля, потерпевшего и др.). Главная задача этапа – сбор информации о гражданах, причастных к правонарушениям. В тех случаях, когда возможна предварительная подготовка к контакту, сбор информации целесообразно вести по следующим направлениям: общая характе- ристика (образование, пол – гендерная ориентация, возраст, социальное положе- ние и т. п.); ближайшее социальное окружение, круг общения (друзья – враги, судимости), образ жизни; склонности, интересы, привычки, манера общения, ре- чевые, мимические, топомимические стереотипы; одобряемые и запретные темы общения и др. В случаях, когда предварительное изучение граждан невозможно, сбор ин- формации может осуществляться в процессе скрытого (в рамках закона об ОРД) или открытого наблюдения за поведением подозреваемых, манерами, реакциями в процессе невербального общения (скрытое наблюдение, видео). На основе собранной информации формируется стратегия предстоящего коммуникативного поведения, т.е.: как и в какой ситуации целесообразнее начать общение сотруднику с данным гражданином. При этом необходимо учи- тывать несколько требований: – начальная фаза действия должна предполагать обязательное развитие пу- тем использования различных приемов аттракции: механизмов расположения к себе собеседника (имя собственное, комплименты, улыбка и др.); – начальная фаза действия должна предполагать обязательное развитие пу- тем использования различных приемов аттракции: механизмов расположения к себе собеседника (имя собственное, комплименты, улыбка и др.); – необходимо формировать у кандидата заинтересованность в общении, предлагать темы для разговора, интересные именно для него; – позиция полицейского в беседе может быть иногда немного ниже той, ко- торая предлагается партнеру (дать возможность гражданину взглянуть на ситуа- цию сверху вниз), это способствует расслаблению напряженности и заинтересо- ванности в продолжении общения. 5 Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) Центр научного сотрудничества «Интерактив плюс» ttps://interactive-plus.ru Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) Центр научного сотрудничества «Интерактив плюс» 2. Основной этап – коммуникативный. Выбор внешних условий, облегча- ющих установление контакта, т.е. времени, места, расстояния, ситуации, опти- мально обеспечивающих реализацию намеченной стратегии поведения. Цель этапа – снять у гражданина настороженность предубеждение и другие барь- еры общения и заинтересовать перспективами. 1. Выявление внешних коммуникативных свойств в начале зрительного контакта, т.е. оценка психического состояния и лживости, отношения кандидата к начавшемуся общению по мимическим, топомимическим знакам, речи, голосу, интонации, т.е.- проводится своего рода диагностическая оценка по невербаль- ным и вербальным признакам. 2. Устранение барьеров в общении, которые могут возникать в ре а) появления у сотрудника или у граждан отрицательной установки к пред- стоящему общению (необходимо – мобилизовать волевые ресурсы, знания, уме- ния, опыт и сломать негативную установку, если она как-то просматривается в движениях, речи или чувствуется в недовольной мимике, демонстративных по- зах); б) непонимания гражданами сути предлагаемого поведения; при формиро- вании конфликтных взаимоотношений предлагается два варианта решения: пер- вый – снять конфликт всеми возможными дозволенными способами; второй – обострить конфликт в редких случаях, когда иное невозможно и есть угроза со- вершения тяжких преступлений; б) непонимания гражданами сути предлагаемого поведения; при формиро- вании конфликтных взаимоотношений предлагается два варианта решения: пер- вый – снять конфликт всеми возможными дозволенными способами; второй – обострить конфликт в редких случаях, когда иное невозможно и есть угроза со- вершения тяжких преступлений; в) в случаях затруднения общения в результате особого эмоционального со- стояния гражданина (депрессия, тревога, фрустрация, агрессия) -необходимо от- ложить общение и переключить внимание на другой объект, тем самым снизить эмоциональный накал (реакции), подключить других специалистов (опытных пе- дагогов психологов или психиатра) для снятия этих состояний. Пример из практики проведения практического занятия деловой игры: Пер- вая ситуация – курсант играет роль человека, манипулирующего окружающими, агрессивного, который руководствуется принципом «… я тут имею власть, я сильнее, ты мне должен подчиняться, во что бы то не стало…». 6 https://interactive plus.ru Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) Scientific Cooperation Center "Interactive plus" Вторая ситуация – курсанты совместно решают как должны строиться от- ношения с асоциальной частью населения, исходя из понятия ассертивности, т.е., проявляя толерантность и уважение к соблюдению интересов населения. Остальные курсанты в роли населения участвуют в полемике: кому дове- рять решение проблемы своих родственников, и как бы поступили сами, при об- щении с тем и другим полицейским – и это будет третья ситуация. Центр научного сотрудничества «Интерактив плюс» Ставится задача: при встрече с правонарушителем в мирной обстановке вести себя так, как, если бы на месте правонарушителя был твой брат, друг, сын, дочь и т. д. И как при этом отстоять интересы полицейского?! Задача встречи с правонарушителем в экстремальной ситуации -рассмат- ривается в рамках критического инцидента отдельно. Поправки и уточнения должен вносить преподаватель по ходу обсуждения. В ходе обсуждения, данное понятие – ассертивного бесконфликтного поведения при общении с населением, для создания позитивного имиджа и успешной адап- тации, сначала заучивается, затем осознается при моделировании ситуации в де- ятельности, и тогда только может надолго запомниться и перейти в умение пра- вильно вести себя при общении с общей популяцией. Далее слушателям и курсантам разъясняются принципы ассертивного пове- дения сотрудников органов внутренних дел при формировании положительного имиджа, разработанные автором в процессе обучения курсантов и предлагается продемонстрировать в общении: 1) стремление найти такой выход из положения, который бы устраивал все задействованные стороны в рамках закона; 1) стремление найти такой выход из положения, который бы устраивал все задействованные стороны в рамках закона; 2) уверенность в себе, в физической подготовленности -защищать обижен- ных, слабых; самостоятельности, самоуважения и уважения к другим при крити- ческих инцидентах; 3) пример прямоты и открытости, щадящая чувства окружающих в рамках нравственности, гуманности и законности; 4) установку выслушать и разобраться по справедливости; 7 Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) Центр научного сотрудничества «Интерактив плюс» ttps://interactive-plus.ru Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) 8 https://interactive-plus.ru Центр научного сотрудничества «Интерактив плюс» 5) презумпцию невиновности с избеганием выражений: «…я посажу тебя… ты получишь срок…» и др. агрессивных выражений, которые провоцируют от- ветную грубость; – честности и ответственности в поведении; 6) профессионально позитивную установку с демонстрацией желания по- мочь в гуманном решении конфликта или другой проблемы; 6) профессионально позитивную установку с демонстрацией желания по- мочь в гуманном решении конфликта или другой проблемы; 7) готовность действовать всегда в паре с умением подстраховать друг друга и выступить в качестве свидетеля, с обязательным использованием технической аппаратуры (видео, аудио и др.). Проигрывание или заучивание каждого принципа с умением демонстриро- вать его и понимать сущность принципа увлекает слушателей и курсантов. При этом разбираются случаи из практики. 3. Заключительный этап – интерактивное общение. Стимулирование инте- реса к продолжению общения (материальное, моральное поощрение и т. п.). За- ключение контракта и другие моменты, которые предполагаются при активности контакта с обеих сторон, заинтересованности в продолжении контактов обеими сторонами. 3. Заключительный этап – интерактивное общение. Стимулирование инте- реса к продолжению общения (материальное, моральное поощрение и т. п.). За- ключение контракта и другие моменты, которые предполагаются при активности контакта с обеих сторон, заинтересованности в продолжении контактов обеими сторонами. Курсанты при этом усваивают общение как процесс, в котором обычно вы- деляются три основные указанные выше стороны: перцептивная – восприятие и познание людьми друг друга в процессе вербального и невербального общения; коммуникативная – обмен важной информацией между общающимися, устране- ние барьеров; интерактивная – взаимодействие между людьми как взаимная ак- тивность между общающимися на равных, влияние друг на друга в процессе об- щения (обмен знаниями, идеями, действиями, воздействиями, эмоциями и т. п.- максимальное использование положительно направленных сторон и приемов об- щения), с целью получения скрываемой достоверной информации. 4. Мы, при обучении курсантов и слушателей, на заключительном этапе предлагаем освоить и четвертую морально-нравственную сторону общения, ос- нованную на принципах законности, гуманизма, равенства сторон, беспристраст- ности и взаимного уважения, соблюдения ассертивности. Это значит, что для по- вышения имиджа сотрудников среди населения, общение сотрудников полиции 4. Мы, при обучении курсантов и слушателей, на заключительном этапе предлагаем освоить и четвертую морально-нравственную сторону общения, ос- нованную на принципах законности, гуманизма, равенства сторон, беспристраст- ности и взаимного уважения, соблюдения ассертивности. Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) Центр научного сотрудничества «Интерактив плюс» Это значит, что для по- вышения имиджа сотрудников среди населения, общение сотрудников полиции 8 https://interactive-plus.ru Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) Scientific Cooperation Center "Interactive plus" с населением должно быть построено на взаимном уважении личности партнера, учете его этнических и религиозных предпочтений; соблюдении его интересов, прав и обязанностей, презумпции невиновности; недопустимости унижающих, оскорбительных оборотов речи, тона, мимики и движений, высокомерия; необ- ходимости соблюдения субординации в профессиональных сферах деятельности и взаимоуважения. Курсантам до сведения доводится, что это актуально в настоящее время в связи с увеличением с каждым годом количества мигрантов в России из ближ- него и дальнего зарубежья и необходимостью более пристального внимания к ним в процессе общения, так как эта среда может быть криминогенной в связи с частой нелегальностью их миграции. При соблюдении принципов морально-нравственной стороны общения со- трудник полиции избегает конфликтов с гражданами и реже попадает в поле зре- ния, жаждущих сенсаций представителей СМИ, пытающихся часто поверх- ностно и не справедливо нарицательно представлять населению имидж полицей- ских, провоцируя их, на ошибки в поведении. При этом в процессе преподавания необходимо подчеркнуть, что повыше- ние имиджа сотрудников полиции одновременно является показателем лучшей их адаптированности к социальной среде. Рассмотрим кратко только преподавание и изучение этой стороны общения, (учитывая заданные рамки статьи), которая имеет познавательное значение как для психологов и педагогов, так и для обучаемых курсантов и слушателей. Сначала курсанты знакомятся с основными положениями по этике и морали коммуникативного поведения сотрудников ОВД, отраженными в следующих ос- новных нормативных и правовых документах: Кодексе чести рядового и началь- ствующего состава органов внутренних дел РФ, Кодексе профессиональной этики сотрудника органов внутренних дел РФ, в Федеральном законе «О поли- ции» от 7 февраля 2011 г. №3-ФЗ, в новом Федеральном законе от 30 ноября 2011 г. №342-ФЗ «О службе в органах внутренних дел Российской Федерации и 9 Центр научного сотрудничества «Интерактив плюс» внесении изменений в отдельные законодательные акты Российской Федерации» и др. инструкциями и нормативными документами. внесении изменений в отдельные законодательные акты Российской Федерации» и др. инструкциями и нормативными документами. 10 https://interactive plus.ru Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) 10 https://interactive-plus.ru https://interactive-plus.ru Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4. https://interactive-plus.ru Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) Центр научного сотрудничества «Интерактив плюс» К практическим занятиям заучиваются некоторые правила поведения, реко- мендованные полицейским кодексом профессиональной этики: – постоянно контролировать свое поведение, чувства и эмоции, не позволяя личным симпатиям или антипатиям, неприязни, недоброму настроению или дру- жеским чувствам влиять на служебные решения, уметь предвидеть последствия своих поступков и действий; – постоянно контролировать свое поведение, чувства и эмоции, не позволяя личным симпатиям или антипатиям, неприязни, недоброму настроению или дру- жеским чувствам влиять на служебные решения, уметь предвидеть последствия своих поступков и действий; – обращаться одинаково корректно с гражданами, независимо от их служеб- ного или социального положения, не проявляя подобострастия к социально успешным и пренебрежения к людям с низким социальным статусом и др. – обращаться одинаково корректно с гражданами, независимо от их служеб- ного или социального положения, не проявляя подобострастия к социально успешным и пренебрежения к людям с низким социальным статусом и др. Выявленные нами при экспериментально-психологическом исследовании данные психологических портретов оперативников и граждан говорят о том, что склонность оперативных сотрудников к доминированию должна контролиро- ваться [6, с. 38]. Этот момент обсуждается на семинарских занятиях. Аффек- тивно-коммуникативные эмоциональные явления, особенно стрессы, не ред- кость в профессиональной деятельности сотрудников ОВД, жизнь которых все- гда сопряжена с риском при встрече с вооруженным преступником или просто с агрессивной толпой. Эти положения обсуждаются на семинарских занятиях в форме докладов и дискуссий. Нам, при проведении экспериментальных исследований, удалось выделить некоторые особенности психологического общения сотрудников полиции с гражданами, относящиеся к морально-нравственной стороне общения, которые должны знать курсанты. Это следующие моменты, которые обязательно включа- ются в дискуссии и тренинги, проводимые на практических и семинарских заня- тиях и, обсуждающиеся, подробно с демонстрацией отрывков из таких кино- фильмов, как «Место встречи изменить нельзя», на этом примере обсуждается не вербальная диагностика по анатомо-физиологическим особенностям внешно- сти человека. Scientific Cooperation Center "Interactive plus" 1. Конспиративность и конфиденциальность оперативно-розыскных дей- ствий – это важнейшая особенность, без соблюдения которой, достичь нужного результата в добывании информации часто бывает невозможно. Она же делает при этом особенной коммуникацию общающихся сторон. Поэтому вытекает необходимость действий, направленных на соблюдение закона, который строго регламентирует использование специфических методов оперативно – розыскной деятельности исключительно в целях борьбы с преступностью, в том числе и в целях охраны неприкосновенности личности, частных и государственных мате- риальных ценностей. Отсюда возникает и необходимость освоения психологи- чески важных коммуникативных качеств морально-нравственного общения по- лиции с населением на основе взаимопонимания и особой доверительности от- ношений при конспирациях. 2. Готовность к применению нравственно упречных (небезупречных) мето- дов вторжения в частную жизнь граждан. Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) Центр научного сотрудничества «Интерактив плюс» Эта особенность требует от полицей- ских и граждан, таких качеств как скрытность и малообщительность. Данные ка- чества требуются, например, при применении так называемых интрузивных ме- тодов – необходимости частичного вторжения в частную жизнь граждан. При проведении мероприятий по прослушиванию телефонных переговоров разраба- тываемого лица, иногда возможно непреднамеренное прослушивание перегово- ров лиц непричастных к совершению преступления (родственники, друзья, дети, секретари, сослуживцы и др.). В этих случаях объективно ограничиваются и нарушаются конституционные права указанных лиц. При проведении таких ме- роприятий излишняя открытость и общительность со стороны граждан и поли- цейских не требуются, при этом необходимы такие морально-нравственные ка- чества как порядочность, сочувствие, честность, верность профессии полицей- ского и др. качества, которые в дальнейшем необходимо изучать и развивать. 3. Рискованность профессионального общения. Это обстоятельство предъ- являет специфические требования к личности сотрудников и граждан, участву- ющих в проведении ОРМ (оперативном внедрении и др.) – сохранять общитель- ность, раскованность, умение применить вербальные и невербальные формы 3. Рискованность профессионального общения. Это обстоятельство предъ- являет специфические требования к личности сотрудников и граждан, участву- ющих в проведении ОРМ (оперативном внедрении и др.) – сохранять общитель- ность, раскованность, умение применить вербальные и невербальные формы 11 11 Центр научного сотрудничества «Интерактив плюс» коммуникации в условиях тяжелых нервно-психических нагрузок, угрозы жизни и здоровью. Решение этой задачи требует от полицейских незаурядно утончен- ной коммуникативной компетентности: умения устанавливать контакты в пси- хологически сложных ситуациях, способности к разумному риску, чтобы усы- пить бдительность правонарушающей стороны общения, владения формами не- вербальной коммуникации для получения и передачи нужной информации на расстоянии и без физических и нравственных потерь. Все эти моменты заучиваются на практических занятиях, в конце занятий для поощрения и закрепления полученных знаний выставляются оценки в жур- нал. Достижение в процессе обучения среднего балла от 4,0 до 4,5 – является од- новременно и методикой оценки полученных нами результатов. Показатели ди- намики психологических негативных коммуникативных установок оценивались по методике В.В. Бойко. По данной методике у курсантов, после изучения курса, определялись: нега- тивный опыт общения при максимальном балле 20, у курсантов-криминалистов выявлено – 10; открытая жестокость – при максимальном балле 45, у курсан- тов – 25; завуалированная жестокость – при максимальном балле 20, у курсан- тов – 12; обоснованный негативизм – при максимальном балле 5, у курсантов – 5; брюзжание – при максимальном балле 10, у курсантов –3. Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) Центр научного сотрудничества «Интерактив плюс» Таким образом, коммуникативная компетентность при обучении курсантов в нашем исследовании рассматривается как совокупность знаний, умений, навы- ков, обеспечивающих эффективность усвоения сути коммуникативного про- цесса обучающимися, для общения в будущем с населением, который является основным коммуникатором, влияющим на адаптацию и имидж полицейских в социуме. Выводы: 1. Результаты изучения и выделение особенностей преподавания курсантам четвертой морально-нравственной стороны общения, требует дальнейших уточнений и детальных разработок методов преподавания курсантам и слуша- телям для развития у них психодиагностических («профайлинговых») способно- стей, формирования стратегии бесконфликтного общения с населением для по- вышения положительного имиджа полицейских в социуме и их успешной адап- тации к все более усложняющимся социальным условиям. 2. Успешности полицейских в профессиональной сфере, на наш взгляд, можно добиться путем развития компетентности и коммуникативных способно- стей и, руководствуясь, в деятельности морально-нравственными принципами. Результаты изучения негативных коммуникативных установок методикой Бойко показали достоверно значимую (р  0,005) положительную динамику по многим показателям, после проведенных занятий. Центр научного сотрудничества «Интерактив плюс» Полученные статистически значимо низкие показатели негативных комму- никативных установок у курсантов в данном случае связаны с одной стороны - отсутствием практических навыков настоящего общения с населением и с дру- гой – положительным влиянием полученных знаний-по коммуникативной ком- петентности. В данном случае мы, преподаватели и профессора, учим будущих полицей- ских «отличать зерна от плевел». Заключение и выводы 12 https://interactive-plus.ru Психологическая составляющая морально-нравственных требований по об- щению, предъявляемых к сотрудникам полиции, имеет большое значение для раскрытия преступлений. Прежде всего, осуществляющие розыск сотрудники, p p Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) Scientific Cooperation Center "Interactive plus" должны знать множество способов установления коммуникативных каналов – источников получения необходимой им информации в рамках правовых и мо- рально-нравственных норм. Для развития этих навыков необходимы не только опыт, знания, умения, но и особая компетентность во владении невербальными методами коммуникации при добывании скрываемой информации. Этого тре- буют и особые условия труда, связанные с риском для жизни и здоровья. При усвоении знаний по этой стороне общения курсанты должны понимать, что они на практике будут вовлекаться к участию в коммуникации с открытой социальной системой, которая подчиняется определенным требованиям закона и морали, в которой эти требования сталкиваются в конфликтных взаимоотноше- ниях со СМИ и отдельными группировками населения, зачастую враждебно настроенными к полицейским. В работе кратко упомянуты некоторые особенности деятельности полицей- ских, которые могут способствовать возникновению конфликтов с населением, чего можно избежать при получении знаний о морально- нравственной стороне общения с населением. Безусловно, все трудности, с которыми сталкиваются со- трудники полиции во многих случаях, связаны с нарушением культуры общения как со стороны полицейских, так и со стороны граждан, -при нехватке, как внеш- них форм коммуникации, так и внутренних -духовных устоев. Каким бы ни был высоким унаследованный коммуникативный потенциал личности, в борьбе с правонарушениями он тускнеет и нивелируется, не выдер- живает напряжения при столкновении с безнравственностью и аморальностью, при отсутствии вооружения в виде знаний. Усвоение курсантами принципов морально-нравственного общения, зна- ний, и умений общаться с населением – является в какой-то степени профилак- тикой развития межличностных конфликтов и эмоционального «выгорания», яв- ляющихся начальной стадией дезадаптации и развития стрессовых расстройств, в результате накопления отрицательных эмоций. Известно, что у воспитанного и эрудированного человека границы толерантности при конфликтах увеличива- ются. 13 Центр научного сотрудничества «Интерактив плюс» https://interactive-plus.ru Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) Список литературы 1. Андреева Г.М. Социальная психология. – М.: Аспект Пресс, 2000. – С. 27. 2. Вердебер Р. Психология общения / Р. Вербедер, К. Вербедер. – СПб.: Прайм-Еврознак, 2003. – 320 с. 3. Петровская Л.А. Компетентность в общении. – М.: Изд-во МГУ, 1989. – 216 с. 3. Петровская Л.А. Компетентность в общении. – М.: Изд-во МГУ, 1989. – 216 с. 4. Човдырова Г.С. Психологическая диагностика и развитие коммуникатив- ной компетентности сотрудников органов внутренних дел: Учебное пособие / Г.С. Човдырова, Т.С. Клименко. – М.: Московский университет МВД России, 2014. – 97 с. Scientific Cooperation Center "Interactive plus" 5. Човдырова Г.С. Психология профессионального общения оперативных сотрудников органов внутренних дел с гражданами: Учебное пособие / Г.С. Човдырова, В.Л. Кубышко, С.К. Клименко, В.В. Борисова, Н.Д. Эриа- швили. – М.: Юнити-дана, 2012. – 126 с. 5. Човдырова Г.С. Психология профессионального общения оперативных сотрудников органов внутренних дел с гражданами: Учебное пособие / Г.С. Човдырова, В.Л. Кубышко, С.К. Клименко, В.В. Борисова, Н.Д. Эриа- швили. – М.: Юнити-дана, 2012. – 126 с. 6. 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https://openalex.org/W4252228708	https://malariajournal.biomedcentral.com/track/pdf/10.1186/s12936-020-3122-2.pdf	English	null	Polymorphic markers for identification of parasite population in Plasmodium malariae	Research Square (Research Square)	2,020	cc-by	9,362	Mathema et al. Malar J (2020) 19:48 https://doi.org/10.1186/s12936-020-3122-2 Mathema et al. Malar J (2020) 19:48 https://doi.org/10.1186/s12936-020-3122-2 Malaria Journal Open Access Abstract Background: Molecular genotyping in Plasmodium serves many aims including providing tools for studying parasite population genetics and distinguishing recrudescence from reinfection. Microsatellite typing, insertion-deletion (INDEL) and single nucleotide polymorphisms is used for genotyping, but only limited information is available for Plasmodium malariae, an important human malaria species. This study aimed to provide a set of genetic markers to facilitate the study of P. malariae population genetics. Methods: Markers for microsatellite genotyping and pmmsp1 gene polymorphisms were developed and validated in symptomatic P. malariae field isolates from Myanmar (N = 37). Fragment analysis was used to determine allele sizes at each locus to calculate multiplicity of infections (MOI), linkage disequilibrium, heterozygosity and construct dendro- grams. Nucleotide diversity (π), number of haplotypes, and genetic diversity (Hd) were assessed and a phylogenetic tree was constructed. Genome-wide microsatellite maps with annotated regions of newly identified markers were constructed. Results: Six microsatellite markers were developed and tested in 37 P. malariae isolates which showed sufficient heterozygosity (0.530–0.922), and absence of linkage disequilibrium (IA S=0.03, p value > 0.05) (N = 37). In addition, a tandem repeat (VNTR)-based pmmsp1 INDEL polymorphisms marker was developed and assessed in 27 P. malariae isolates showing a nucleotide diversity of 0.0976, haplotype gene diversity of 0.698 and identified 14 unique variants. The size of VNTR consensus repeat unit adopted as allele was 27 base pairs. The markers Pm12_426 and pmmsp1 showed greatest diversity with heterozygosity scores of 0.920 and 0.835, respectively. Using six microsatellites mark- ers, the likelihood that any two parasite strains would have the same microsatellite genotypes was 8.46 × 10−4 and was further reduced to 1.66 × 10−4 when pmmsp1 polymorphisms were included. Conclusions: Six novel microsatellites genotyping markers and a set of pmmsp1 VNTR-based INDEL polymorphisms markers for P. malariae were developed and validated. Each marker could be independently or in combination employed to access genotyping of the parasite. The newly developed markers may serve as a useful tool for inves- tigating parasite diversity, population genetics, molecular epidemiology and for distinguishing recrudescence from reinfection in drug efficacy studies. Keywords: Genotyping, INDEL, Nucleotide diversity, Tandem repeats © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background Over the past decade, the world has experienced signifi- cant reduction in global falciparum malaria burden, but this decline was less prominent for the other human Plas- modium species [1]. Plasmodium malariae is endemic throughout parts of South America, Africa, Asia, and the Correspondence: mallika.imw@mahidol.ac.th †Vivek Bhakta Mathema and Supatchara Nakeesathit contributed equally to this work 1 Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand Full list of author information is available at the end of the article Polymorphic markers for identification of parasite population in Plasmodium malariae Vivek Bhakta Mathema1†, Supatchara Nakeesathit2†, Watcharee Pagornrat2, Frank Smithuis2,3,4, Nicholas J. White2,4, Arjen M. Dondorp2,4 and Mallika Imwong1 Vivek Bhakta Mathema1†, Supatchara Nakeesathit2†, Watcharee Pagornrat2, Frank Smithuis2,3,4, Nicholas J. White2,4, Arjen M. Dondorp2,4 and Mallika Imwong1* malariae genotyping markers [13, 14] showed promising potential and could identify up to 10 distinct alleles for a single locus. y p g Recent developments in high-throughput sequencing technology has enabled Insertion-deletions (INDEL)- based gene polymorphisms to construct high-resolution genetic maps and evaluate parasite population structures [33]. The approach has been used for investigating poly- morphisms in genes encoding gametocyte antigen, duffy binding protein and merozoite surface proteins (msp) [34–36]. Previous studies in P. vivax and P. falciparum have utilized gene polymorphisms in msp and circum- sporozoite protein to study the parasite diversity [37–39]. The msp genes are part of a larger family that encodes the major surface antigens for invasive forms of Plasmodium during erythrocytic stages [40–42]. Functions for some of these proteins have described for P. vivax [43] and P. falciparum [44, 45]. Compared to microsatellite-based genotyping, the utility of surface protein gene-based markers are often limited due to reduced polymorphisms as a result of selective pressure by the host immune sys- tem [46, 47], the members of the msp family have been widely used as genotyping markers [36, 48]. The vari- able number of tandem repeats (VNTR)-related INDEL polymorphisms contributes to msp gene diversity [40, 48, 49]. The msp genes consists of exons as amino acid coding blocks interspersed between their conserved and semi-conserved blocks (Fig. 1). Sequences within these semi-conserved regions can be highly variable and thus are potential genetic markers. In this context, the perfor- mance of pfmsp1, pfmsp2, pvmsp1 and pvmsp3α genes to discriminate between recrudescence and reinfection has been studied [50, 51]. The homologue gene P. malariae merozoite surface protein 1 (pmmsp1) is equally poly- morphic and a candidate for parasite genotyping [52, 53]. In particular, high allelic frequency and genetic diver- sity in msp1 were observed for symptomatic P. falcipa- rum malaria patients in Africa [54], Thailand [38] and Burkina-Faso [55]. Apart from a few previous studies reported with limited samples for P. malariae infections in human [56] and monkey [57, 58], only sparse informa- tion is available on pmmsp1 INDEL gene polymorphisms. The present study developed and validated six microsat- ellite genotyping markers as well as markers for pmmsp1 polymorphisms, which can serve as a tool for epidemio- logical and other studies. Microsatellite markers remain an important technique in population genetics because of its high level of poly- morphisms and fidelity to discriminate variants within parasite population [13, 14, 22]. © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Mathema et al. Malar J (2020) 19:48 Mathema et al. Malar J (2020) 19:48 Page 2 of 12 Page 2 of 12 Western Pacific [2–4]. It has been argued that malaria elimination programmes largely focused on Plasmodium falciparum and Plasmodium vivax may have under- mined P. malariae and Plasmodium ovale endemicity [5]. Plasmodium malariae deploys a different transmission strategy, with a large parasite reservoir in asymptomatic carriers with low parasitaemia. These chronic P. malariae infections can occasionally cause anaemia and nephrotic syndrome [4, 6–8]. Use of molecular techniques such as qPCR has enabled more sensitive detection of parasite carriage compared to microscopy [3, 9]. Previous stud- ies conducted in Cameroon [10] and Equatorial Guinea [9] using qPCR based species identification revealed that P. falciparum was the dominating species responsible for over 80.0% of malaria positive cases, followed by P. malariae (≥ 12.0%). In the Colombian Amazon region, the P. malariae infections were reported as high as 43.2% [11]. Nevertheless, P. malariae is understudied compared to the other human Plasmodium species [12], although there are recently increased efforts to describe the epi- demiology of P. malariae [13–17]. However, important molecular tools for this are currently lacking, which will facilitate the description of the course of natural infec- tions, multiplicity of infection (MOI) and anti-malarial drug resistance [18, 19]. In clinical trials on drug efficacy, genetic markers are important to distinguish between recrudescence and reinfection [20, 21]. The previously identified few P. Microsatellites are short tandem repeats of nucleotides usually consisting of 1 to 10 base pair (bp) unit motifs [23]. These are abundant in non-coding regions of the genome, which are gener- ally not under selection pressure, and their molecular origin and evolution result from improper alignment, mispairing and strand-slippage events [22, 24, 25]. Het- erozygosity refers to observation of two different alleles at a locus which forms fundamental bases for investiga- tion of genetic variation in population. Since blood-stage Plasmodium is haploid, a single-genotype infection is expected to have single allele at each location while a multiple-genotype infection is expected to carry mul- tiple alleles [14, 20]. Defining potential microsatellite markers requires assessment of their polymorphisms, and evaluation of heterozygosity and linkage disequilib- rium [20, 26]. High precision fragment analyzers based on capillarity electrophoresis have a resolution of 1–2 bp. This technology in combination with analysis software like GeneScan™ provides a powerful tool for analyses of microsatellite markers. Few adequate microsatellite markers have been identified for P. malariae compared to P. falciparum the [21, 27–29] and P. vivax [20, 30–32]. Study site, DNA sampling and reference sequences ICB, interspecies conserved block; CB, Conserved block. b Schematic representation of the primary (NF1, NR1) and secondary (NF2, NR2) oligonucleotide primers used to sequence partial region of the pmmsp1 gene cgi-bin/batchprimer3/batchprimer3.cgi). The selection criteria of primers were set as: (i) PCR product should be unique to the target flanking sequence containing the microsatellite; (ii) The variation in length of PCR prod- uct should be result of deviation in microsatellite length; (iii) Microsatellites with higher copy numbers, followed by manual inspection of each primer products to avoid selection of primers that could potentially associate with experimentally verified protein coding regions of the parasite or any interspecies cross reactivity; (iv) Prefer- ence is given to primers located at physically different chromosomes with no linkage disequilibrium. Likewise, during validation steps the factors such as ease of ampli- fication, no starter peaks and absence of non-specific bands were also considered for selection of microsatel- lite markers. To investigate polymorphisms in pmmsp1 gene, the partial regions of pmmsp1 genes were amplified using semi-nested PCR with specifically designed primer sets (Table 2). Allelic frequencies were determined based on length of consensus sequences for variable number of tandem repeats (VNTR) in semi-conserved regions interspersed between coding blocks (Fig. 1). Only the well-amplified VNTR in majority of samples with copy numbers greater than 2.0 were considered as alleles for INDEL polymorphisms analysis. therapy regimen [59]. Parasite DNA was extracted using QIAamp DNA mini kit (Qiagen, Germany). All samples were confirmed as P. malariae using 18 small-subunit ribosomal RNA (18S rRNA)-based PCR [60] following the Standards for the Reporting of Diagnostic Accuracy (STARD) [61–63]. Ethical approval for the study was obtained from the ethical review board of the Faculty of Tropical Medicine, Mahidol University. Reference sequences for whole genomes of P. malariae UG01, P. fal- ciparum 3D7, P. vivax SAL-1, P. ovale curtisi GH01 and P. knowlesi STRAIN-H were downloaded from PlasmoDB [64] repository (http://plasmodb.org/common/down- loads/release-36/). The reference sequence for pmmsp1 gene (Accession no. FJ824669) was obtained from NCBI nucleotide database. Study site, DNA sampling and reference sequences Study site, DNA sampling and reference sequences Pl di l i l f t Study site, DNA sampling and reference sequences Plasmodium malariae samples from symptomatic patients were collected in 2008 in Rakhine state of Myanmar (N = 37) as part of a larger study to compare the effectiveness of five artemisinin-based combination Mathema et al. Malar J (2020) 19:48 Page 3 of 12 Fig. 1 Representative block diagram of merozoite surface protein 1 [40]. a The block diagram for msp1 gene is based on conserved amino acid sequences in Plasmodium vivax, P. falciparum and Plasmodium yoelii (inner block). The outer solid and dotted hollow blocks represents conserved and semi-conserved regions based on P. falciparum sequence. The solid block represents ICB with more than 48% identical sequences among three species. The hatched block represents more than 50% identical (conserved) sequences between P. falciparum and P. vivax. Open regions represents less than 45% identity among three species. ICB, interspecies conserved block; CB, Conserved block. b Schematic representation of the primary (NF1, NR1) and secondary (NF2, NR2) oligonucleotide primers used to sequence partial region of the pmmsp1 gene Fig. 1 Representative block diagram of merozoite surface protein 1 [40]. a The block diagram for msp1 gene is based on conserved amino acid sequences in Plasmodium vivax, P. falciparum and Plasmodium yoelii (inner block). The outer solid and dotted hollow blocks represents conserved and semi-conserved regions based on P. falciparum sequence. The solid block represents ICB with more than 48% identical sequences among three species. The hatched block represents more than 50% identical (conserved) sequences between P. falciparum and P. vivax. Open regions represents less than 45% identity among three species. ICB, interspecies conserved block; CB, Conserved block. b Schematic representation of the primary (NF1, NR1) and secondary (NF2, NR2) oligonucleotide primers used to sequence partial region of the pmmsp1 gene Fig. 1 Representative block diagram of merozoite surface protein 1 [40]. a The block diagram for msp1 gene is based on conserved amino acid sequences in Plasmodium vivax, P. falciparum and Plasmodium yoelii (inner block). The outer solid and dotted hollow blocks represents conserved and semi-conserved regions based on P. falciparum sequence. The solid block represents ICB with more than 48% identical sequences among three species. The hatched block represents more than 50% identical (conserved) sequences between P. falciparum and P. vivax. Open regions represents less than 45% identity among three species. Development of genotyping markersi Identification of genome-wide perfect and imperfect microsatellites was performed using Phobos version 3.3.11 [65, 66]. The detection criteria for tandem repeat was restricted to evaluation of perfect and imperfect repeats with unit motifs of 1–10 bp having a minimum threshold repeat number of 14, 7, 5, 4, 4, 4, 4, 4, 4 and 4 for mono-, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nonan-, and deca-nucleotide microsatellites, respectively. The genome wide distribution of microsatellites in ref- erence sequences were summarized (Additional file 1: Table S1). Validation of markers using PCR analysis Primers for microsatellite genotyping markers were labelled with 6-Carboxyfluorescein (6-FAM) and vali- dated using PCR (Table 1) followed by fragment analy- sis. PCR were performed in Eppendorf Mastercycler® pro (Eppendorf, Germany) with a total volume of 20 μL Sequences of six potential microsatellite genotyping markers together with 150 bp flanking sequences were extracted and primers were designed (Table 1) using BatchPrimer3 version 1.0 (https://probes.pw.usda.gov/ Mathema et al. Malar J (2020) 19:48 Page 4 of 12 Table 1 List of candidate genotyping microsatellite markers for Plasmodium malariae UG01 a Novel microsatellite markers for Plasmodium malariae based on fragment analysis of PCR products (N = 37) b Forward primers were labelled with 6-FAM for fragment analysis. Thermal cyclin profile: initial denaturation at 94 °C for 5 min, 40 cycles of 94 °C for 1 min, 58–63 °C f 1 i d 72 °C f 1 i f ll d b fi l t i t 72 °C f 5 i S. no. Locusa Primer Primer sequence Tm (°C) Location 1 Pm05_707 Forwardb GGTAGAAGGAGCAACGGACA 63.5 Chromosome 5 Reverse CGCTCGGGTCATCGTTATTA 61.3 2 Pm06_506 Forwardb TTGTGCGTATGCAACCTTTC 57.6 Chromosome 6 Reverse CAAAAGGGAAGGAGCACAAA 57.0 3 Pm07_429 Forwardb TTCCTTTTCATCCTCTGCAA 59.1 Chromosome 7 Reverse CGAATGAGAGTAGTGCGGAAA 62.8 4 Pm09_801 Forwardb TGACTTCGGTTAGAATATGTTTGC 60.7 Chromosome 9 Reverse TCACACTCCTTTCAATTTCTCA 59.4 5 Pm12_426 Forwardb TTCGTGTTCTCGCTTTCCTC 62.0 Chromosome 12 Reverse GATCACTTCGCACGGGATAG 61.9 6 Pm13_110 Forwardb TCAAGTGGAATAACCGCAAG 60.0 Chromosome 13 Reverse CAGACGAGGACTTTCATTTCG 60.4 Table 1 List of candidate genotyping microsatellite markers for Plasmodium malariae UG01 Table 2 Primary and secondary primers used for amplification of pmmsp1 gene located on chromosome seven using semi-nested PCR Thermal cycling profile: initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 2 min and extension at 72 °C for 2 min then last extension step at 72 °C for 5 min. 2 µL of each primary reaction was used as template for the 100 µL secondary PCR reaction. Thermal cycling profile: Initial denaturation step at 95 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 58 °C for 2 min and extension at 72 °C for 2 min then final extension step at 72 °C for 5 min a Primary and bSecondary set of primers were used to amplify the pmmsp1 gene segment of Plasmodium malariae S. no. Validation of markers using PCR analysis Locus Primer Primer sequence Tm (°C) 1 Primarya PMMSP1full_F2 (N1F) GAATTGTCGAAAGCATTGGT 54.2 PMMSP1full_OR2 (N1R) TCAACTTCTTTCTTTTCTGCTTC 55.0 2 Secondaryb PMMSP1VNTR_1F (NF2) CCAAGCATACGGAACAGGAG 58.8 PMMSP1VNTR_1R (NR2) CAAATCTAATTGGTCGCACTTC 56.2 primers used for amplification of pmmsp1 gene located on chromosome seven using Table 2 Primary and secondary primers used for amplification of pmmsp1 gene located on chro semi-nested PCR MgCl2, 125 µM dNTPs, 125 nM of each primers and 0.4 units of Platinum Taq Polymerase (Table 2). Sequences of amplified products were obtained using high-fidelity capillary electrophoresis conducted by Macrogen. Mono infection of P. malariae DNA verified using 18S rRNA- based PCR was taken as positive control. The PCR mas- ter mix with nuclease free water instead of parasite DNA was taken as control. containing 1× PCR Buffer II (Mg2+ free), 2–3 mM MgCl2, 125 μM dNTPs, 0.25 μM primers and 0.4 U/20 μL of Platinum Taq Polymerase (Invitrogen, USA). Gel elec- trophoresis was used to detect the amplified products on 3% agarose gel. Fragment analysis of the 6-FAM-labelled PCR products was conducted using gel capillary elec- trophoresis by Macrogen (Macrogen Inc., South Korea). During fragment analysis, presence of a distinct expected single peak with a minimum of 100 relative fluorescent units (RFU) were accepted as cut-off value. If multiple peaks were detected, then one-third height of dominant peak with minimum of corresponding proportionate RFUs were taken as selection threshold for scoring the multiple recessive microsatellite alleles per locus. Multiplicity of infections As the blood-stage malaria parasites are haploid, the presence of multiple peaks during evaluation of frag- ment size or VNTR analysis for one or more alleles at target locus was inferred as co-infection with two or more genetically distinct variants. This was referred to as multiplicity of infections (MOI) for the same isolate [20, 21, 67]. The mean MOI for positive samples was calcu- lated independently for each marker by dividing the total number of P. malariae clones identified by the number For pmmsp1 polymorphisms, semi-nested PCR reac- tion was conducted (Table 2) to increase sensitivity. Pri- mary and secondary PCR products were generated using corresponding volumes of 20 and 100 µL reaction in the presence of 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 2 mM Mathema et al. Malar J (2020) 19:48 Page 5 of 12 analysis of the primers sequences against the NCBI data- bases and in silico PCR using default settings of UGENE version 1.30 [73] against whole genome reference data- base [64] of all available human infecting Plasmodium species. All markers did not belong or cross-aligned with any regions of other Plasmodium species. (ii) The sam- ples used were verified for mono-infection of P. malariae pre-confirmed using 18S rRNA-based PCR specific. Dur- ing PCR validation, all markers were specific to P. malar- iae only and had no cross reactivity to P. falciparum and P. vivax. (iii) Screening for sequence polymorphisms in the pmmsp1 gene by comparing to reference sequence or fragments analysis of amplified products. Non-specific amplification was not observed for any markers during assessment of qPCR products by gel electrophoresis. For microsatellite markers, inspection of potential contami- nation was safeguarded by routine insertion of known negative samples in each PCR run to access true nega- tive results. For all six microsatellite markers except one sample for Pm06_506 (1/37), the target amplification was positive for all symptomatic P. malariae patients (N = 37), thus making sensitivity and specificity ~ 100% (Additional file 1: Table S3). For primers targeting the pmmsp1 gene polymorphisms, all well-sequenced samples (N = 27) exhibited VNTR polymorphisms and non-specific ampli- fication or false positive results were not identified (Addi- tional file 1: Table S3). Additional inspection of potential contamination involved cross-comparison of the ampli- fied region with known sequences from P. falciparum and P. vivax. Multiplicity of infections To avoid cross-contamination of samples during DNA addition and PCR processing steps, negative con- trols consisting of only water were added in each run. of samples PCR positive for the parasite. For microsatel- lites, the single or predominant locus at each allele was considered for evaluating allele frequencies. The allele fragment size was interpreted using GeneScan™ version 3.1. Additional alleles were scored only if the peak height was at least one-third the height of the major peak during fragment analysis. For pmmsp1 gene, sequence data were interpreted using Bioedit version 7.0.4. Allelic frequen- cies for pmmsp1 gene were determined based on length of consensus sequences for variable number of tandem repeats (VNTR) in semi-conserved regions interspersed between coding blocks (Fig. 1). Identification of novel markers The likelihood of two infections by same genotype were deduced by combining individual probabilities of two or more unlinked genetic markers and designated as com- bined probabilities (πPi), where πPi = P1 × P2 × ⋯Pi, where Pi = p2 i is individual probabilities for each markers being utilized. The assumption was based on each infections being independent and the probability of reinfection by same genotype were products of probabili- ties for individual markers [72]. Figure 2 summarizes the approach we used to develop and validate genetic markers in P. malariae. To determine suitable P. malariae genotyping markers, the prevalence of polymorphisms for potential markers in 37 sympto- matic P. malariae-positive blood samples from Myanmar was evaluated (Additional file 1: Table S2). Six promis- ing genotyping markers, namely, Pm05_707, Pm06_506, Pm07_429, Pm09_801, Pm12_426 and Pm13_110 (Table 1) were identified. For all markers polymorphic fragments could be generated in most P. malariae-posi- tive test samples (Additional file 1: Table S3). Blastx anal- ysis showed that the regions covered by these markers were not linked to known CDS or experimentally verified protein expressing regions of the parasite genome. For the pmmsp1 gene, a gene sequence could be generated in 27 out of the 37 parasite samples, in which VNTR-related INDEL polymorphisms could be evaluated. The major- ity of the VNTRs belonged to a semi-conserved region in Measures of diversityh The expected heterozygosity (HE) was calculated using FSTAT version 1.2 based on previously described formula HE = n n −1 1 −n i = 1p2 where p is the frequency of the ith allele and n is the number of alleles sampled [26]. LIAN version 3.7 was used for analyzing overall multilocus linkage disequilibrium (LD) implementing a standardized index of association ( IS A ). LD for candidate genotyping markers with p-values < 0.05 was considered as significant [68]. Dendogram for microsatellite frag- ment analysis data was constructed using ClustVis [69]. Blastx (https://blast.ncbi.nlm.nih.gov/Blast.cgi?LINK_ LOC=blasthome&PAGE_TYPE=BlastSearch&PROGR AM=blastx) without low-complexity filter was used for identification of regions targeted by primers. Tandemly repeated sequences and copy numbers were identified by using TRF version 4.09 [70]. The number of haplotypes (H), haplotype diversity (Hd) and pairwise nucleotide diversity (π) were evaluated using DnaSP v5 [34]. Phylo- genetic tree was constructed using Mega version 7.0 [71]. Sensitivity and specificityhi The specificity of primers was assessed on samples from symptomatic P. malariae patients (Additional file 1: Table S2) compared to the reference nested-PCR method targeting the 18S rRNA [60]. Specificity of all primer products were checked for amplification of unspecific products to access true positive results. The specificity of the markers was tested by two approaches: (i) BLAST Mathema et al. Malar J (2020) 19:48 Page 6 of 12 Fig. 2 Schematic overview of the Plasmodium malariae molecular marker development and validation used in the current study and their potential implementation absence of a linkage disequilibrium. Sequence analysis of VNTR-based allelic frequencies in semi-conserved regions of pmmsp1 gene interspersed between cod- ing blocks identified 14 distinct alleles (Fig. 3g). A copy number of 2.4 with consensus sequence “GAACAA GCAGAAACAACGGGAACAACA” located at pmmsp1 nucleotide position 2312–2374 bp was the most frequent observed VNTR (40%). Considerable nucleotide diversity (π = 0.0976) and haplotype gene diversity (Hd = 0.698) were observed. Linkage disequilibrium was significant (p-value < 0.01) for segregating sites. The combined like- lihood that any two samples by chance would have the same genotypes was as low as 8.46 × 10−4 for six micros- atellites markers and further reduced to 1.68 × 10−4 when pmmsp1 polymorphisms were included (Table 3). block 8 of pmmsp1 gene (Fig. 1) and contained imperfect repeats of the consensus sequence with a length of 27 bp and with copy numbers in range 2.4–23.9 resulting in allele length in range 64–645 bp (Additional file 2: Sup- plementary information). In the remaining samples, lack of PCR efficiency was the most likely cause of failure to amplify the pmmsp1 gene. Allelic frequencies and measures of marker diversityh Allelic frequencies and measures of marker diversity The six microsatellite markers, namely, Pm05_707, Pm06_506, Pm07_429, Pm09_801, Pm12_426 and Pm13_110 displayed corresponding HE (distinct alleles) of 0.649(5), 0.542(9), 0.764(10), 0.530(5), 0.922(13) and 0.623(5), respectively. Observed allele size ranged from 146 to 269 bp resulting in high diversity of allele frequen- cies (Fig. 3a–f). Candidate genotyping markers were situ- ated on different chromosomes (Table 1, Fig. 4). Linkage analysis showed a IA S = 0.03 (p-value > 0.05), suggesting Mathema et al. Malar J (2020) 19:48 Page 7 of 12 Fig. 3 Genetic richness and allele frequency. Allele frequencies observed for each microsatellite markers (a) Pm05_707, (b) Pm06_506, (c) Pm07_429, (d) Pm09_801, (e) Pm12_426 and (f) Pm13_11, respectively. g Allele frequency based on VNTR observed for P. malariae msp1 gene. VNTR, variable number of tandem repeats; msp, merozoite surface protein Fig. 3 Genetic richness and allele frequency. Allele frequencies observed for each microsatellite markers (a) Pm05_707, (b) Pm06_506, (c) Pm07_429, (d) Pm09_801, (e) Pm12_426 and (f) Pm13_11, respectively. g Allele frequency based on VNTR observed for P. malariae msp1 gene. VNTR, variable number of tandem repeats; msp, merozoite surface protein Fig. 3 Genetic richness and allele frequency. Allele frequencies observed for each microsatellite markers (a) Pm05_707, (b) Pm06_506, (c) Pm07_429, (d) Pm09_801, (e) Pm12_426 and (f) Pm13_11, respectively. g Allele frequency based on VNTR observed for P. malariae msp1 gene. VNTR, variable number of tandem repeats; msp, merozoite surface protein Identification of MOI by genetic markersi genotyping markers for P. malariae, supplement the widely used microsatellite markers developed for P. fal- ciparum [21, 28] and P. vivax [20, 74]. Unlike some of the previous markers [13], all presently identified mark- ers were located in different chromosomes and did not show any significant linkage disequilibrium, suggest- ing better discriminative potentials of these markers. Microsatellite markers are valuable tools for multilocus genotyping, and newly identified markers were able to clearly discriminate multiple P. malariae genotypes in naturally acquired symptomatic infections. Compari- son of discriminative power associated with different genotyping markers in separate studies is complicated and often incomparable since heterozygosity values for a single marker can greatly vary between study sites [20, 72]. The choice of marker, genotyping technique and underlying genetic diversity of study groups affect outcomes of each study. Nonetheless, the heterozygo- sity displayed by six newly identified (0.542–0.922) and Genotyping markers identified up to 13 distinct alleles for microsatellite markers and 14 for pmmsp1 mark- ers (Fig. 3). Pm12_426 displayed the highest mean MOI (1.216) followed by Pm05_707 (Table 4). Because of their high allelic diversity and heterozygosity, the Pm12_426 and pmmsp1 markers were most potent for detecting MOI (Fig. 3, Table 3). Discussionh The genetic epidemiology of P. malaria is largely unknown. Availability of a comprehensive set of appropriate genetic markers is a prerequisite for advancing this field. The present study identifies six genotyping microsatellites and a set of VNTR-based pmmsp1 INDEL polymorphism markers in P. malariae. In contrast, allelic frequencies of microsatellites are generally higher and more evenly distributed due to absence of selective pressure. The newly identified six Mathema et al. Malar J (2020) 19:48 Page 8 of 12 Fig. 4 Location of the genotyping markers in Plasmodium malariae UG01. Different features indicated by the map for microsatellite distribution from outermost to innermost ring can be interpreted as: chromosome 1-14 (I-XIV) in Mbp, scatter plota for genomic microsatellite distribution based on unit motif length which corresponds to the height of spot from base of its ring, line plot with peaks indicating regions with long repeat length and heatmapb corresponding to the aggregate genomic microsatellite for the region. aSpots and regions in scatter plot and bheatmap may appear overlapped due to high density but are physically apart in sequence. The approximate location of newly identified genotyping markers are indicated with white circles and labeled accordingly; Mbs, mega base pairs Fig. 4 Location of the genotyping markers in Plasmodium malariae UG01. Different features indicated by the map for microsatellite distribution from outermost to innermost ring can be interpreted as: chromosome 1-14 (I-XIV) in Mbp, scatter plota for genomic microsatellite distribution based on unit motif length which corresponds to the height of spot from base of its ring, line plot with peaks indicating regions with long repeat length and heatmapb corresponding to the aggregate genomic microsatellite for the region. aSpots and regions in scatter plot and bheatmap may appear overlapped due to high density but are physically apart in sequence. The approximate location of newly identified genotyping markers are indicated with white circles and labeled accordingly; Mbs, mega base pairs P. vivax [67, 75] and P. falciparum [28, 76] which gener- ally involves use of 10–13 sets of microsatellite markers. In particular, the newly identified marker Pm12_426 expressed HE value of 0.922 which was noticeably higher than previously reported highest HE of 0.811 [13]. The observed HE are promising, since the samples six previously reported (0.192–0.849) microsatellite markers for P. malariae [13, 14] could be used together to access higher polymorphisms with samples involving geographically large regions. Discussionh Moreover, the combined set of markers would facilitate microsatellite genotype of P. malariae populations in similar ranks to those of Mathema et al. Malar J (2020) 19:48 Page 9 of 12 Table 3 Probabilities of the coincidence of the same Plasmodium malariae genotype a Ordered according to increasing power of P for microsatellite genotyping markers except for pmmsp1 due to difference in marker type b Combined cumulative probabilities πPi calculated as πPi = P1 × P2 × …Pi S. no. Molecular markera Probability ( P = p2 i ) for each marker Combined probabilities Combined probability values (πPi)b 1 Pm12_426 0.102 P1 = PPm12_426 1.02000 ×10−1 2 Pm07_429 0.255 P2 = P2 × PPm07_429 2.60100 ×10−2 3 Pm05_707 0.369 P3 = P3 × PPm05_707 9.59769 ×10−3 4 Pm13_110 0.394 P4 = P4 × PPm13_110 3.78149 ×10−3 5 Pm06_506 0.462 P5 = P5 × PPm06_506 1.74705 ×10−3 6 Pm09_801 0.484 P6 = P6 × PPm09_801 8.45571 ×10−4 7 pmmsp1 0.196 P7 = P7 × Ppmmsp1 1.65732 ×10−4 Table 3 Probabilities of the coincidence of the same Plasmodium malariae genotype Table 4 Characteristics of polymorphic microsatellite loci detected in Plasmodium malariae (N = 37) a The repeat number of each microsatellite unit motif b Total numbers of alleles including both dominant and minor alleles detected c Expected Heterozygosity (HE) was calculated from a restricted data set containing only the dominant allele in each sample d The allele based on consensus sequence of variable number of tandem repeats S. no. Locus Repeat unita Allele size range (bp) Total no. of alleles detectedb No. of distinct alleles Expected heterozygosity (HE)c Mean MOI 1 Pm05_707 (AAT)9 162–180 42 5 0.649 1.135 2 Pm06_506 (ACAT)40 175–280 41 9 0.542 1.081 3 Pm07_429 (AAT)14 203–269 40 10 0.764 1.081 5 Pm09_801 (ACAT)9 210–266 40 5 0.530 1.081 4 Pm12_426 (ATC)21 164–208 45 13 0.922 1.216 6 Pm13_110 (AAT)10 146–158 39 5 0.623 1.054 7 pmmsp1 VNTRd 64–645 27 14 0.835 1.000 Table 4 Characteristics of polymorphic microsatellite loci detected in Plasmodium malariae (N = 37) b Total numbers of alleles including both dominant and minor alleles detected c Expected Heterozygosity (HE) was calculated from a restricted data set containing only the dominant allele in each sample d The allele based on consensus sequence of variable number of tandem repeats is relatively inexpensive compared to INDEL and SNP analysis [79, 80]. Discussionh Moreover, the amplification and geno- typing stages might be adopted to multiplex different loci, saving costs, time and facilitating large scale stud- ies [23, 25]. involved for validation were collected within same year and from geographically small region. Such features with high heterozygosity tend to make these markers suitable candidate for linkage mapping, which requires highly polymorphic markers [77]. The mean MOI for newly identified microsatellite markers (1.2 ± 0.1), was appreciable compared to previously published mark- ers with mean MOI 1.12–1.32 [13]. Observed allelic diversity, high sensitivity and specificity (≥ 97%) for all six microsatellite markers suggests promising potency for population structure and epidemiological studies. All markers were unlikely to be in coding regions and were positioned within highly repetitive and AT-rich regions of the genome, which increases likelihood of these markers achieving higher heterozygosity in larger population size [78]. Likewise, the combined likelihood enhances the sensitivity of these markers by highly reducing identification of same genotype by chance, which enables utility of the markers in low transmis- sion settings. The microsatellite genotyping method VNTR-targeted INDEL analysis indicated promising ability of pmmsp1 gene polymorphisms to identify vari- ants within the study population. The most frequently identified VNTRs with high heterozygosity were situ- ated in pmmsp1 semi-conserved variable block 8, making it a potential genetic marker for P. malariae population study. The msp1 gene block 8 in previous study for P. fal- ciparum and P. vivax had less than 45% interspecies iden- tity 1 [40]. Presence of polymorphisms for msp1 block-2 have been reported for P. falciparum [81, 82], however, information on the P. malariae homologue is scarce. In present study, the pmmsp1 polymorphisms marker showed highly imbalanced distribution of its most com- mon allele (40% for the A64 fragment) which might be explained by natural selection. The observed linkage Mathema et al. Malar J (2020) 19:48 Mathema et al. Malar J (2020) 19:48 Page 10 of 12 disequilibrium for pmmsp1 polymorphisms is likely caused by SNPs grouped into haplotype blocks which often harbour limited number of distinct haplotypes [83]. Unlike microsatellites, the larger differences arising due to variation in copy numbers from VNTR in the ampli- fied region is easily visualized by gel electrophoresis facilitating easy interpretation. Ethics approval and consent to participate Ethical approval for the study was obtained from the ethical review board of the Faculty of Tropical Medicine, Mahidol University (TMEC 11-019). Consent for publication Not applicable. Consent for publication Not applicable. Funding Th The research was funded by postdoctoral research sponsorship of Mahidol University, Thailand, Thailand Science Research and Innovation (TSRI), Grant No. RTA6280006 and the Wellcome Trust of Great Britain, UK. Abbreviations bp: base pair; CDS: protein-coding-regions; HE: heterozygosity; Hd: genetic diversity; INDEL: insertion deletion; Kbp: kilo base pair; Mbp: million base pair; msp: merozoite surface protein; ORFs: open reading frame; RFU: relative fluo- rescent unit; SNP: single nucleotide polymorphisms; VNTR: variable number tandem repeat. Author details 1 Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. 2 Mahidol–Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol Univer- sity, Bangkok, Thailand. 3 Medical Action Myanmar, Yangon, Myanmar. 4 Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK. Competing interests h h d l h The authors declare that they have no competing interests. Discussionh However, template DNA required for INDEL analysis was more vulnerable to low DNA template quality which likely related to the larger amplicon size, and resulted in a smaller number of sam- ple with successful amplification of the pmmsp1 gene. Nonetheless, the high allelic diversity and heterozygosity observed for well-sequenced samples indicated feasibil- ity of this polymorphisms to be exploited for popula- tion genetic studies. The mean MOI for newly identified microsatellite markers were in range 1.10–1.20, similar to previously reported markers with mean MOI range of 1.12–1.32 [13]. The differences in observed genotypes and MOI by Pm12_426 and pmmsp1 markers for same population could indicate either greater transmission intensities or merely differences in the resolution of these molecular markers. Results from dendrograms and phy- logenetic tree suggested similar outcomes while utilizing these markers to cluster population (Additional file 3: Fig. S1) and identify highly segregating variants. VNTR, variable number of tandem repeats; pmmsp1, Plasmodium malariae merozoite surface protein 1. VNTR, variable number of tandem repeats; pmmsp1, Plasmodium malariae merozoite surface protein 1. Additional file 3: Figure S1. Dendrogram and phylogenetic tree for P. malariae samples (A) Dendrogram was constructed for microsatellite markers with rows clustered using correlation distance and complete link- age. The columns were clustered using correlation distance and average linkage (N = 37). (B) Phylogenetic tree constructed ws constructed using neighbor joining method for pmmsp1 gene sequence INDEL polymor- phisms (N = 27). Figure A and B are not equivalent representative of the population clustering as type of genetic markers and sample size used for analysis are different. Acknowledgements We would like to acknowledge the postdoctoral research sponsorship of Mahidol University for providing necessary funding for the research. We thank Miss. Kanokon Suwannasin, and Mr. Chanon Kunason for their help. Conclusions I In summary, the newly developed genotyping micros- atellite markers and pmmsp1 gene polymorphisms may provide an important tool for studies in P. malariae. Practical applications include discrimination between disease recrudescence and reinfection in drug efficacy trials, studies on gene flow, parasite selection, population relatedness, signatures of selection and genetic diversity as a measure of transmission intensity and other genetic epidemiological questions. In addition to microsatellite typing, the VNTR-associated polymorphisms observed in semi-conserved block 8 of pmmsp1 gene are useful for assessing genetic diversity in P. malariae. Supplementary information Supplementary information accompanies this paper at https://doi. org/10.1186/s12936-020-3122-2. Supplementary information ac org/10.1186/s12936-020-3122-2. Additional file 1: Table S1. Genome-wide coverage and density of microsatellites in genome of five human malaria causing Plasmodium spe- cies. Table S2. Number and geographical origin of Plasmodium species and strains used in the present study. Table S3. Sensitivity and specificity estimates of the markers in method development. Received: 20 November 2019 Accepted: 13 January 2020 Received: 20 November 2019 Accepted: 13 January 2020 Authors’ contributions VBM and MI designed the study. VBM and SN preformed the experiments and data analysis and wrote first draft of the manuscript. WP, SN and MI assisted in part of data analysis. FS provided samples. MI, NJW and AMD assisted in logistic support and manuscript preparation. All authors read and approved the final manuscript. Availability of data and materials The dataset generated during the current study are available from corre- sponding author on reasonable request. The dataset generated during the current study are available from corre- sponding author on reasonable request. 1. Lo E, Nguyen K, Nguyen J, Hemming-Schroeder E, Xu J, Etemesi H, et al. Plasmodium malariae prevalence and csp gene diversity, Kenya, 2014 and 2015. Emerg Infect Dis. 2017;23:601–10. 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https://openalex.org/W4391880180	https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1308543/pdf	English	null	Serum alpha-fetoprotein response as a preoperative prognostic indicator in unresectable hepatocellular carcinoma with salvage hepatectomy following conversion therapy: a multicenter retrospective study	Frontiers in immunology	2,024	cc-by	8,182	Serum alpha-fetoprotein response as a preoperative prognostic indicator in unresectable hepatocellular carcinoma with salvage hepatectomy following conversion therapy: a multicenter retrospective study Kong-Ying Lin 1,2†, Jian-Xi Zhang 2,3†, Zhi-Wen Lin 1,2†, Qing-Jing Chen 1,2†, Liu-Ping Luo 1†, Jin-Hong Chen 4, Kui Wang 5, Sheng Tai 6, Zhi-Bo Zhang 2, Shi-Feng Wang 7, Jing-Dong Li 8, Kai Wang 9, Lu Zheng 10, Si-Ming Zheng 11, Meng-Meng Wu 1, Ke-Can Lin 1, Tian Yang 5* and Yong-Yi Zeng 1,2* Serum alpha-fetoprotein response as a preoperative prognostic indicator in unresectable hepatocellular carcinoma with salvage hepatectomy following conversion therapy: a multicenter retrospective study OPEN ACCESS EDITED BY John Gibbs, Hackensack Meridian Health, United States REVIEWED BY Qingqiang Ni, Shandong Provincial Hospital, China Abdullah Esmail, Houston Methodist Hospital, United States CORRESPONDENCE Yong-Yi Zeng lamp197311@126.com Tian Yang yangtiandfgd@hotmail.com †These authors have contributed equally to this work RECEIVED 06 October 2023 ACCEPTED 02 February 2024 PUBLISHED 16 February 2024 CITATION Lin K-Y, Zhang J-X, Lin Z-W, Chen Q-J, Luo L-P, Chen J-H, Wang K, Tai S, Zhang Z-B, Wang S-F, Li J-D, Wang K, Zheng L, Zheng S-M, Wu M-M, Lin K-C, Yang T and Zeng Y-Y (2024) Serum alpha- fetoprotein response as a preoperative prognostic indicator in unresectable hepatocellular carcinoma with salvage hepatectomy following conversion therapy: a multicenter retrospective study. Front. Immunol. 15:1308543. doi: 10.3389/fimmu.2024.1308543 OPEN ACCESS EDITED BY John Gibbs, Hackensack Meridian Health, United States REVIEWED BY Qingqiang Ni, Shandong Provincial Hospital, China Abdullah Esmail, Houston Methodist Hospital, United States CORRESPONDENCE Yong-Yi Zeng lamp197311@126.com Tian Yang yangtiandfgd@hotmail.com †These authors have contributed equally to this work RECEIVED 06 October 2023 ACCEPTED 02 February 2024 PUBLISHED 16 February 2024 CITATION Lin K-Y, Zhang J-X, Lin Z-W, Chen Q-J, Luo L-P, Chen J-H, Wang K, Tai S, Zhang Z-B, Wang S-F, Li J-D, Wang K, Zheng L, Zheng S-M, Wu M-M, Lin K-C, Yang T and Zeng Y-Y (2024) Serum alpha- fetoprotein response as a preoperative prognostic indicator in unresectable hepatocellular carcinoma with salvage hepatectomy following conversion therapy: a multicenter retrospective study. Front. Immunol. 15:1308543. doi: 10.3389/fimmu.2024.1308543 CITATION Lin K-Y, Zhang J-X, Lin Z-W, Chen Q-J, Luo L-P, Chen J-H, Wang K, Tai S, Zhang Z-B, Wang S-F, Li J-D, Wang K, Zheng L, Zheng S-M, Wu M-M, Lin K-C, Yang T and Zeng Y-Y (2024) Serum alpha- fetoprotein response as a preoperative prognostic indicator in unresectable hepatocellular carcinoma with salvage hepatectomy following conversion therapy: a multicenter retrospective study. Front. Immunol. 15:1308543. TYPE Original Research PUBLISHED 16 February 2024 DOI 10.3389/fimmu.2024.1308543 TYPE Original Research PUBLISHED 16 February 2024 DOI 10.3389/fimmu.2024.1308543 Abbreviations: HCC, hepatocellular carcinoma; RFS, recurrence-free survival; TKIs, tyrosine kinase inhibitors; PD-1, programmed death-1; mRECIST, modiﬁed Response Evaluation Criteria in Solid Tumors; HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; HCV, hepatitis C virus; TACE, transarterial chemoembolization; HAIC, hepatic arterial infusion chemotherapy; AFP, alpha-fetoprotein; CR, complete response; PR, partial response; SD, stable disease; MVI, microvascular invasion; BCLC, Barcelona Clinic Liver Cancer. Serum alpha-fetoprotein response as a preoperative prognostic indicator in unresectable hepatocellular carcinoma with salvage hepatectomy following conversion therapy: a multicenter retrospective study Kong-Ying Lin 1,2†, Jian-Xi Zhang 2,3†, Zhi-Wen Lin 1,2†, Qing-Jing Chen 1,2†, Liu-Ping Luo 1†, Jin-Hong Chen 4, Kui Wang 5, Sheng Tai 6, Zhi-Bo Zhang 2, Shi-Feng Wang 7, Jing-Dong Li 8, Kai Wang 9, Lu Zheng 10, Si-Ming Zheng 11, Meng-Meng Wu 1, Ke-Can Lin 1, Tian Yang 5* and Yong-Yi Zeng 1,2* The median RFS was not reached for AFP responders, with 1-year and 2-year RFS rates of 81.3% and 70.8%, respectively. In contrast, AFP non-responders had a median RFS of 7.43 months, with 1-year and 2-year RFS rates at 37.1% and 37.1%, respectively. Multivariate Cox regression analysis identiﬁed AFP response as an independent predictor of RFS. Integrating AFP response with radiologic tumor response facilitated further stratiﬁcation of patients into distinct risk categories: those with radiologic remission experienced the most favorable RFS, followed by patients with partial response/stable disease and AFP response, and the least favorable RFS among patients with partial response/stable disease but without AFP response. Sensitivity analyses further conﬁrmed the association between AFP response and improved RFS across various cutoff values and in patients with AFP ≥200 ng/mL at diagnosis (all P<0.05). Conclusion: The “20-80” rule based on AFP response could be helpful for clinicians to preoperatively stratify the risk of patients undergoing salvage hepatectomy, enabling identiﬁcation and management of those unlikely to beneﬁt from this procedure. KEYWORDS hepatocellular carcinoma, salvage resection, alpha-fetoprotein response, recurrence- free survival, conversion therapy Results: AFP responders demonstrated signiﬁcantly better postoperative RFS compared to non-responders (P<0.001). The median RFS was not reached for AFP responders, with 1-year and 2-year RFS rates of 81.3% and 70.8%, respectively. In contrast, AFP non-responders had a median RFS of 7.43 months, with 1-year and 2-year RFS rates at 37.1% and 37.1%, respectively. Multivariate Cox regression analysis identiﬁed AFP response as an independent predictor of RFS. Integrating AFP response with radiologic tumor response facilitated further stratiﬁcation of patients into distinct risk categories: those with radiologic remission experienced the most favorable RFS, followed by patients with partial response/stable disease and AFP response, and the least favorable RFS among patients with partial response/stable disease but without AFP response. Sensitivity analyses further conﬁrmed the association between AFP response and improved RFS across various cutoff values and in patients with AFP ≥200 ng/mL at diagnosis (all P<0.05). Conclusion: The “20-80” rule based on AFP response could be helpful for clinicians to preoperatively stratify the risk of patients undergoing salvage hepatectomy, enabling identiﬁcation and management of those unlikely to beneﬁt from this procedure. KEYWORDS hepatocellular carcinoma, salvage resection, alpha-fetoprotein response, recurrence- free survival, conversion therapy Serum alpha-fetoprotein response as a preoperative prognostic indicator in unresectable hepatocellular carcinoma with salvage hepatectomy following conversion therapy: a multicenter retrospective study Kong-Ying Lin 1,2†, Jian-Xi Zhang 2,3†, Zhi-Wen Lin 1,2†, Qing-Jing Chen 1,2†, Liu-Ping Luo 1†, Jin-Hong Chen 4, Kui Wang 5, Sheng Tai 6, Zhi-Bo Zhang 2, Shi-Feng Wang 7, Jing-Dong Li 8, Kai Wang 9, Lu Zheng 10, Si-Ming Zheng 11, Meng-Meng Wu 1, Ke-Can Lin 1, Tian Yang 5* and Yong-Yi Zeng 1,2* doi: 10.3389/fimmu.2024.1308543 1Department of Hepatopancreatobiliary Surgery, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, China, 2Department of Hepatopancreatobiliary Surgery, First Afﬁliated Hospital of Fujian Medical University, Fuzhou, China, 3Department of Hepatobiliary Surgery, Xiamen Hospital, Beijing University of Chinese Medicine, Xiamen, China, 4Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Fudan University, Shanghai, China, 5Department of Hepatobiliary Surgery, Eastern Hepatobiliary Surgery Hospital, Navy Medical University (Second Military Medical University), Shanghai, China, 6Department of Hepatopancreatobiliary Surgery, Second Afﬁliated Hospital of Harbin Medical University, Harbin, China, 7Department of Hepatopancreatobiliary Surgery, Ganzhou Fifth People’s Hospital of Gannan Medical University, Ganzhou, China, 8Department of Hepatopancreatobiliary Surgery, The Afﬁliated Hospital of North Sichuan Medical College, Sichuan, China, 9Department of Hepatopancreatobiliary Surgery, Second Afﬁliated Hospital of Nanchang University, Nanchang, China, 10Department of Hepatopancreatobiliary Surgery, Second Afﬁliated Hospital of Army Medical University, Chongqing, China, 11Department of Hepatopancreatobiliary Surgery, First Afﬁliated Hospital of Ningbo University, Ningbo, China Front. Immunol. 15:1308543. doi: 10.3389/fimmu.2024.1308543 © 2024 Lin, Zhang, Lin, Chen, Luo, Chen, Wang, Tai, Zhang, Wang, Li, Wang, Zheng, Zheng, Wu, Lin, Yang and Zeng. This is an open-access article distributed under the terms © 2024 Lin, Zhang, Lin, Chen, Luo, Chen, Wang, Tai, Zhang, Wang, Li, Wang, Zheng, Zheng, Wu, Lin, Yang and Zeng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Background: This study evaluates the efﬁcacy of alpha-fetoprotein (AFP) response as a surrogate marker for determining recurrence-free survival (RFS) in patients with unresectable hepatocellular carcinoma (uHCC) who undergo salvage hepatectomy following conversion therapy with tyrosine kinase inhibitor (TKI) and anti-PD-1 antibody-based regimen. Methods: This multicenter retrospective study included 74 patients with uHCC and positive AFP (>20 ng/mL) at diagnosis, who underwent salvage hepatectomy after treatment with TKIs and anti-PD-1 antibody-based regimens. The association between AFP response—deﬁned as a ≥80% decrease in ﬁnal AFP levels before salvage hepatectomy from diagnosis—and RFS post-hepatectomy was investigated. 01 Frontiers in Immunology frontiersin.org Lin et al. 10.3389/fimmu.2024.1308543 10.3389/fimmu.2024.1308543 Results: AFP responders demonstrated signiﬁcantly better postoperative RFS compared to non-responders (P<0.001). Conventional regimens deﬁnition of AFP response, with cutoff values often ranging between 20% and 50%, its utility as an alternative indicator for disease progression or reaction to various treatments—including radical resection, transarterial chemoembolization, tyrosine kinase inhibitors, immune checkpoint inhibitors, and combination therapies—has been widely acknowledged (22–25). The role of AFP response in risk stratiﬁcation and treatment response evaluation continues to attract signiﬁcant scientiﬁc interest. As a retrospective observational study, no interventions were made to the patient’s treatment protocols. All conversion plans adhered to the latest evidence for effectiveness and safety in antitumor therapy for HCC, established through shared decision- making with the patients. All patients received at least one cycle of TKI combined with a- PD-1 therapy, with almost all receiving concomitant transcatheter intra-arterial therapy including transarterial chemoembolization (TACE) or hepatic arterial infusion chemotherapy (HAIC). For patients who received transcatheter intra-arterial therapies, initiation of TKIs and a-PD-1 agents typically followed within one week post-treatment. The TKIs utilized in this study are all recommended by the Chinese primary liver cancer treatment guidelines for the ﬁrst-line treatment of advanced HCC (lenvatinib and sorafenib), except for apatinib, which is recommended in combination with camrelizumab (a-PD-1) as the ﬁrst-line treatment for advanced HCC by Chinese guidelines. The a-PD-1 agents in this study encompassed camrelizumab, tislelizumab, pembrolizumab, toripalimab, and sintilimab. The selection of each therapeutic agent was inﬂuenced by its availability, institutional preferences, and the ﬁnancial circumstances of the patients. Administration of TKIs and a-PD- 1 agents, including dosages, frequencies, and adjustments, adhered strictly to the guidelines speciﬁed on the respective drug labels. In this multicenter, retrospective study, we enrolled 74 patients with initially unresectable HCC and positive AFP at diagnosis (> 20 ng/mL) who underwent salvage hepatectomy following treatment with a combination regimen of tyrosine kinase inhibitors (TKIs) and anti-PD-1 antibodies (a-PD-1). Our objective was to assess the utility of AFP response as a preoperative surrogate marker for recurrence-free survival after salvage hepatectomy. To our knowledge, this is the ﬁrst study to explore the application of AFP response in patients undergoing salvage hepatectomy, potentially offering valuable insights for clinical decision-making in this context. Introduction However, postoperative recurrence constitutes the principal hindrance to a favorable prognosis post-salvage hepatectomy (11, 12). Current clinical practice faces challenges due to the lack of preoperative criteria guiding salvage hepatectomy, such as identifying suitable patients or determining the optimal timing for the procedure (13). Given the invasive nature of salvage hepatectomy, there is a pressing necessity for dependable, replicable, and non-invasive preoperative prognostic markers that can accurately identify patients unlikely to beneﬁt from this surgical approach. Liver resection constitutes the primary therapeutic modality for hepatocellular carcinoma (HCC) (1–4). Nevertheless, a considerable subset of patients is often precluded from liver resection in clinical settings, owing to extensive tumor burden, compromised liver function, or the presence of extrahepatic metastases. The emergence of multidisciplinary therapeutic strategies, anchored in surgical resection, offers potential curative avenues for those initially deemed inoperable for HCC (5–7). Numerous studies highlight the importance of salvage hepatectomy for patients who become eligible for surgical resection (8–10). Conceptually, Salvage hepatectomy has the potential to ensure total tumor removal, effectively circumventing the resistance and side effects associated with systemic or localized therapies, thereby enhancing long-term oncological outcomes. Alpha-fetoprotein (AFP), a straightforward, rapid, and routinely employed clinical biomarker, is extensively utilized for HCC screening, diagnosis, and risk stratiﬁcation (14–16). Serum AFP levels are recognized as an ideal surrogate biomarker for tumor burden, with higher levels often indicative of more aggressive tumor biology. The prognostic stratiﬁcation capacity of AFP levels has been substantiated through numerous studies, leading to its incorporation as a pivotal variable in esteemed HCC prognostic scoring systems, such as the Cancer of the Liver Italian Program score (17), Chinese University Prognostic Index (18), Metroticket 2.0 model (15), and French AFP model (16). In addition to these roles, dynamic changes in AFP levels, commonly referred to as AFP response, have been validated as effective in monitoring the efﬁcacy of anti-tumor treatments, where decreases post-treatment suggest an encouraging overall therapeutic response (19–21). Despite the lack of consensus in previous research regarding the precise 02 Frontiers in Immunology frontiersin.org Lin et al. Lin et al. 10.3389/fimmu.2024.1308543 Study population A retrospective analysis was performed on standardized data of 101 patients who underwent salvage hepatectomy following conversion therapy based on TKI and a-PD-1 for initially uHCC between November 25, 2019, and August 31, 2022, from 10 tertiary academic hospitals, including Mengchao Hepatobiliary Hospital of Fujian Medical University, Eastern Hepatobiliary Surgery Hospital of the Naval Medical University, Huashan Hospital Afﬁliated to Fudan University, First Afﬁliated Hospital of Fujian Medical University, Ganzhou Fifth People’s Hospital of Gannan Medical University, Second Afﬁliated Hospital of Harbin Medical University, Second Afﬁliated Hospital of Nanchang University, First Afﬁliated Hospital of Ningbo University, Second Afﬁliated Hospital of Army Medical University, and The Afﬁliated Hospital of North Sichuan Medical College. HCC diagnoses were based on the American Association for the Study of Liver Diseases guidelines and Chinese Guidelines for the Diagnosis and Treatment of Primary Liver Cancer (2, 3). Tumor unresectability was deﬁned according to surgical technique limitations and/or oncological outcomes (13). Technical unresectability pertains to the inability to perform a safe radical resection (R0 resection), due to inadequate residual liver volume (<40% for cirrhotic patients and <30% for non-cirrhotic patients) or anatomical constraints associated with the tumor’s proximity to major vascular structures. Oncological unresectability involves advanced disease characterized by aggressive tumor features such as major vascular invasion, multiple lesions beyond the up-to-seven criteria, or extrahepatic metastases. The ethical committees of all involved medical centers approved this study, adhering to the Declaration of Helsinki guidelines. All patients provided written informed consent before initiating conversion therapy and undergoing salvage hepatectomy. Experienced interventional radiologists at each participating center performed both HAIC and TACE procedures. The HAIC protocol adhered to the FOLFOX-HAIC regimen, comprising Oxaliplatin (130 mg/m2) infused over 2-3 hours, followed by Leucovorin (400 mg/m2) over 1-2 hours, then a 5-Fluorouracil bolus (400 mg/m2), and a continuous infusion of 2400 mg/m2 over 23 hours (26). This procedure was repeated every three weeks, with each session involving temporary catheter insertion. For TACE, after achieving super-selective catheterization of the tumor’s arterial supply, a mixture of epirubicin and iodized oil (up to 30ml) was gradually introduced under ﬂuoroscopic guidance. This continued until sufﬁcient deposition of iodized oil in the tumor lesions and a noticeable reduction in the blood supply were achieved. This was followed by the injection of gelatin sponge particles, continuing until blood ﬂow at the microcatheter tip ceased completely. The TACE procedure was repeated as deemed necessary. Statistics contraindications for liver resection. Prior to undergoing salvage hepatectomy, patients had tyrosine kinase inhibitors (TKIs) withdrawn at least one week preoperatively, and a-PD-1 inhibitors discontinued for a minimum of two weeks. Descriptive statistics were used to analyze the demographic and clinical data of patients. Continuous variables were expressed as mean ± standard deviation or median (interquartile range), and differences were tested using Student’s t-test or the Mann-Whitney U-test. Categorical variables were expressed as frequencies (percentages) and tested using chi-square tests or Fisher’s exact tests. RFS after salvage hepatectomy was depicted using the Kaplan- Meier method, and differences between groups were compared using the log-rank test. A multivariable Cox proportional hazards model was used to assess the preoperative prognostic value of AFP response, adjusting for potential confounding clinical variables. Preoperative clinical variables with a P value less than 0.1 in univariate analysis were included in the multivariable Cox proportional hazards regression analysis to determine the ﬁnal independent preoperative risk factors. All statistical analyses were two-tailed, and differences with a P value less than 0.05 were considered statistically signiﬁcant. Statistical analyses were performed using SPSS version 20 (SPSS, Inc., Chicago, IL, USA) and R version 4.1.1 (R Project, Vienna, Austria). For complex hepatectomies, preoperative 3D reconstruction was routinely utilized to facilitate the development of a tailored surgical plan. This plan encompassed both anatomical and partial liver resections, determining the extent of resection based on factors such as tumor location, size, number, and the patient’s underlying liver condition. Laparoscopic or open hepatectomy was conducted as previously reported, with a preference for anatomical liver resection where applicable. Extent of resection is divided into major hepatectomy and minor hepatectomy. Major hepatectomy is deﬁned as the resection of 3 or more Couinaud segments, while minor hepatectomy is deﬁned as the resection of less than 3 Couinaud segments (27). Postoperative follow-up After discharge, patients were monitored in outpatient clinics using a standardized surveillance protocol for HCC recurrence. Follow-up visits were scheduled every 2-3 months during the ﬁrst 2 years post-hepatectomy, followed by biannual visits thereafter. This follow-up regimen encompassed liver function assessments, serum AFP testing, chest computed tomography (CT) scans, abdominal ultrasounds, and liver contrast-enhanced CT or magnetic resonance imaging. Additional diagnostic procedures, such as bone scans or positron emission tomography scans, were employed as indicated for patients with suspected recurrence. Patient characteristics, conversion regimens, and salvage hepatectomy A total of 74 patients with positive serum AFP levels (deﬁned as > 20 ng/mL) at diagnosis were included in the study (Figure 1). The mean age was 52.6 ± 12.3 years, with the majority being male (60, 81.1%). Hepatitis B virus infection was the predominant etiology (65, 87.8%). Liver cirrhosis was present in 44 (59.5%) patients, and 70 (94.6%) were classiﬁed as Child-Pugh A. Regarding tumor characteristics, 34 (45.9%) patients had a single lesion, the mean tumor size was 9.83 ± 4.36 cm, and 58 (78.4%) presented with macrovascular invasion (Table 1). Almost all patients (73, 98.6%) received conversion therapy comprising TKI combined with a-PD-1 and concomitant transcatheter intra-arterial therapy. The median conversion time was 3.44 months (interquartile range [IQR], 2.44, 5.55). Radiological tumor response, assessed using the modiﬁed Response Evaluation Criteria in Solid Tumors, showed that 15 (20.3%) patients achieved complete response, 45 (60.8%) achieved partial response, and 14 (18.9%) had stable disease. The primary surgical approaches for salvage hepatectomy were open surgery (54, 73.0%), and most patients (49, 66.2%) underwent major liver resection. Postoperative pathological evaluation revealed that 18 patients (24.3%) achieved a pathological complete response (Table 1). Salvage hepatectomy During conversion therapy, patients’ general condition and tumor resectability were dynamically assessed. Eligibility for salvage hepatectomy was determined based on the following criteria: a) Feasibility of achieving a safe R0 resection; b) Maintenance of an adequate residual liver volume (≥30% for non-cirrhotic patients, ≥40% for cirrhotic patients); c) Sustained complete or partial response, or stable disease for a minimum of two months in intrahepatic lesions; d) Lack of continuous and severe adverse reactions during conversion therapy; e) Absence of explicit Frontiers in Immunology 03 frontiersin.org Lin et al. Lin et al. 10.3389/fimmu.2024.1308543 10.3389/fimmu.2024.1308543 Deﬁnition of AFP response Data on AFP levels at the time of diagnosis and at the ﬁnal assessment were collected, each measured within one week prior to conversion therapy and salvage hepatectomy, respectively. The formula for calculating AFP variation (%) is deﬁned as: (AFP at diagnosis - AFP ﬁnal)/AFP at diagnosis *100 (%). The optimal cutoff for 80% AFP variation was established using Xtile software, with recurrence-free survival following salvage hepatectomy as the endpoint event (28). Based on this threshold, patients were categorized into two groups: AFP responders (AFP variation ≥80%) and non-responders (AFP variation <80%). To minimize cohort heterogeneity, sensitivity analysis was conducted using previously published AFP response cutoffs of 50% or 20%, to comprehensively assess the clinical relevance of AFP response in predicting recurrence-free survival following salvage hepatectomy (20, 21). Additionally, given the potential for non-tumor-related AFP elevations or ﬂuctuations, particularly in patients with concomitant cirrhosis or hepatitis (typically not exceeding 200ng/ mL), a further sensitivity analysis was performed on a subgroup with baseline AFP levels ≥200ng/mL (21, 29, 30). Frontiers in Immunology AFP variations and AFP response The primary endpoint of this study was recurrence-free survival (RFS) after salvage hepatectomy, which was deﬁned as the time interval from the date of salvage hepatectomy to tumor recurrence, death, or the last follow-up, whichever occurred earliest. The median AFP levels at diagnosis and at the ﬁnal assessment were 1,210 ng/mL (IQR, 292, 8,620) and 44.9 ng/mL (IQR, 7.78, 835), respectively. As depicted in Figure 2, at diagnosis, AFP levels 04 frontiersin.org Lin et al. Lin et al. 10.3389/fimmu.2024.1308543 FIGURE 1 Selection of the study population. HCC hepatocellular carcinoma, PD-1, programmed death 1. GURE 1 election of the study population. HCC hepatocellular carcinoma, PD-1, programmed death 1. TABLE 1 Patient baseline characteristics. AFP variations and AFP response Characteristics Total cohort (n = 74) AFP response, <80% (n = 22) AFP response, ≥80% (n = 52) P Value Age, years, Mean (SD) 52.6 (12.3) 49.1 (10.9) 54.0 (12.6) 0.096 Gender, Male/Female 60 (81.1%)/14 (18.9%) 15 (68.2%)/7 (31.8%) 45 (86.5%)/7 (13.5%) 0.103 Etiology, HBV/HCV/Non-viral 65 (87.8%)/1 (1.4%)/8 (10.8%) 18 (81.8%)/0 (0%)/4 (18.2%) 47 (90.4%)/1 (1.9%)/4 (7.7%) 0.459 Child-Pugh class, A/B 70 (94.6%)/4 (5.4%) 20 (90.9%)/2 (9.1%) 50 (96.2%)/2 (3.8%) 0.577 Liver cirrhosis, Present/Absent 44 (59.5%)/30 (40.5%) 12 (54.5%)/10 (45.5%) 32 (61.5%)/20 (38.5%) 0.575 TKIs, Sorafenib/Lenvatinib/Apatinib 15 (20.3%)/51 (68.9%)/8 (10.8%) 3 (13.6%)/15 (68.2%)/ 4 (18.2%) 12 (23.1%)/36 (69.2%)/4 (7.7%) 0.379 a-PD-1, Camrelizumab/Tislelizumab/ Toripalimab/Sintilimab/Pembrolizumab 35 (47.3%)/6 (8.1%)/4 (5.4%)/16 (21.6%)/13 (17.6%) 11 (50.0%)/3 (13.6%)/1 (4.5%)/ 5 (22.7%)/2 (9.1%) 24 (46.2%)/3 (5.8%)/3 (5.8%)/ 11 (21.2%)/11 (21.2%) 0.627 Combine TACE 61 (82.4%) 19 (86.4%) 42 (80.8%) 0.742 Combine HAIC 12 (16.2%) 3 (13.6%) 9 (17.3%) 1.000 Conversion time, Median (IQR), months 3.44 (2.44, 5.55) 3.28 (2.23, 6.08) 3.44 (2.57, 5.25) 0.799 Radiological response, CR/PR/SD 15 (20.3%)/45 (60.8%)/ 14 (18.9%) 2 (9.1%)/14 (63.6%)/6 (27.3%) 13 (25.0%)/31 (59.6%)/ 8 (15.4%) 0.233 AFP at diagnosis, ng/ml Median (IQR), ng/ml 1210 (292, 8620) 1210 (159, 2460) 1770 (561, 22900) 0.016 <1000/≥1000, ng/ml 31 (41.9%)/43 (58.1%) 10 (45.5%)/12 (54.5%) 21 (40.4%)/31 (59.6%) 0.686 Final AFP, ng/ml Median (IQR), ng/ml 44.9 (7.78, 835) 1040 (110, 4030) 13.2 (6.31, 115) 0.081 <1000/≥1000, ng/ml 57 (77.0%)/17 (23.0%) 11 (50.0%)/11 (50.0%) 46 (88.5%)/6 (11.5%) <0.001 Operative time, Median (IQR), minutes 215 (184, 258) 223 (182, 281) 214 (189, 245) 0.420 Surgical type, Open/Laparotomy 54 (73.0%)/20 (27.0%) 15 (68.2%)/7 (31.8%) 39 (75.0%)/13 (25.0%) 0.546 Intraoperative blood loss, Median (IQR), ml 325 (200, 800) 375 (200, 800) 325 (200, 625) 0.962 Intraoperative blood transfusion, Yes/No 21 (28.4%)/53 (71.6%) 8 (36.4%)/14 (63.6%) 13 (25.0%)/39 (75.0%) 0.322 Extend of hepatectomy, Major/Minor 49 (66.2%)/25 (33.8%) 15 (68.2%)/7 (31.8%) 34 (65.4%)/18 (34.6%) 0.816 (Continued) 05 Frontiers in Immunology frontiersin.org Lin et al. Association of AFP response and AFP levels with RFS for 21 (28.4%) patients ranged from 20 to 400 ng/mL, 10 (13.5%) patients were within the 400 to 1000 ng/mL range, and 43 (58.1%) patients were at or above 1000 ng/mL. Following conversion therapy, there was a notable reduction in ﬁnal AFP levels: 30 (40.5%) patients had levels of ≤20 ng/mL, 24 (32.4%) patients ranged from 20 to 400 ng/mL, 3 (4.1%) patients were within the 400 to 1000 ng/mL range, and 17 (23.0%) patients were at or above 1000 ng/mL. for 21 (28.4%) patients ranged from 20 to 400 ng/mL, 10 (13.5%) patients were within the 400 to 1000 ng/mL range, and 43 (58.1%) patients were at or above 1000 ng/mL. Following conversion therapy, there was a notable reduction in ﬁnal AFP levels: 30 (40.5%) patients had levels of ≤20 ng/mL, 24 (32.4%) patients ranged from 20 to 400 ng/mL, 3 (4.1%) patients were within the 400 to 1000 ng/mL range, and 17 (23.0%) patients were at or above 1000 ng/mL. The median follow-up duration in this study was 14.88 months (IQR, 10.57-24.38). The median RFS for the entire cohort was not reached (95% conﬁdence interval [CI], 22.2, not reached), and the 1- and 2-year RFS rates were 68.0% (95% CI, 57.7%, 80.2%) and 60.0% (95% CI, 48.0%, 75.1%), respectively (Supplementary Figure S1). Survival analysis showed that AFP responders had better postoperative RFS than non-responders (P<0.001, Figure 3A). The median postoperative RFS was not reached in the AFP responder group, with 1-year and 2-year RFS rates of 81.3% (95%CI, 70.8%, 93.2%) and 70.8% (95%CI, 57.1%, 87.8%), respectively. In contrast, the AFP non-responder group had a median postoperative RFS of 7.43 months (95%CI, 4.9, not reached), with 1-year and 2-year RFS rates at 37.1% (95%CI, 20.8%, 66.3%) and 37.1% (95%CI, 20.8%, 66.3%), respectively. The median AFP variation, calculated using the methodological formula, was 94.1% (IQR, 53.2%, 99.2%). The optimal cutoff value of AFP variation determined by Xtile was 80%, dividing patients into AFP responders (n=52, AFP variation ≥80%) and non- responders (n=22, AFP variation <80%). As shown in Table 1, clinical characteristics were generally balanced between the two groups, with the exception of AFP level at diagnosis, ﬁnal AFP levels, and pathological complete response. AFP variations and AFP response 10.3389/fimmu.2024.1308543 TABLE 1 Continued Characteristics Total cohort (n = 74) AFP response, <80% (n = 22) AFP response, ≥80% (n = 52) P Value Final AFP, ng/ml Postoperative hospital day, Median, (IQR), days 11.0 (9.00, 14.0) 10.5 (9.25, 13.8) 11.0 (8.75, 14.0) 0.206 Tumor number, Solitary/Multiple 34 (45.9%)/40 (54.1%) 11 (50.0%)/11 (50.0%) 23 (44.2%)/29 (55.8%) 0.649 Tumor diameter, Mean (SD), cm 9.83 (4.36) 10.3 (4.73) 9.64 (4.23) 0.600 Macrovascular invasion, Present/Absent 58 (78.4%)/16 (21.6%) 15 (68.2%)/7 (31.8%) 43 (82.7%)/9 (17.3%) 0.218 MVI, Present/Absent 21 (28.4%)/53 (71.6%) 8 (36.4%)/14 (63.6%) 13 (25.0%)/39 (75.0%) 0.322 Pathological complete response, Yes/No 18 (24.3%)/56 (75.7%) 1 (4.5%)/21 (95.5%) 17 (32.7%)/35 (67.3%) 0.009 BCLC staging system, A/B/C 4 (5.4%)/12 (16.2%)/58 (78.4%) 2 (9.1%)/5 (22.7%)/15 (68.2%) 2 (3.8%)/7 (13.5%)/43 (82.7%) 0.299 HBV, hepatitis B virus; HCV, hepatitis C virus; TACE, transarterial chemoembolization; HAIC, hepatic arterial infusion chemotherapy; TKI, tyrosine Kinase Inhibitor; PD-1, programmed death 1; AFP, alpha-fetoprotein; CR, complete response; PR, partial response; SD, stable disease; MVI, microvascular invasion; BCLC, Barcelona Clinic Liver Cancer; SD, standard deviation; IQR interquartile range. Bold indicates statistical signiﬁcance. HBV, hepatitis B virus; HCV, hepatitis C virus; TACE, transarterial chemoembolization; HAIC, hepatic arterial infusion chemotherapy; TKI, tyrosine Kinase Inhibitor; PD-1, programmed death 1; AFP, alpha-fetoprotein; CR, complete response; PR, partial response; SD, stable disease; MVI, microvascular invasion; BCLC, Barcelona Clinic Liver Cancer; SD, standard deviation; IQR interquartile range. Bold indicates statistical signiﬁcance. Association of AFP response and AFP levels with RFS The AFP responders had higher median AFP levels at diagnosis (1770 ng/mL vs 1210 ng/ mL, P=0.016), a higher proportion of ﬁnal AFP levels < 1000ng/mL (88.5% vs 50.0%, P<0.001), and a higher rate of pathologic complete response (32.7% vs 4.5%, P=0.009). Additionally, we explored the association between AFP levels at diagnosis, ﬁnal AFP levels, and postoperative RFS. Patients were categorized into high and low AFP groups using previously documented cutoff values of 200 ng/mL, 400 ng/mL, and 1,000 ng/mL. The results revealed that elevated ﬁnal AFP levels correlated with poor RFS, while AFP levels at diagnosis were not indicative of prognosis (Figure 3B, Supplementary Figures S2, S3). FIGURE 2 Histogram of AFP status at diagnosis and ﬁnal. AFP, alpha-fetoprotein. FIGURE 2 Histogram of AFP status at diagnosis and ﬁnal. AFP, alpha-fetoprotein. Univariable and multivariable Cox proportional hazard regression models were performed to explore the independent preoperative predictive signiﬁcance of AFP response and AFP levels on postoperative RFS. Univariable analysis revealed that only AFP response (hazard ratio [HR], 0.264; 95% conﬁdence interval [CI], 0.119 to 0.589; P=0.001) and ﬁnal AFP levels (HR, 2.395; 95% CI, 1.047 to 5.477; P=0.039) were statistically signiﬁcant variables (Table 2). In the multivariable analysis, after adjustment for other preoperative variables with a P value < 0.1, AFP response remained an independent preoperative predictor for postoperative RFS (HR, 0.332; 95% CI, 0.129 to 0.857; P=0.023, Table 2). FIGURE 2 Histogram of AFP status at diagnosis and ﬁnal. AFP, alpha-fetoprotein. Histogram of AFP status at diagnosis and ﬁnal. AFP, alpha-fetoprotein. frontiersin.org 06 Frontiers in Immunology Lin et al. 10.3389/fimmu.2024.1308543 Lin et al. APF response combined with radiological tumor response A B FIGURE 3 Cumulative recurrence-free survival curves comparison for patients with and without AFP response (≥80%) (A), and for patients with ﬁnal AFP ≥1000 ng/mL and < 1000 ng/mL (B). AFP, alpha-fetoprotein. A The Kaplan-Meier curve analysis for radiological response is depicted in Figure 4A. Patients who achieved complete response exhibited better postoperative RFS compared to those with partial response or stable disease (Figure 4A). Integrating AFP response with radiological response facilitated the further stratiﬁcation of patients into distinct risk groups: those with complete response experienced the most favorable postoperative RFS, followed by patients with partial response/stable disease and AFP response, whereas patients with partial response/stable disease but lacking AFP response demonstrated the least favorable postoperative RFS (P=0.001, Figure 4B). B FIGURE 3 B antigen; TACE, transarterial chemoembolization; CR, Complete response; PR, Partial response; SD, Stable disease; AFP, alpha-fetoprotein; HR, hazard ratio; CI antigen; TACE, transarterial chemoembolization; CR, Complete response; PR, Partial response; SD, Stable disease; AFP, alpha-fetoprotein; HR, hazard ratio; CI niﬁcance. Sensitivity analysis of AFP response To mitigate potential heterogeneity in the 80% cutoff value chosen by Xtile, which might be speciﬁc to our present cohort, we explored alternative cutoff values of 50% and 20%, as reported in previous literature, for deﬁning AFP responses. We also conducted analyses using these AFP response cutoffs (20%, 50%, and 80%) in a subgroup with initial AFP levels exceeding 200 ng/ mL. As demonstrated in Table 3, Supplementary Figures S4, S5, AFP response consistently correlated with improved postoperative RFS across all examined cutoffs, including in the subgroup with an AFP level greater than 200 ng/mL at diagnosis (all P<0.05). GU 3 Cumulative recurrence-free survival curves comparison for patients with and without AFP response (≥80%) (A), and for patients with ﬁnal AFP ≥1000 ng/mL and < 1000 ng/mL (B). AFP, alpha-fetoprotein. TABLE 2 Univariable and multivariable analysis of risk factors related to RFS. Variables Univariable Analysis Multivariable Analysis HR (95% CI) P Value HR (95% CI) P Value Age, ≥65 vs <65, years 0.341 (0.080, 1.447) 0.145 Gender, male vs female 0.759 (0.304, 1.897) 0.555 HBsAg, positive vs negative 4.490 (0.608, 33.173) 0.141 Liver cirrhosis, present vs absent 1.022 (0.463, 2.255) 0.958 Combine TACE, yes vs no 0.477 (0.199, 1.138) 0.095 0.557 (0.208, 1.494) 0.245 Conversion time, months 0.833 (0.667, 1.041) 0.108 Radiological response, CR vs PR/SD 0.251 (0.059, 1.063) 0.061 0.336 (0.077, 1.470) 0.148 Tumor diameter, cm 0.963 (0.882, 1.050) 0.391 Tumor number, multiple vs solitary 1.425 (0.646, 3.143) 0.381 Macrovascular invasion, present vs absent 0.466 (0.202, 1.075) 0.073 0.545 (0.224, 1.330) 0.183 AFP level at diagnosis, ≥1000 vs <1000, ng/ml 0.800 (0.369-1.732) 0.571 Final AFP level, ≥1000 vs <1000, ng/ml 2.395 (1.047, 5.477) 0.039 1.147 (0.419, 3.141) 0.789 AFP response, ≥80% vs <80% 0.264 (0.119, 0.589) 0.001 0.332 (0.129, 0.857) 0.023 HBsAg, hepatitis B surface antigen; TACE, transarterial chemoembolization; CR, Complete response; PR, Partial response; SD, Stable disease; AFP, alpha-fetoprotein; HR, hazard ratio; CI, ﬁd i t l 07 Frontiers in Immunology frontiersin.org Lin et al. Lin et al. 10.3389/fimmu.2024.1308543 TABLE 3 Sensitivity analysis. Sensitivity analysis of AFP response Groups HR 95%CI P value AFP > 20ng/ml cohort AFP response vs AFP non-response, 50% as the cutoff 0.374 0.166, 0.841 0.017 AFP response vs AFP non-response, 20% as the cutoff 0.423 0.185, 0.965 0.041 AFP≥200ng/ml cohort AFP response vs AFP non-response, 80% as the cutoff 0.183 0.073, 0.462 <0.001 AFP response vs AFP non-response, 50% as the cutoff 0.277 0.109, 0.702 0.007 AFP response vs AFP non-response, 20% as the cutoff 0.324 0.126, 0.835 0.020 AFP, alpha-fetoprotein; HR, hazard ratio; CI, conﬁdence intervals. Bold indicates statistical signiﬁcance. A B FIGURE 4 Cumulative recurrence-free survival curves comparison for patients with radiographic responses of CR, PR, and SD (A), and for patients with CR, patients with PR/SD and AFP response, and patients with PR/SD without AFP response (B). AFP, alpha-fetoprotein; CR, complete response; PR, partial response; SD, stable disease. A B FIGURE 4 B aligns with the distinct characteristics of our patient cohort, who underwent salvage hepatectomy following successful conversion therapy. The choice of a higher cutoff is justiﬁed by our cohort’s median AFP level at diagnosis, which was notably high at 1,210 ng/ mL and then signiﬁcantly reduced to 44.9 ng/mL before hepatectomy. Furthermore, our selection of this threshold is based on the statistical rationale provided by Xtile, offering a more methodologically robust alternative to the arbitrary 50% or 20% cutoffs employed in previous studies (28). In our context, the 80% cutoff offers the advantage of accurately reﬂecting true AFP changes attributable to HCC, while reducing the inﬂuence of varying liver disease conditions and inter-laboratory discrepancies. Cumulative recurrence-free survival curves comparison for patients with radiographic responses of CR, PR, and SD (A), and for patients with CR, patients with PR/SD and AFP response, and patients with PR/SD without AFP response (B). AFP, alpha-fetoprotein; CR, complete response; PR, partial response; SD, stable disease. Discussion Radiological evaluation for salvage hepatectomy is a routine clinical practice for assessing potential treatment efﬁcacy. Radiological assessments present speciﬁc limitations, including subjectivity, interobserver heterogeneity, and challenges arising from cirrhosis, post-treatment scarring, vascular occlusion, and even contrast agent injection timing (23, 32). A primary concern with radiological assessment is its inability to accurately predict prognosis (33, 34). However, our ﬁndings show that patients who achieve complete radiological response have better prognoses than those with partial response or stable disease, suggesting that preoperative imaging continues to be a reliable predictor for salvage hepatectomy prognosis. Additionally, our results suggest that integrating AFP response with preoperative radiological tumor response could offer potential advantages for prognostic evaluation. Radiological tumor response contributes to the assessment of tumor resectability and plays a vital role in devising comprehensive surgical resection strategies. However, it represents only macroscopic tumor reduction, not microscopic liver metastases, which are often the source of postoperative recurrence. Conversely, AFP response may reﬂect the overall subclinical disease burden and provide further information on the overall improvement in tumor burden. As a result, the potential beneﬁts of combining AFP response with radiological tumor response in the salvage hepatectomy population merit further investigation. In this multicenter retrospective study, we systematically assessed the association between serum AFP level variations and postoperative RFS of patients with initially unresectable HCC who undergoing salvage hepatectomy after conversion therapy regimens based on TKIs combined with a-PD-1. Our ﬁndings highlight that a reduction of AFP by ≥80% following conversion therapy is closely linked with improved postoperative RFS in patients with baseline AFP levels above 20 ng/mL. This “20-80” rule enriches the clinical decision-making framework for salvage hepatectomy following TKI and a-PD-1 based conversion therapy, particularly in light of the procedure’s invasive nature. It potentially aids clinicians and patients in evaluating the efﬁcacy of preoperative therapy and in making informed choices about proceeding with surgery. AFP, recognized as a key biomarker in HCC management, has shown renewed signiﬁcance in predicting treatment outcomes through its dynamic variations, commonly referred to as the “AFP response” (19, 23, 24, 30, 31). This concept gained attention with two pivotal studies in 2009, published in the Journal of Clinical Oncology, which demonstrated the utility of AFP response, using thresholds of 50% and 20%, as a marker post-locoregional and systemic chemotherapy for HCC, respectively. Our study, however, adopts a higher cutoff value of 80% for AFP response. Frontiers in Immunology Author contributions K-YL: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Resources, Software, Supervision, Visualization, Writing – original draft, Writing – review & editing. J-XZ: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Supervision, Writing – original draft. Z-WL: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Project administration, Supervision, Writing – original draft. Q-JC: Conceptualization, Data curation, Investigation, Methodology, Project administration, Resources, Software, Supervision, Writing – original draft. L-PL: Data curation, Investigation, Methodology, Project administration, Resources, Writing – original draft. J-HC: Investigation, Methodology, Project administration, Resources, Software, Supervision, Writing – original draft. KuW: Conceptualization, Data curation, Validation, Writing – review & editing. ST: Data curation, Validation, Writing – review & editing. Z-BZ: Data curation, Writing – review & editing. S-FW: Data curation, Methodology, Writing – review & editing. J-DL: Data curation, Formal Analysis, Supervision, Writing – review & editing. KaW: Data curation, Writing – review & editing. LZ: Data curation, Writing – review & editing. S-MZ: Data curation, Writing – review & editing. M-MW: Data curation, Writing – review & editing. K- CL: Data curation, Writing – review & editing. TY: Writing – review & editing. Y-YZ: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Project administration, Software, Supervision, Validation, Writing – review & editing. Our study has several limitations. Firstly, its retrospective nature introduces the possibility of selection bias. Secondly, the limited sample size of our study posed constraints on the statistical power. Consequently, there is a pressing need for larger, multi- center prospective studies to conﬁrm the correlation between AFP response and postoperative prognosis in salvage hepatectomy and to establish a standardized AFP response cutoff. Thirdly, the relatively short follow-up duration of our study limits our ability to assess the association between AFP response and postoperative overall survival. This limitation highlights the necessity for future research to more comprehensively explore the relationship between AFP response and overall survival, given the critical importance of long-term survival outcomes in evaluating the effectiveness of salvage hepatectomy. Fourthly, our study focused exclusively on AFP without including other relevant tumor markers, such as protein induced by vitamin K absence or antagonist-II. Lastly, limited by the retrospective non-interventional nature of our study, we exclusively included patients who underwent salvage hepatectomy, lacking a comparable cohort that met resection criteria but did not proceed with salvage hepatectomy, largely due to the inherent difﬁculties in data collection. Discussion This decision Frontiers in Immunology frontiersin.org 08 Lin et al. 10.3389/fimmu.2024.1308543 Ethics statement All procedures performed in studies involving human participants were by the ethical standards of the institutional and/ or national research committee. And this research was conducted ethically by the World Medical Association Declaration of Helsinki. Data availability statement The present study is noteworthy for several reasons. Firstly, to our knowledge, this is the ﬁrst study assessing AFP response as a preoperative surrogate marker for RFS after salvage hepatectomy. Secondly, a critical advantage of utilizing AFP response in our study is its role in providing preoperative guidance. This application is distinct from its conventional use in assessing the effectiveness of systemic or local treatments for HCC, which is typically evaluated one month post-treatment initiation (19–21). Our approach advances the understanding of AFP response, offering valuable insights prior to the commencement of salvage hepatectomy, thereby enhancing the decision-making process in clinical settings. Thirdly, the ﬁndings of our study have signiﬁcant implications for neoadjuvant therapy in HCC. While clinical trials in this area are extensive, beneﬁting from the integration of novel systemic antitumor agents, there remains a pressing need for an effective, straightforward preoperative efﬁcacy evaluation indicator. Our ﬁndings may illuminate a novel direction suggesting that attention may be warranted toward the AFP response in future clinical trials of neoadjuvant therapy for HCC, with the aim to ascertain its capacity to serve as an efﬁcacious marker of the oncological advantage derived from neoadjuvant therapy. The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors. 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https://openalex.org/W4392808561	https://www.mdpi.com/2305-6290/8/1/29/pdf?version=1710412331	English	null	A Multi-Objective Model for Designing a Sustainable Closed-Loop Supply Chain Logistics Network	Logistics	2,024	cc-by	18,198	Citation: Arab Momeni, M.; Jain, V.; Bagheri, M. A Multi-Objective Model for Designing a Sustainable Closed-Loop Supply Chain Logistics Network. Logistics 2024, 8, 29. https://doi.org/10.3390/ logistics8010029 Article Mojtaba Arab Momeni 1 , Vipul Jain 2,* and Mehdi Bagheri 1 1 Department of Industrial Engineering, Jam Faculty of Engineering, Persian Gulf University, Bushehr 7516913817, Iran; mojtaba.mam.sut@gmail.com (M.A.M.); mbagheri.pgu@gmail.com (M.B.) 2 Wellington School of Business and Government, Victoria University of Wellington, Wellington 6140, New Zealand * Correspondence: vipul.jain@vuw.ac.nz 1 Department of Industrial Engineering, Jam Faculty of Engineering, Persian Gulf University, Bushehr 7516913817, Iran; mojtaba.mam.sut@gmail.com (M.A.M.); mbagheri.pgu@gmail.com (M.B.) 2 Wellington School of Business and Government, Victoria University of Wellington, Wellington 6140, New Zealand * Correspondence: vipul.jain@vuw.ac.nz Abstract: Background: The growing concern for environmental and social issues has led to a focus on designing sustainable supply chains and increasing industrial responsibility towards society. In this paper, a multi-objective mixed-integer programming model is presented for designing a sustainable closed-loop supply chain. The model is aimed at the minimization of the total cost with the total used facilities, the negative environmental impacts, and the maximization of the positive social impacts. Methods: The epsilon-constraint method is utilized for solving the model and further extracting the Pareto solutions. Results: The result of the research clearly shows an optimal trade-off between the conflicting objectives, where, by paying more attention to the social and environmental aspects of sustainability, the total costs are increased or by optimizing the number of facilities, a better balance between the dynamics associated with the short-term and long-term goals is reached. The results of the sensitivity analysis also show that increasing the demand of the supply chain has the greatest impact on the supply chain costs compared to other objectives. Conclusions: Consequently, investigating such comprehensive sustainable objectives provides better insights into the impact of design variables on the expectations of stakeholders. Keywords: closed-loop supply chain; sustainable design; epsilon-constraint method logistics logistics logistics logistics 1. Introduction The increase in customer pressure for timely and quality products, as well as the increase in global competition, has forced the organizations involved in the production of products and services to have an integrated and coordinated supply chain network. However, with the increase in the negative effects of industrial activities on the environment and the increase in awareness of its impacts, as well as the pressure of legislator institutions, the traditional supply chain has been pushed towards the green supply chain (GSC). In fact, the GSC concentrates on eco-friendly aspects of the supply chain operations to bring the greenness concept to all processes throughout the supply chain including product design, raw material sourcing, manufacturing and distribution processes, etc. It can be seen recently that corporate pressures on organizations, such as the need to assess the viability of vendors and employees, has put forward a broader concept of the supply chain as sustainable supply chain management (SSCM). SSCM amounts to the economic, environmental, and social consequences of the supply chain operation, known as the triple bottom of sustainability. SSCM measures make the supply chain accountable to public and social concerns such as the social and ethical issues around strategic procurement. Received: 10 January 2024 Revised: 15 February 2024 Accepted: 5 March 2024 Published: 13 March 2024 Received: 10 January 2024 Revised: 15 February 2024 Accepted: 5 March 2024 Published: 13 March 2024 Copyright: © 2024 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/). Today, due to the increasing attention to social and environmental issues, reverse logis- tics has become an important strategy to increase customer satisfaction. Reverse logistics originates from the perspective of waste management. However, its implementation is complicated by the presence of uncertainty, conflicting objectives, and difficulty of measur- ing environmental and social impacts [1]. However, recently, reverse logistics has attracted https://www.mdpi.com/journal/logistics Logistics 2024, 8, 29. https://doi.org/10.3390/logistics8010029 Logistics 2024, 8, 29 2 of 25 the attention of many manufacturers through activities related to the process of collecting and retrieving products with a life cycle span [2,3] and of customers through collection centers for the purpose of reconstruction, recycling, and green disposal [4]. In this regard, many companies have begun to use integrated environmental strategies to improve their business models and obtain a competitive advantage through reuse and product recovery operations. Accordingly, governments have enacted laws to address the pollution problems and the ways by which factories deal with recycled products [5]. On the other hand, the concept of reverse logistics has been used for the entire product life cycle, from design to consumption, return to the factory, and the collection and transportation processes [6]. Moreover, researchers have proposed some classifications for reverse logistic processes such as “collection, separation, reproduction, disposal, and redistribution”, “reuse, service, reproduction, and recycling”, and “remanufacturing, recycling, and disposal” [7]. In this paper, the last classification is considered for forward and reverse logistics or, equivalently, the closed-loop supply chain. p pp y It is to be noted that the lack of environmental resources and the growing rate of environmental pollutants have threatened human lives and faced them with many chal- lenges [8]. Although the development of industries and employment growth lead to the improvement in the economic situation of societies and provides many benefits to individ- uals and organizations, they have also created many environmental and social problems. These challenges have led to the emergence of the sustainability concept in recent years. Therefore, given that every decision in the supply chain has special economic, environ- mental, and social effects, sustainability has become more important in the design and implementation of supply chains [9]. Furthermore, green logistics has also created a new revolution in sustainability. 2. Literature Review In this section, some relevant papers that address the reverse and closed-loop sustain- able design of supply chains are reviewed. The design of a sustainable closed-loop supply chain was studied in [15] by minimizing costs and risks and providing additional benefits using the conversion of the returned products to fertilizer. g p In [16], the design of a green supply chain network under stochastic demand and carbon price was investigated. To show the effects of different parameters on the logistic results, a sensitivity analysis was also carried out, where the carbon price and budget avail- ability had positive effects on greening the supply chain. Zohal and Soleimani suggested a multi-objective model for designing a closed-loop supply chain with four forward material flows and three reverse flows in the gold industry. They followed the least amount of car- bon emission besides the minimum cost in designing the supply chain [17]. Zarbakhshnia and Jaghdani have developed a multi-objective and multi-product model for a forward and reverse logistics network focusing on economic and environmental goals. They used the ε-constraint method to solve their model and showed that demand changes lead to more changes in the cost objective function than the social and environmental objective functions [18]. Taleizadeh et al. explored a multi-objective, single-product, multi-period closed-loop supply chain by considering pricing and discount policies on returned prod- ucts and modeling it by exploring economic, social, and environmental objectives [9]. Tehrani and Gupta used a multi-objective mixed integer programming model to design a sustainable green closed-loop supply chain. The sensitivity analysis conducted in their paper indicated that if by increasing the demand rate, the number of facilities and their capacity level are increased appropriately, further increase in the total profit of the supply chain will be achieved [19]. In [20], the authors focused more on environmental aspects of a closed-loop supply chain and pursued minimizing the total cost, energy consumption, CO2 emission, and waste generation of supply chains with a focus on disruption risk. They proposed a multi-objective model to cope with these goals and used the non-dominated sorting genetic algorithm (NSGA-II) to extract Pareto solutions. They demonstrated that by moving from a forward supply to a closed-loop one, both cost and environment indicators are improved. p Investigating newer and broader objective functions has been a common trend in research in the context of sustainable supply chain management in recent years. Tirkolaee et al. In fact, the main goal of green logistics is to coordinate all activities in an efficient way, so that a balance between economic, environmental, and social dimensions is provided [10]. This also stimulates organizations to care to set environment protection objectives such as reducing pollution, water usage, and waste generation while they still pursue the economic objective of maximizing their profits [11]. This consequently leads to increases in the organization’s effectiveness and sustainable production and sup- ply [4]. Combining the economic and environmental problems such as environmentally friendly design, green packaging, green design (environmental design), and sustainable production forms the green production structure [11]. In green management, reverse logistics plays a key role. This provides opportunities for firms to increase their rate of return on the end-of-life products and improve their perfor- mance [12]. Moreover, the return products are remanufactured as second-hand products or recycled to extract the raw materials required to produce new products [13]. In this regard, there is a need for a sustainable design that integrates economic, environmental, and social goals in all processes of the supply chain, which could ensure its growth and survival in the long term and hence, increase its acceptability from society’s point of view. Consequently, in this research, comprehensive objectives addressing the real-life complexities and chal- lenging expectations of stakeholders are considered in the sustainable design of the reverse supply chain. The developed model presents various solutions, each of which reflects one desire of the decision makers toward the sustainable goals, so the model also provides high flexibility for the configuration of the supply chain. For designing sustainable logistics, a multi-objective, multi-period, and multi-product model is proposed in this paper. For addressing the economic dimension of sustainability, the model seeks to optimize the operation, processes, transportation, and set-up costs; employee support schemes; as well as the number of required facilities of the supply chain. The environmental objectives of the model also include the minimization of carbon emission, wastewater, and energy consumption. Finally, as the social concerns of the model, the positive effects of social programs such as employment will also be optimized. Furthermore, given that the proposed model is a multi-objective one, the epsilon-constraint (ε-constraint) method is used to solve the model and extract the Pareto optimal solutions. Logistics 2024, 8, 29 3 of 25 This method has been shown to have a great ability to explore the solution space and extract many Pareto solutions [14]. The paper is organized as follows. The next Section is the literature review to identify and highlight the gaps in the literature covered by the model in this paper. Section 3 presents the assumption, the notation, the objective functions, and the ε-constraints of the proposed model as well as the ε-constraint method for solving the model. The numerical results of the paper are described in Section 4. Finally, the paper’s conclusion and some opportunities for future research are presented in the last Section. 2. Literature Review added the reliability of the supply chain and the value of products regarding the priority of the suppliers to the most common objective function of the supply chain management, i.e., the minimization of the cost. They first used the DEMATEL method to identify the importance weights of the objective functions and, second, solved the proposed multi-objective model using the GAMS/CPLEX solver [21]. They concluded that the demand and purchase prices from suppliers are the most sensitive factors in the introduced objective functions. In [22], the market value-added was introduced as a measure of the accumulated economic performance and the installation of plants and distribution centers in regions with low human development as a measure for pursuing social dimensions of sustainability. In that paper, the configuration of the supply chain in terms of the percentages of flows between facilities was revealed to be influenced by adding the social and environmental dimensions of sustainability. The maximization of employees’ safety is another example of social objectives investigated in [23]. Jaigirdar et al. designated a sustainable supply chain for perishable products to lessen the annual supply chain cost and cold storage set-up cost and enhance the freshness of Logistics 2024, 8, 29 4 of 25 perishables [24]. They used the weighted sum method to handle the multi-objective model and underlined that improved distribution planning could be regarded as a strategic decision that better aligns the supply chain toward a sustainable one. Other multi-objective functions including minimization of the total cost and rejected and late delivery units and maximization of the assessment score of the selected suppliers were explored in [25] to determine order allocation and facility location of the supply chain network. perishables [24]. They used the weighted sum method to handle the multi-objective model and underlined that improved distribution planning could be regarded as a strategic decision that better aligns the supply chain toward a sustainable one. Other multi-objective functions including minimization of the total cost and rejected and late delivery units and maximization of the assessment score of the selected suppliers were explored in [25] to determine order allocation and facility location of the supply chain network. The closed-loop supply chain also was raised as an effective waste management solution [26]. 2. Literature Review The authors proposed a comprehensive CLSC network that optimizes envi- ronmental, economic, and social footprints through a multi-objective optimization approach and solved their model using the weighted sum method and several improving heuristics. Increasing the reverse logistics profits and reducing the worker salary were determined as changes that seek more sustainable goals, while increasing uncertainty affects all objectives inversely. It has also been shown that regulatory policies affect the configuration of the closed-loop supply chain network when sustainability goals are considered. The effects of such policies alongside the technical innovation of increasing the lifetime of perishable products were researched in [27], where the latter exhibited a positive effect on the incorpo- rated sustainable objective function. Planning facilities for eco-industrial parks [28] and modeling wood biomass supply chains [29] are among the new applications of sustainable design. The authors also presented the method of fuzzy analytic hierarchy process for determining the weight of the sustainable goals in their proposed mixed integer linear programming models. p g g In Table 1, the reviewed papers and the differences between them and the current research are summarized. Regarding the conducted studies, the broader sustainable ob- jectives of the paper outweigh the innovations of the paper. These objectives not only cover the traditional objective function such as the cost function of supply chains, but also introduce the cost of employee support schemes and social self-sufficiency alongside the environmental protection goals, such as minimum carbon emission and water and energy consumption, which together were paid less attention in the previous research. Minimizing the number of used facilities besides the costs of the supply chain is also a recent objective function and as will be discussed in the results section. It could explore a proper balance between short- and long-term goals of the supply chain. Finally, the ε-constraint method employed in this study, unlike methods such as the weighted sum method, has the ability to extract various Pareto solutions. This prepares sophisticated information to analyze the results, shows the effectiveness of the multi-objective method in providing solutions that pay attention to all dimensions of sustainability, asnd leads to useful managerial insight for policy making. Table 1. The summary of the reviewed papers and their differences from the current research. Table 1. The summary of the reviewed papers and their differences from the current research. Table 1. 3. Model Description In this paper, a multi-objective model is proposed for minimizing the total costs and environmental and social effects as well as the number of facilities in a closed-loop supply chain. For the environmental objectives, the reduction in carbon emissions, water wastages, and energy usages are targeted. Moreover, increasing employment support schemes and self-sufficiency are also studied as the social goals in this article. In the supply chain network and the forward flow, the suppliers send raw materials to the manufacturing centers, and next, the manufacturers send the finished products to the customers or keep them properly in storage for future use. On the other hand, disposal and recycling centers are the first centers in the reverse flow of the supply chain. In this direction, the returned products collected in the supply chain are divided into manufacturable, recyclable, and unusable products first, and next, they are sent in turn to remanufacturing, recycling, and disposal centers, respectively. The repaired items in remanufacturing centers can either be sent to the manufacturing centers to be used in new products or be sent to suppliers to be used as spare parts, while the recycled items could be employed as raw materials in the manufacturing centers or other suppliers.l Figure 1 shows the forward and reverse flows of the closed-loop supply chain under consideration. In this supply chain, the distributing and collecting centers have the role of both distributing products to local customers and collecting used returned products from them. The used returned products are then spread between the remanufacturing, recycling, and disposal centers based on predetermined rates. In the remanufacturing centers, limited processes such as cleaning are performed on the used returned products to prepare them for sending to the manufacturing plants to be further processed and converted to the final products. However, if they exceed the capacity of the remanufacturing centers, they are transported to the supply centers to be used as raw materials for final products. Also, some of the used returned products do not have the competency to be transformed into the final product after processing in remanufacturing and manufacturing centers. They might be processed in recycling centers to extract the raw materials from them. These raw materials are sent to the supplier centers again to be used in the final products. 2. Literature Review Source Product Period Objective Method Sustainable Dimensions Total Costs Number of Facili- ties CO2 Emis- sion Water Usage Energy Con- sump- tion Employee Support Schemes Self- Sufficiency Others [24] multi single multi Weighted sum * * [25] multi single multi MOPSO * * [26] single single multi Weighted sum * * * * [27] multi multi multi Weighted sum * * * [28] single single multi Weighted sum * * * This paper multi multi multi ε-constraint * * * * * * * The notation * indicates that the topic is covered by the mentioned study. Based on the above discussion, the main research objectives of the paper are the following: 1. Presenting a multi-objective model to handle new and realistic sustainable goals. 2. Presenting the ε-constraint method to extract various Pareto solutions. Based on the above discussion, the main research objectives of the paper are the following: Based on the above discussion, the main research objectives of the paper are the following: 1. Presenting a multi-objective model to handle new and realistic sustainable goals. 2. Presenting the ε-constraint method to extract various Pareto solutions. 3. Providing clear interpretations of solutions and the results of the model. Based on the above discussion, the main research objectives of the paper are the following: 1. Presenting a multi-objective model to handle new and realistic sustainable goals. 2. Presenting the ε-constraint method to extract various Pareto solutions. 3. Providing clear interpretations of solutions and the results of the model. Based on the above discussion, the main research objectives of the paper are the following: 1. Presenting a multi-objective model to handle new and realistic sustainable goals. 2. Presenting the ε-constraint method to extract various Pareto solutions. 3. Providing clear interpretations of solutions and the results of the model. 2. Literature Review The summary of the reviewed papers and their differences from the current research. Source Product Period Objective Method Sustainable Dimensions Total Costs Number of Facili- ties CO2 Emis- sion Water Usage Energy Con- sump- tion Employee Support Schemes Self- Sufficiency Others [15] multi multi multi Weighted sum * * * [17] single single multi Ant colony * * [18] multi single multi ε-constraint * * * [19] multi multi multi TH (Torabi–Hassini) * * * * [21] multi multi multi Weighted Goal Programming * * [22] multi multi single Heuristic * [23] single multi multi Lagrangian relaxation * * * [20] multi single multi NSGA-II * * * * Source Product Period Objective Method Sustainable Dimensions Total Costs Number of Facili- ties CO2 Emis- sion Water Usage Energy Con- sump- tion Employee Support Schemes Self- Sufficiency Others [15] multi multi multi Weighted sum * * * [17] single single multi Ant colony * * [18] multi single multi ε-constraint * * * [19] multi multi multi TH (Torabi–Hassini) * * * * [21] multi multi multi Weighted Goal Programming * * [22] multi multi single Heuristic * [23] single multi multi Lagrangian relaxation * * * [20] multi single multi NSGA-II * * * * Logistics 2024, 8, 29 5 of 25 Table 1. Cont. Table 1. Cont. Source Product Period Objective Method Sustainable Dimensions Total Costs Number of Facili- ties CO2 Emis- sion Water Usage Energy Con- sump- tion Employee Support Schemes Self- Sufficiency Others [24] multi single multi Weighted sum * * [25] multi single multi MOPSO * * [26] single single multi Weighted sum * * * * [27] multi multi multi Weighted sum * * * [28] single single multi Weighted sum * * * This paper multi multi multi ε-constraint * * * * * * * The notation * indicates that the topic is covered by the mentioned study. Based on the above discussion, the main research objectives of the paper are the following: 1. Presenting a multi-objective model to handle new and realistic sustainable goals. 2. Presenting the ε-constraint method to extract various Pareto solutions. 3. Providing clear interpretations of solutions and the results of the model. one type of customer. The shortage is not allowed • The shortage is not allowed. The shortage is not allowed. Notably, in some real-world problems, some parameters, such as demand, are uncer- in, in which case the proposed model needs fundamental adjustments. Also, we have sumed that all parameters have a predetermined value, but clearly, to estimate some rameters, such as the transportation costs between facilities, strong information sources ch as geographic information systems (GIS) can be used [29]. Moreover, historical data e needed to determine the distribution rates of returned used products among remanu- cturing, recycling, and disposal centers to establish the model. These rates are mainly ﬂuenced by the quality of such products and dependent on the consumption and return lture of local customers and, therefore, might vary over time. Thus, in real-world prob- Notably, in some real-world problems, some parameters, such as demand, are un- certain, in which case the proposed model needs fundamental adjustments. Also, we have assumed that all parameters have a predetermined value, but clearly, to estimate some parameters, such as the transportation costs between facilities, strong information sources such as geographic information systems (GIS) can be used [29]. Moreover, histori- cal data are needed to determine the distribution rates of returned used products among remanufacturing, recycling, and disposal centers to establish the model. These rates are mainly influenced by the quality of such products and dependent on the consumption and return culture of local customers and, therefore, might vary over time. Thus, in real-world problems, adjustments are necessary to measure and update these parameters. 3. Model Description However, the returned used products that do not have enough quality for recycling and remanufacturing are sent to the disposal centers to be discarded as garbage. Logistics 2024, 8, 29 6 of 25 ecy- gure 1. The forward and reverse ﬂows in the closed-loop supply chain. Figure 1. The forward and reverse flows in the closed-loop supply chain. gure 1. The forward and reverse ﬂows in the closed-loop supply chain. Figure 1. The forward and reverse flows in the closed-loop supply chain. Modeling Structure 1 Model Assumptions 3.1. Model Assumptions Model Assumptions The model assumptions are the following: The model assumptions are the following: . Model Assumptions The model assumptions are the following: The model assumptions are the following: The model assumptions are the following: Th d l i lti i d d lti d t • The model is multi-period and multi-product. The model is multi-period and multi-product. Th t d d d th b f t d d t i t i • The customer demand and the number of returned products is certain. The customer demand and the number of returned products is certain. Th lit f l d d t i diﬀ t f th t f d t • The quality of recycled products is different from that of new products. p The quality of recycled products is diﬀerent from that of new products. The sale point of recycled and new products is the same, so the model considers only • The quality of recycled products is different from that of new products. • The sale point of recycled and new products is the same, so the model considers only one type of customer. The quality of recycled products is diﬀerent from that of new products. The sale point of recycled and new products is the same, so the model considers only q y y p p • The sale point of recycled and new products is the same, so the model considers only one type of customer. Optimizing objective functions: ms, adjustments are necessary 3.2. The Mathematical Model 2. The Mathematical Model Before describing the proposed model in mathematical terms its simpliﬁed summary Before describing the proposed model in mathematical terms, its simplified summary is presented as Table 2: g p p presented as Table 2: Table 2. The overview of the proposed model. presented as Table 2: Table 2. The overview of the proposed model. is presented as Table 2: Table 2. The overview of the proposed model. Modeling Structure Descriptions Optimizing objective functions: Minimizing the total costs: Fixed costs of facilities Transportation costs Operation costs Costs of employee support schemes Holding cost Minimizing the negative environmental impacts: Carbon emission Water wasted Energy consumption Maximizing the positive social impacts: Job creation Self-sufficiency Minimizing the number of facilities is presented as Table 2: Table 2. The overview of the proposed model. Modeling Structure Descriptions Optimizing objective functions: Minimizing the total costs: Fixed costs of facilities Transportation costs Operation costs Costs of employee support schemes Holding cost Minimizing the negative environmental impacts: Carbon emission Water wasted Energy consumption Maximizing the positive social impacts: Job creation Self-sufficiency Minimizing the number of facilities Logistics 2024, 8, 29 7 of 25 g o 5 Table 2. Cont. Modeling Structure Descriptions Considering the supply chain constraints: Meet all customers’ demand Operate all returned products Flow constraints between facilities Inventory constraints in warehouses and manufacturing centers Capacity constraints of active facilities Capacity constraints of transportation vehicles Ensure at least one facility of each type will be opened Budget constraints of employee support schemes Furthermore, the model notations are provided below: Indices and Sets Description s ∈S Index of supplier centers (s = 1, 2, . . . , S) m ∈M Index of manufacturing centers (m = 1, 2, . . . , M) dc ∈DC Index of distributing and collecting centers (dc = 1, 2, . . . , DC) lc ∈LC Index of local customer area (lc = 1, 2, . . . , LC) rm ∈RM Index of remanufacturing and revival centers (rm = 1, 2, . . . , RM) rc ∈RC Index of recycling centers (rc = 1, 2, . . . , RC) ds ∈DS Index of disposal centers (ds = 1, 2, . . . , DS) p ∈P Index of products (p = 1, 2, . . . , P) t ∈T Index of time periods (t = 1, 2, . . . , T) v ∈V Index of available vehicles (v = 1, 2, . . . , V) es ∈ES Index of employee support scheme (es = 1, 2, . . . , ES) Parameter Description dp,t lc Demand for item p in local customer area lc in period t (units). presented as Table 2: Table 2. The overview of the proposed model. upp,t k Number of returned items p from local customer area lc in period t (units). rrmp,t dc Rate of sending item p from distributing and collecting center dc to the remanufacturing and revival centers in period t. rrcp,t dc Rate of sending item p from distributing and collecting center dc to the recycling centers in period t. rdsp,t dc Rate of sending item p from distributing and collecting center dc to the disposal centers in period t. f ct dc Fixed cost of establishing distributing and collecting center dc (USD). f ct rm Fixed cost of establishing remanufacturing and revival center rm (USD). f ct rc Fixed cost of establishing recycling centers rc (USD). f ct ds Fixed cost of establishing disposal center ds (USD). tct s,m Cost of transporting a unit of raw material from supplier center s to manufacturing center m in period t (USD/unit). tcp,t m,dc Cost of transporting a unit of item p from manufacturing center m to distributing and collecting center dc in period t (USD/unit). tcp,t m,m Cost of transporting a unit of item p from manufacturing center m to its warehouse m in period t (USD/unit). tcp,t dc,lc Cost of transporting a unit of item p from distributing and collecting center dc to local customer area lc in period t (USD/unit). tcp,t m,dc Cost of transporting a unit of item p from manufacturing center’s warehouse m to distributing and collecting center dc in period t (USD/unit). tcp,t lc,dc Cost of transporting a unit of returned item p from local customer area lc to distributing and collecting center dc in period t (USD/unit). tcp,t dc,rm Cost of transporting a unit of returned item p from distributing and collecting center dc to remanufacturing and revival center rm in period t (USD/unit). tcp,t dc,rc Cost of transporting a unit of returned item p from distributing and collecting center dc to recycling center rc in period t (USD/unit). tcp,t dc,ds Cost of transporting a unit of returned item p from distributing and collecting center dc to disposal center ds in period t (USD/unit). tcp,t rm,m Cost of transporting a unit of recovered item p from remanufacturing and revival center rm to manufacturing center m in period t (USD/unit). tcp,t rm,s Cost of transporting a unit of recovered item p from remanufacturing and revival center rm to supplier center s in period t (USD/unit). presented as Table 2: Table 2. The overview of the proposed model. tcp,t rc,s Cost of transporting a unit of recovered item p from recycling center rc to supplier center s in period t (USD/unit). pcp,t m Operation cost per unit of item p in manufacturing center m in period t (USD/unit). pcp,t dc Operation cost per unit of item p in distributing and collecting center dc in period t (USD/unit). pcp,t rm Operation cost of a unit of returned item p in remanufacturing and revival center rm in period t (USD/unit). Table 2. Cont. Modeling Structure Descriptions Considering the supply chain constraints: Meet all customers’ demand Operate all returned products Flow constraints between facilities Inventory constraints in warehouses and manufacturing centers Capacity constraints of active facilities Capacity constraints of transportation vehicles Ensure at least one facility of each type will be opened Budget constraints of employee support schemes Furthermore, the model notations are provided below: Indices and Sets Description s ∈S Index of supplier centers (s = 1, 2, . . . , S) m ∈M Index of manufacturing centers (m = 1, 2, . . . , M) dc ∈DC Index of distributing and collecting centers (dc = 1, 2, . . . , DC) lc ∈LC Index of local customer area (lc = 1, 2, . . . , LC) rm ∈RM Index of remanufacturing and revival centers (rm = 1, 2, . . . , RM) rc ∈RC Index of recycling centers (rc = 1, 2, . . . , RC) ds ∈DS Index of disposal centers (ds = 1, 2, . . . , DS) p ∈P Index of products (p = 1, 2, . . . , P) t ∈T Index of time periods (t = 1, 2, . . . , T) v ∈V Index of available vehicles (v = 1, 2, . . . , V) es ∈ES Index of employee support scheme (es = 1, 2, . . . , ES) Logistics 2024, 8, 29 8 of 25 Parameter Description pcp,t rc Operation cost per unit of returned item p in recycling center rc in period t (USD/unit). pcp,t ds Operation cost per unit of returned item p in disposal center ds in period t (USD/unit). cop 2m Amount of CO2 emission for a unit of item p in manufacturing center m (g/unit). cop 2rm Amount of CO2 emission for a unit of recovered item p in remanufacturing and revival center rm (g/unit). presented as Table 2: Table 2. The overview of the proposed model. cop 2rc Amount of CO2 emission for a unit of item p in recycling centers rc (g/unit). cop 2dc Amount of CO2 emission for a unit of returned item p in disposal center ds (g/unit). co2v Amount of CO2 emission for using vehicle v per mile (g/mile). hp,t m Cost of stocking a unit of item p in warehouse of manufacturing center m (USD/unit). cas Capacity of supplier center s (units). cam Capacity of manufacturing center m (units). caim Capacity of manufacturing center’s warehouse m (units). cadc Capacity of distributing and collecting center dc for new items (units). cardc Capacity of distributing and collecting center dc for recovered items (units). carm Capacity of remanufacturing and revival center rm (units). carc Capacity of recycling center rc (units). cads Capacity of disposal center ds (units). cav Capacity of vehicle v (units). vdc Number of machines in production lines of distributing and collecting center dc (numbers). vrm Number of machines in production lines of remanufacturing and revival center rm (numbers). vrc Number of machines in production lines of recycling center rc (numbers). vds Number of machines in production lines of disposal center ds (numbers). cst es Cost of employee support scheme es in period t (USD) mbs Maximum allocated budget for employees’ support (USD). ncs Number of created job opportunities at supplier center s per unit of item p (numbers). ncm Number of created job opportunities at manufacturing center m per unit of item p (numbers). ncrm Number of created job opportunities at remanufacturing and revival center rm per unit of item p (numbers). ncrc Number of created job opportunities at recycling center rc per unit of item p (numbers). ncds Number of created job opportunities at disposal center ds per unit of item p (numbers). ecp m Amount of used energy per unit of item p in manufacturing center m (kWh/unit). ecp dc Amount of used energy per unit of item p in distributing and collecting center dc (kWh/unit). ecp rm Amount of used energy per unit of item p at remanufacturing and revival center rm (kWh/unit). ecp ds Amount of used energy per unit of item p in recycling center rc (kWh/unit). wvp m Amount of used energy per unit of item p in disposal center ds (kWh/unit). wvp rc Wasted volume of water per unit of item p in at remanufacturing and revival center rc (kWh/unit). presented as Table 2: Table 2. The overview of the proposed model. wvp rm Wasted volume of water per unit of item p in recycling centers rm (liters/unit). wvp dc Wasted volume of water per unit of item p in disposal center ds (liters/unit). pvp Volume of each item p unit (liters/unit). w1 Weight of CO2 emission in the environmental objective function. w2 Weight of wasted water in the environmental objective function. w3 Weight of energy usage in the environmental objective function. w4 Weight of created job opportunities in the social objective function. w5 Weight of self-sufficiency in the social objective function. Variable Description Tp,t s,m Quantity of raw material transported from supplier center s to manufacturing center m in period t (units). Tp,t m,dc Quantity of product p transported from manufacturing center m to distributing and collecting center dc in period t (units). Tp,t dc,lc Quantity of product p transported from distributing and collecting center dc to local customer area lc in period t (units). Tp,t lc,dc Quantity of returned used product p transported from local customer area lc to distributing and collecting center dc in period t (units). Tp,t dc,rm Quantity of returned product p transported from distributing and collecting center dc to remanufacturing and revival center rm in period t (units). Tp,t dc,rc Quantity of returned product p transported from distributing and collecting center dc to recycling centers rc in period t (units). Tp,t dc,ds Quantity of returned product p transported from distributing and collecting center dc to disposal center ds in period t (units). Tp,t rm,m Quantity of recovered product p transported from remanufacturing and revival center rm to manufacturing center m in period t (units). Tp,t rm,s Quantity of recovered product p transported from remanufacturing and revival center rm to supplier center s in period t (units). Tp,t rc,s Quantity of raw material p transported from recycling centers rc to supplier center s in period t (units). Logistics 2024, 8, 29 9 of 25 Variable Description TIp,t m,m Quantity of product p transported from manufacturing center m to its warehouse m in period t (units). TIp,t m,dc Quantity of product p transported from manufacturing center’s warehouse m to distributing and collecting center dc in period t (units). QMp,t m Quantity of manufactured product p in manufacturing center m (units). QPp,t m Quantity of product p in manufacturing center’s warehouse m (units). QRMp,t rm Quantity of recovered product p in remanufacturing and revival center rm (units). presented as Table 2: Table 2. The overview of the proposed model. QDSp,t ds Quantity of disposed products in disposal center ds (units). QRCp,t rc Quantity of recycled product p in recycling centers rc (units). NVt v,s,m An integer variable representing the number of vehicles v used to transport raw material p from supplier center s to manufacturing center m in period t (numbers). NVp,t v,m,dc An integer variable representing the number of vehicles v used to transport product p from manufacturing center m to distributing and collecting center dc in period t (numbers). NVp,t v,dc,lc An integer variable representing the number of vehicles v used to transport product p from distributing and collecting center dc to local customer area lc in period t (numbers). NVp,t v,dc,rm An integer variable representing the number of vehicles v used to transport product p from distributing and collecting center dc to remanufacturing and revival center rm in period t (numbers). NVp,t v,dc,rc An integer variable representing the number of vehicles v used to transport product p from distributing and collecting center dc to recycling center rc in period t (numbers). NVp,t v,dc,ds An integer variable representing the number of vehicles v used to transport product p from distributing and collecting center dc to disposal center ds in period t (numbers). Up,t m 1 = if the inflow to the warehouse m for product p is activated in period t. 0 = if otherwise the outflow from the warehouse m for product p is activated in period t. Xdc 1 = if distributing and collecting center ds is established. 0 = otherwise. Xrm 1 = if remanufacturing and revival center rm is established. 0 = otherwise. Xrc 1 = if recycling centers rc is established. 0 = otherwise. Xds 1 = if disposal center ds is established. 0 = otherwise. Ot es 1 = if employee support scheme es is chosen in period t. 0 = otherwise. Description Quantity of product p transported from manufacturing center m to its warehouse m in period t (units). Quantity of product p transported from manufacturing center’s warehouse m to distributing and collecting center dc in period t (units). 1 = if remanufacturing and revival center rm is established. 0 = otherwise. 1 = if remanufacturing and revival center rm is established 1 = if disposal center ds is established. h 1 = if employee support scheme es is chosen in period t. 0 = otherwise. presented as Table 2: Table 2. The overview of the proposed model. Taking into account the notations of this paper, the proposed model is introduced in the following. In doing so, first, the objective functions of the model are presented and then, the model constraints are explained. As mentioned, the model objective functions include the cost function, environment function, social function, and function related to the number of facilities and machines. 3.2.1. The Cost Functions The total cost of the model is equal to the fixed costs of facilities + the transportation costs + the processes and operation costs + the costs of employee support schemes + the holding costs. • The fixed costs of facilities (FC) • The fixed costs of facilities (FC) The fixed costs of facilities including distributing and collecting centers, remanufactur- ing and revival centers, recycling centers, and disposal centers are as Equation (1): FC = DC ∑ dc=1 T ∑ t=1 f ct dc.Xdc + RM ∑ rm=1 T ∑ t=1 f ct rm.Xrm + RC ∑ rc=1 T ∑ t=1 f ct rc.Xrc + DS ∑ ds=1 T ∑ t=1 f ct ds.Xds (1) (1) • The costs of employee support schemes (SC) According to the Global Reporting Initiative Standard (GRI), employee support schemes can include payment of facilities with low or free wages, overtime pay, hous- ing, educational and training grants, and so on. Given that the cost of scheme es in period t is SCes,t, the total cost of support schemes is according to Equation (4). SC = ES ∑ es=1 T ∑ t=1 sct es.Ot es (4) (4) • The holding cost (HC) • The transportation costs (TC) • The transportation costs (TC) The transportation cost between different facilities in the supply chain network is calculated as Equation (2). Logistics 2024, 8, 29 10 of 25 TC = S ∑ s=1 M ∑ m=1 T ∑ t=1 Tt s,m .tcs,m + P ∑ p=1 M ∑ m=1 DC ∑ dc=1 T ∑ t=1 Tt,p m,dc .tct,p m,dc + P ∑ p=1 DC ∑ dc=1 LC ∑ lc=1 T ∑ t=1 Tt,p dc,lc .tct,p dc,lc+ P ∑ p=1 DC ∑ dc=1 DS ∑ ds=1 T ∑ t=1 Tt,p dc,ds .tct,p dc,ds + P ∑ p=1 M ∑ m=1 DC ∑ dc=1 T ∑ t=1 TIt,p m,dc .tct,p m,dc + S ∑ s=1 M ∑ m=1 T ∑ t=1 TIt,p m,m .tct,p m,m+ P ∑ p=1 LC ∑ lc=1 DC ∑ dc=1 T ∑ t=1 Tt,p lc,dc .tct,p lc,dc + P ∑ p=1 DC ∑ dc=1 RC ∑ rc=1 T ∑ t=1 Tt,p dc,rc .tct,p dc,rc + P ∑ p=1 DC ∑ dc=1 RM ∑ rm=1 T ∑ t=1 Tt,p lc,dc .tct,p dc,rm+ P ∑ p=1 RM ∑ rm=1 M ∑ m=1 T ∑ t=1 Tt,p rm,m .tct,p rm,m + P ∑ p=1 RM ∑ rm=1 S ∑ s=1 T ∑ t=1 Tt,p rm,s .tct,p rm,s + P ∑ p=1 RC ∑ rc=1 S ∑ s=1 T ∑ t=1 Tt,p rc,s .tct,p rc,s (2) (2) • The holding cost (HC) Considering that the inventories could only be stored in the manufacturing centers, the total holding cost is determined as Equation (5). HC = T ∑ t=1 P ∑ p=1 M ∑ m=1 hp,t m .QPp,t m (5) (5) Therefore, taking into account Equations (1)–(4), the first objective function Z1, i.e., the minimization of the total cost, is specified by Equation (6): Min Z1 = FC + TC + OC + SC + HC (6) (6) • The operation costs (OC) • The operation costs (OC) The operation cost comprises the production cost and the processing costs in the distributing and collecting centers, remanufacturing and revival centers, recycling centers, and disposal centers. This cost is shown in Equation (3). OC = T ∑ t=1 P ∑ p=1 M ∑ m=1 pcp,t m .QMp,t m + T ∑ t=1 P ∑ p=1 DC ∑ dc=1 pcp,t dc .QDCp,t dc + T ∑ t=1 P ∑ p=1 RC ∑ rc=1 pcp,t rc .QRCp,t rc + T ∑ t=1 P ∑ p=1 RM ∑ rm=1 pcp,t rm.QRMp,t rm + T ∑ t=1 P ∑ p=1 DS ∑ ds=1 pcp,t ds .QDSp,t ds (3) (3) • The costs of employee support schemes (SC) • The costs of employee support schemes (SC) • The costs of employee support schemes (SC) 3.2.2. Environmental Function The three environmental destructive effects studied in the present study include (1) the carbon emissions, (2) the water wasted, and (3) the energy consumption. • The water wasted (WW) The generated wastewater as a result of manufacturing, remanufacturing, recycling, and disposing processes is stipulated by Equation (9). WW = P ∑ p=1 M ∑ m=1 wvp m T ∑ t=1 QMp,t m + P ∑ p=1 RM ∑ rm=1 wvp rm T ∑ t=1 QRMp,t rm + P ∑ p=1 RC ∑ rc=1 wvp rc T ∑ t=1 QRCp,t rc + P ∑ p=1 DS ∑ ds=1 wvp ds T ∑ t=1 QDSp,t m (9) (9) • The energy consumption (EC) • The energy consumption (EC) • The energy consumption (EC) Energy consumption is another environmental indicator indicating the efficiency of energy usage according to GRI standards. In this paper, the sum of all the energy consumed on all products in all supply chain activities is considered as an energy indicator and is calculated using Equation (10). EC = P ∑ p=1 M ∑ m=1 ecp m T ∑ t=1 QMp,t m + P ∑ p=1 DC ∑ dc=1 ecp dc T ∑ t=1 QDCp,t dc + P ∑ p=1 RC ∑ rc=1 ecp rc T ∑ t=1 QRCp,t rc + P ∑ p=1 RM ∑ rm=1 ecp rm T ∑ t=1 QRMp,t m + P ∑ p=1 DS ∑ ds=1 ecp ds T ∑ t=1 QDSp,t ds (10) (10) Now, regarding the above effects and considering the importance weights w1, w2, and w3 for the carbon emissions, wastewater, and energy consumption, respectively, the second objective of the proposed model (Z2) is expressed by Equation (11). min Z2 = w1.(QCO2 + NVCO2) + w2.WW + w3.EC (11) (11) 3.2.3. The Social Function In this paper, the social effects of the closed-loop supply chain are considered on job creation and self-sufficiency. • The carbon emissions (CE) According to the GRI guidelines, toxic greenhouse gases have a significant effect on air temperature and climate change. Through the supply chain, carbon could be generated by two resources. The first resource is the processes of the supply chain, including the production processes, the distribution and collection processes, the remanufacturing processes, the recycling processes, and the disposal processes. The carbon emissions Logistics 2024, 8, 29 11 of 25 11 of 25 of these processes, denoted by QCO2, is shown in Equation (7). The second source of carbon emissions is transportation between the supply chain facilities. For this purpose, the transportation of raw material to the manufacturing centers, finished products to the distribution centers and from there to the customers, the returned products to the collecting centers, and then transferring of them to the remanufacturing, recycling, and disposal centers are included. Equation (8) represents the carbon emissions stemming from the transportation activities (NvCO2). QCO2 = P ∑ p=1 M ∑ m=1 cop 2m T ∑ t=1 QMp,t m + P ∑ p=1 DC ∑ dc=1 cop 2dc T ∑ t=1 QDCp,t dc + P ∑ p=1 RM ∑ rm=1 cop 2rm T ∑ t=1 QRMp,t rm + P ∑ p=1 RC ∑ rc=1 cop 2rc T ∑ t=1 QRCp,t rc + P ∑ p=1 DS ∑ ds=1 cop 2ds T ∑ t=1 QDSp,t ds (7) (7) NVCO2 = S ∑ s=1 M ∑ m=1 dts,m v ∑ v=1 P ∑ p=1 co2vNVp v,s,m + S ∑ s=1 M ∑ m=1 dtm,dc v ∑ v=1 P ∑ p=1 co2vNVp v,m,dc + DC ∑ dc=1 LC ∑ lc=1 dtdc,lc v ∑ v=1 P ∑ p=1 co2vNVp v,dc,lc + DC ∑ dc=1 RM ∑ rm=1 dtdc,rm v ∑ v=1 P ∑ p=1 co2vNVp v,dc,rm+ DC ∑ dc=1 RC ∑ rc=1 dtdc,rc v ∑ v=1 P ∑ p=1 co2vNVp v,dc,rc + DC ∑ dc=1 DS ∑ ds=1 dtdc,ds v ∑ v=1 P ∑ p=1 co2vNVp v,dc,ds (8) (8) • The water wasted (WW) • The water wasted (WW) • The job creation (JC) One of the positive effects of manufacturing, distributing, collecting, remanufacturing, recycling, and disposing of processes is the creation of job opportunities. Job creation will Logistics 2024, 8, 29 12 of 25 12 of 25 also have positive effects on society, such as satisfaction and the reduction in migration. In this paper, job creation is determined with respect to the number of products processed throughout the supply chain in all periods. Hence, Equation (12) is used to describe the job creation of the supply chain. JC = M ∑ m=1 ncm . P ∑ p=1 T ∑ t=1 QMp,t m ! + DC ∑ dc=1 ncdc . P ∑ p=1 T ∑ t=1 QDCp,t dc ! + RC ∑ rc=1 ncrc . P ∑ p=1 T ∑ t=1 QRCp,t rc ! + RM ∑ rm=1 ncrm . P ∑ p=1 T ∑ t=1 QRMp,t rm ! + DS ∑ ds=1 ncds . P ∑ p=1 T ∑ t=1 QDSp,t ds ! (12) (12) • The self-sufficiency (SS)i • The self-sufficiency (SS)i 3.2.4. The Number of Facilities The final objective of the model is the minimization of the facilities used in the supply chain. Due to the fact that the useful life of the facilities might be different with the number of periods in the planning horizon, the cost of facilities itself does not appropriately reflect the effective cost of employing facilities. Instead, minimizing the number of facilities separately does not disturb the planning horizon used for measuring other cost components and helps pursue one of the considered goals of supply chain management. Given the notation of the paper, this objective function can be quantified by Equation (15). min Z4 = DC ∑ dc=1 vdc .Xdc + RM ∑ rm=1 vrm .Xrm + RC ∑ rc=1 vrc .Xrc + DS ∑ ds=1 vds .Xds (15) (15) • The self-sufficiency (SS) As the number of domestically manufactured products is increased, the self-efficiency increases, and this has positive effects on the other social concerns such as reduced inflation and exchange rate. The self-sufficiency impact can be specified by Equation (13). SS = θ . T ∑ t=1 P ∑ p=1 M ∑ m=1 QMp,t m (13) (13) Accordingly, the social function of the model (Z3) is characterized by considering the weights of w4 and w5 for the job creation and self-sufficiency objectives, respectively, according to Equation (14): min Z3 = −w4 .JC −w5 .SS (14) (14) 3.2.5. The Model Constraints After describing the objectives, the model constraints are described. The first constraint set is shown in Equations (16) and (17). DC ∑ dc=1 Tp,t dc,lc = dp,t k ∀lc ∈LC, p ∈P, t ∈T (16) DC ∑ dc=1 Tp,t lc,dc = upp,t k ∀lc ∈LC, p ∈P, t ∈T (17) (16) DC ∑ dc=1 Tp,t lc,dc = upp,t k ∀lc ∈LC, p ∈P, t ∈T (17) (17) Equations (16) and (17), respectively, ensure that all customers’ demands are answered in the forward logistics and all returned products are collected by the collecting centers in the reverse logistics. Therefore, customers’ demands and returned products should be equal to the product flows from distributing and collecting centers to the customers and product flows from the customers to the distributing and collecting centers. However, as shown in Equations (18) and (19), the incoming flows to the remanufacturing centers and recycling centers should be equal to their outgoing flows. Logistics 2024, 8, 29 13 of 25 DC ∑ dc=1 Tp,t dc,rm = M ∑ m=1 Tp,t rm,m + S ∑ s=1 Tp,t rm,s ∀rm ∈RM, p ∈P, t ∈T (18) DC ∑ dc=1 Tp,t dc,rc = S ∑ s=1 Tp,t rc,s ∀rc ∈RC, p ∈P, t ∈T (19) DC ∑ dc=1 Tp,t dc,rm = M ∑ m=1 Tp,t rm,m + S ∑ s=1 Tp,t rm,s ∀rm ∈RM, p ∈P, t ∈T (18) (18) DC ∑ dc=1 Tp,t dc,rc = S ∑ s=1 Tp,t rc,s ∀rc ∈RC, p ∈P, t ∈T (19) (19) In addition to the demand constraints, flow balance constraints should also be satisfied. These constraints state that the sum of the inflows to each node in the logistics network is equal to the sum of the outflows from that node. These constraints for the remanufac- tured products in the distributing and collecting centers, for the recycled products in the distributing and collecting centers, for the disposed of products in the distributing and collecting centers, for the finished products transferred from the manufacturing centers and their warehouses to the distributing and collecting centers, for the finished products in the manufacturing centers, and for the raw materials in the supplier centers are shown in Equations (20)–(25), respectively. Also, as explained, the distribution rates of returned products to the remanufacturing, recycling, and disposal centers are practical rates based on historical data. 3.2.5. The Model Constraints For example, if in the distributing and collecting center dc, it has been experienced that 50% of the returned products p are prone to remanufacturing, while 30% of them have a lower quality and are only suitable for recycling, then RRMp,t dc = 0.5, RRCp,t dc = 0.3, and RDSp,t dc = 0.2 for all t. Another point is that the set of warehouses is included in the manufacturing centers, so the inventory constraints are just added for them. RM ∑ rm=1 Tp,t dc,rm = LC ∑ lc=1 rrmp,t dc .Tp,t lc,dc ∀dc ∈DC, p ∈P, t ∈T (20) DC ∑ dc=1 Tp,t dc,rc = LC ∑ lc=1 rrcp,t dc .Tp,t lc,dc ∀dc ∈DC, p ∈P, t ∈T (21) DC ∑ dc=1 Tp,t dc,ds = LC ∑ lc=1 rdsp,t dc .Tp,t lc,dc ∀dc ∈DC, p ∈P, t ∈T (22) M ∑ m=1 (Tp,t m,dc + TIp,t m,dc) = LC ∑ lc=1 Tp,t dc,lc ∀dc ∈DC, p ∈P, t ∈T (23) S ∑ s=1 Tp,t s,m + RM ∑ rm=1 Tp,t rm,m = DC ∑ dc=1 Tp,t m,dc + TIp,t m,m ∀m ∈M, p ∈P, t ∈T (24) RC ∑ rc=1 Tp,t rc,s + RM ∑ rm=1 Tp,t rm,s ≤ M ∑ m=1 Tp,t s,m ∀s ∈S, p ∈P, t ∈T (25) (20) (21) (22) (23) S ∑ s=1 Tp,t s,m + RM ∑ rm=1 Tp,t rm,m = DC ∑ dc=1 Tp,t m,dc + TIp,t m,m ∀m ∈M, p ∈P, t ∈T (24) (24) RC ∑ rc=1 Tp,t rc,s + RM ∑ rm=1 Tp,t rm,s ≤ M ∑ m=1 Tp,t s,m ∀s ∈S, p ∈P, t ∈T (25) (25) We also need constraints that establish the relationship between the production and the storage. Equations (26)–(29) are used for this purpose. We also need constraints that establish the relationship between the production and the storage. Equations (26)–(29) are used for this purpose. 3.2.5. The Model Constraints QMp,t m = DC ∑ dc=1 Tp,t m,dc + TIp,t m,m ∀m ∈M, p ∈P, t ∈T (26) (26) QPp,t m = QPp,t−1 m + TIp,t m,m − DC ∑ dc=1 TIp,t m,dc ∀m ∈M, p ∈P, t ∈T (27) TIp,t m,m ≤BigM .Up,t m ∀m ∈M, p ∈P, t ∈T (28) (27) (28) DC ∑ dc=1 TIp,t m,dc ≤BigM.(1 −Up,t m ) ∀m ∈M, p ∈P, t ∈T (29) (29) Equation (26) indicates that the manufactured products in each period are sent to the distributing and collecting centers or stored in the warehouses. On the other hand, the inventory level of warehouses in each period is equal to the remaining inventory level Logistics 2024, 8, 29 14 of 25 from the previous period plus the transferred products from the related manufacturing center to the warehouse in that period minus the sum of the products that are sent from the warehouse to the distributing and collecting centers (as shown in Equation (27)). Equations (28) and (29) do not allow the products to be transported from the manufacturing centers to their warehouse and simultaneously, to be sent from their warehouse to the customers. In other words, the products will only be allowed to be transferred to the warehouse if the manufactured products in each period exceed the demanded products of the distributing and collecting centers in that period. g g p Equations (30)–(40) confirm that the flow of products is only allowed between the active facilities, the outflows from a facility are less than the processed products in that facility, and the processed products in each facility cannot exceed the capacity of the facility. We denote such constraints as capacity constraints. Therefore, the capacity constraints of facilities are Equations (30) and (31) for the manufacturing centers, Equations (32) and (33) for the distributing and collecting centers, Equations (34) and (35) for the remanufacturing centers, Equations (36) and (37) for the disposal centers, Equations (38) and (39) for the recycling centers, and Equation (40) for the capacity of warehouses in the manufacturing centers. 3.2.5. The Model Constraints DC ∑ dc=1 Tp,t m,dc ≤QMp,t m ∀m ∈M, p ∈P, t ∈T (30) P ∑ p=1 QMp,t m ≤cam ∀m ∈M, t ∈T (31) LC ∑ lc=1 Tp,t dc,lc ≤QDCp,t dc ∀dc ∈DC, p ∈P, t ∈T (32) P ∑ p=1 QDCp,t dc ≤(cadc + cradc) .Xdc ∀dc ∈DC, t ∈T (33) DC ∑ dc=1 Tp,t dc,rm ≤QRMp,t rm ∀rm ∈RM, p ∈P, t ∈T (34) P ∑ p=1 QRMp,t rm ≤carm .Xrm ∀rm ∈RM, t ∈T (35) DC ∑ dc=1 Tp,t dc,ds ≤QDSp,t ds ∀ds ∈DS, p ∈P, t ∈T (36) P ∑ p=1 QDSp,t ds ≤cads .Xds ∀ds ∈DS, t ∈T (37) DC ∑ dc=1 Tp,t dc,rc ≤QRCp,t rc ∀rc ∈RC, p ∈P, t ∈T (38) P ∑ p=1 QRCp,t rc ≤carc .Xrc ∀rc ∈RC, t ∈T (39) P ∑ p=1 QPp,t m ≤caim ∀m ∈M, t ∈T (40) C ∑ =1 Tp,t m,dc ≤QMp,t m ∀m ∈M, p ∈P, t ∈T (30) (30) (31) (32) P ∑ p=1 QDCp,t dc ≤(cadc + cradc) .Xdc ∀dc ∈DC, t ∈T (33) (33) DC ∑ dc=1 Tp,t dc,rm ≤QRMp,t rm ∀rm ∈RM, p ∈P, t ∈T (34) (38) (39) (40) Furthermore, the number of employed vehicles in the supply chain should be identi- fied and the amounts of products transported through the supply chain should be limited to their capacity. For this purpose, Equations (41)–(46) have been introduced, which describe, respectively, the transportable products between the suppliers and the manufacturers, the manufacturers and the distributing and collecting centers, the distributing and collecting centers and the customers, the customers and the distributing and collecting centers, the Logistics 2024, 8, 29 15 of 25 distributing and collecting centers and the recycling centers, and the distributing and collecting centers and the remanufacturing centers. distributing and collecting centers and the recycling centers, and the distributing and collecting centers and the remanufacturing centers. 3.2.5. The Model Constraints pvp .Tp,t s,m ≤ V ∑ v=1 cap v .NVp,t v,s,m ∀s ∈S, m ∈M, p ∈P, t ∈T (41) pvp .Tp,t m,dc ≤ V ∑ v=1 cap v .NVp,t v,m,dc ∀m ∈M, dc ∈DC, p ∈P, t ∈T (42) pvp .Tp,t dc,lc ≤ V ∑ v=1 cap v .NVp,t v,dc,lc ∀dc ∈DC, lc ∈LC, p ∈P, t ∈T (43) pvp .Tp,t lc,dc ≤ V ∑ v=1 cap v .NVp,t v,lc,dc ∀lc ∈LC, dc ∈DC, p ∈P, t ∈T (44) pvp .Tp,t dc,rc ≤ V ∑ v=1 cap v .NVp,t v,dc,rc ∀dc ∈DC, rc ∈RC, p ∈P, t ∈T (45) pvp .Tp,t dc,rm ≤ V ∑ v=1 cap v .NVp,t v,dc,rm ∀dc ∈DC, rm ∈RM, p ∈P, t ∈T (46) (41) (42) (45) (46) According to Equation (47), the maximum employee support schemes that should be selected for increasing the abilities of employees in the supply chain are restricted to the budget considered for this purpose. ES ∑ es=1 CSt es .Ot es ≤MBS ∀t ∈T (47) (47) Equations (48)–(51) imply that for the completion of the closed-loop supply chain functions in both the forward and reverse directions, at least one facility of each distributing and collecting center, remanufacturing center, recycling center, and disposing center should be established. DC DC ∑ dc=1 Xdc ≥1 (48) RM ∑ rm=1 Xrm ≥1 (49) RC ∑ rc=1 Xrc ≥1 (50) DS ∑ ds=1 Xds ≥1 (51) (48) (49) (50) (51) Finally, Equations (52)–(54) represent the type of variables used in the proposed model. NVt v,s,m, NVp,t v,m,dc, NVp,t v,dc,lc, NVp,t v,dc,rm, NVp,t v,dc,rc, NVp,t v,dc,ds ∈Z+ ∀v ∈V, s ∈S, m ∈M, dc ∈DC, lc ∈LC, rm ∈RM, rc ∈RC, ds ∈DS, p ∈P, t ∈T (52) (52) Tp,t m,dc, TIp,t m,m, Tp,t dc,lc, TIp,c m,dc, Tp,t lc,dc, Tp,t dc,rm, Tp,t dc,ds, Tp,t dc,rc, Tp,t rm,m, Tp,t rc,s, Tp,t rm,s, QPp,t m , QMp,t m , TIp,t m,dc, QRMp,t rm, QDSp,t ds , QRCp,t rc ≥0 ∀v ∈V, s ∈S, m ∈M, dc ∈DC, lc ∈LC, rm ∈RM, rc ∈RC, ds ∈DS, p ∈P, t ∈T (53) (53) Xdc, Xrm, Xrc, Xds ∈{0, 1} (54) (54) 3.3. The Solution Approach Given the importance of the Pareto solutions in multi-objective problems, any solution approach that can extract more and more diverse solutions is more efficient.i One of the most efficient solution approaches in the context of the multi-objective problems is the ε-constraint method [31]. Some of the advantages of this method over some other widely used approaches in solving multi-objective problems such as the weighting method are as follows [31]: 1. Find the non-extreme points in the Pareto front in addition to the extreme points (Utopia points) in that front. 1. Find the non-extreme points in the Pareto front in addition to the extreme points (Utopia points) in that front. 2. The different scales of the objective functions have no effects on exploring the solution space in this method. 3. This method can detect Pareto points when there are several Pareto fronts in the solution space. To explain the ε-constraint method, the following multi-objective model that aims to minimizing the objective functions Z1(x), Z2(x), . . ., and Zn(x) is considered: To explain the ε-constraint method, the following multi-objective model that aims to minimizing the objective functions Z1(x), Z2(x), . . ., and Zn(x) is considered: Min (Z1(x), Z2(x), . . . , Zn(x)) t Min (Z1(x), Z2(x), . . . , Zn(x)) s. t. (55) Min (Z1(x), Z2(x), . . . , Zn(x)) Min (Z1(x), Z2(x), . . . , Zn(x)) s. t. x ∈S (55) (55) x ∈S where x is the vector of decision variables and S denotes the solution space. The ε-constraint method finds the Pareto solutions in successive iterations. In each iteration, one of the objective functions is considered as the objective function of a single-objective model wherein other objective functions are passed to the constraints by considering an upper bound for them. In an iteration of the ε-constraint method where Zi(x) has been chosen as the single objective, the following model should be solved: where x is the vector of decision variables and S denotes the solution space. The ε-constraint method finds the Pareto solutions in successive iterations. In each iteration, one of the objective functions is considered as the objective function of a single-objective model wherein other objective functions are passed to the constraints by considering an upper bound for them. 3.3. The Solution Approach In multi-objective problems, a desired solution should have good performance in the achievement of all objective functions. However, the considered objective functions usually conflict with each other, and optimizing one objective function does not lead to Logistics 2024, 8, 29 16 of 25 the optimization of other objective functions. Indeed, if all the objectives were in line with each other, it would no longer be necessary to study the problem using a multi-objective model, and it would be enough to just optimize one of the objective functions to achieve the optimal solution. Hence, most of the solution approaches proposed for multi-objective problems try to obtain a balance for different objective functions, and in this regard, Pareto solutions have been introduced. A Pareto solution is a solution against which there is no other feasible solution with better values for all the objective functions. The set of all Pareto solutions form the Pareto front in the solution space. Each of the Pareto solutions can be considered as the optimal solution because they are definitely better than other non-Pareto solutions. However, the selection among the Pareto-optimal solutions is left to the ultimate decision maker to choose the most desirable solution among them [30]. Given the importance of the Pareto solutions in multi-objective problems, any solution approach that can extract more and more diverse solutions is more efficient. the optimization of other objective functions. Indeed, if all the objectives were in line with each other, it would no longer be necessary to study the problem using a multi-objective model, and it would be enough to just optimize one of the objective functions to achieve the optimal solution. Hence, most of the solution approaches proposed for multi-objective problems try to obtain a balance for different objective functions, and in this regard, Pareto solutions have been introduced. A Pareto solution is a solution against which there is no other feasible solution with better values for all the objective functions. The set of all Pareto solutions form the Pareto front in the solution space. Each of the Pareto solutions can be considered as the optimal solution because they are definitely better than other non-Pareto solutions. However, the selection among the Pareto-optimal solutions is left to the ultimate decision maker to choose the most desirable solution among them [30]. 4. The Numerical Results To examine the performance and efficiency of the model, three examples of the forward and reverse supply chain in the context of automobile battery products are considered. In Tables 3 and 4, a description of the examples, including the number of facilities and the parameters’ values, is presented. Table 3. The dimensions of the case studies. Table 3. The dimensions of the case studies. Table 3. The dimensions of the case studies. Examples P V ES LC DS RC RM DC M S T The small-size problem 2 3 2 2 2 1 1 2 1 2 2 The medium-size problem 5 5 4 5 3 3 2 3 2 3 4 The large-size problem 10 10 6 8 4 6 5 4 6 5 5 Table 4. The parameter values in the examples. Table 4. The parameter values in the examples. 3.3. The Solution Approach In an iteration of the ε-constraint method where Zi(x) has been chosen as the single objective, the following model should be solved: min Zi(x) s. t. Z1(x) ≤ε1 ... Zi−1(x) ≤εi−1 Zi+1(x) ≤εi+1 ... Zn(x) ≤εn x ∈S (56) (56) Indeed, the term ε in this method indicates that in each iteration, each objective function, except the one that is intended to be optimized, is allowed to deviate from its ideal value by a certain amount, namely,ε, and as proven [31], this yields Pareto solutions. Also, to find proper values for εi, first, the ideal solution of objective function Zi(x) is introduced as the vector x∗ i . x∗ i indicates the vector of solutions that optimizes the objective function Zi(x). In other words, x∗ i satisfies the relationship Zi(x∗ i ) ≤Zi(x) for all x ∈S. Furthermore, Z∗ i and Z− i are defined by Equations (57) and (58). Logistics 2024, 8, 29 17 of 25 Z∗ i = Zi(x∗ i ) (57) Z∗ i = Zi(x∗ i ) Z∗ i = Zi(x∗ i ) (57) Z− i = Max(Zi(x∗ 1), Zi(x∗ 2), . . . , Zi(x∗ n)) (58) (58) Taking into account the definitions of Z∗ i and Z− i , the values of εi could be specified using Equation (59): t Taking into account the definitions of Z∗ i and Z− i , the values of εi could be specified using Equation (59): t εi = Z∗ i + t r(Z− i −Z∗ i ) t = 1, 2, . . . , r (59) (59) It is also notable that the higher the value for t, the more Pareto points are expected to be explored and identified by the ε-constraint method. It is also notable that the higher the value for t, the more Pareto points are expected to be explored and identified by the ε-constraint method. 4. The Numerical Results 4.1. Objective Function Evaluations and Model Complexity The Small-Size Problem The Medium-Size Problem The Large-Size Problem Objective Function Pareto Solution Z1 Z2 Z3 Z4 Z1 Z2 Z3 Z4 Z1 Z2 Z3 Z4 1 165,320 43,918 3735 10 493,640 130,690 10,490 11 1,630,951 639,500 34,050 21 2 140,540 81,725 3333 10 418,140 220,750 9377 13 1,474,490 826,440 30,030 24 3 122,160 119,528 2930 11 367,310 310,810 8261 15 1,328,780 1,013,400 26,010 27 4 104,510 157,337 2529 12 281,765 490,919 6029 18 1,183,165 1,200,300 21,980 30 5 89,297 195,136 2125 11 240,850 580,990 4913 20 1,036,904 1,387,300 17,960 33 6 74,089 232,945 1723 13 83,549 941,240 450 27 897,926 1,574,200 13,930 36 7 58,902 270,749 1318 14 - - - - 762,711 1,761,210 9909 39 8 44,060 308,554 917 14 - - - - 506,381 2,013,510 1884 45 9 29,219 346,359 513 15 - - - - - - - - 10 14,016 384,146 112 16 - - - - - - - - Next, taking into account the values in Table 5 and choosing r = 10, Equation (59) is employed to derive the values of εi for each objective function. Doing so and using the ε-constraint model in Equation (57), the Pareto solutions are extracted, as shown in Table 6. It is notable that in this table, the duplicate values for different values of εi have been removed. Also, Figures 2–4 include triple combinations of the objective functions to make it possible to display them in 3D spaces. Next, taking into account the values in Table 5 and choosing r = 10, Equation (59) is employed to derive the values of εi for each objective function. Doing so and using the ε-constraint model in Equation (57), the Pareto solutions are extracted, as shown in Table 6. It is notable that in this table, the duplicate values for different values of εi have been removed. Also, Figures 2–4 include triple combinations of the objective functions to make it possible to display them in 3D spaces. Table 6. The Pareto solutions for the case problems. 4. The Numerical Results Parameter Description Parameter Description Parameter Description dp,t lc Uniform (200, 500) vds 2 pcp,t rc Uniform (0.7, 1.5) upp,t k Uniform (5, 8) cst es Uniform (10, 27) pcp,t ds Uniform (0.7, 1.5) rrmp,t dc Uniform (0.2, 0.8) dts,m Uniform (7, 15) cas 1000 rrcp,t dc Uniform (0.2, 0.8) dtm,dc Uniform (7, 15) cam 1100 rdsp,t dc Uniform (0.2, 0.8) dtdc,lc Uniform (7, 15) caim 1000 f ct dc Uniform (1000, 3500) dtdc,rm Uniform (7, 15) cadc 1200 f ct rm Uniform (1000, 3500) dtdc,rc Uniform (7, 15) cardc 1000 f ct rc Uniform (1000, 3500) dtdc,ds Uniform (7, 15) carm 1300 f ct ds Uniform (1000, 3500) mbs 10000 carc 1300 tct s,m Uniform (0.1, 0.2) ncs 0.01 cads 800 tcp,t m,dc Uniform (0.1, 0.2) ncm 0.014 cav 2000 cqp,t m,m Uniform (0.1, 0.2) ncrm 0.012 pvp 2 tcp,t dc,lc Uniform (0.1, 0.2) ncrc 0.01 w1 0.4 tcp,t m,dc Uniform (0.1, 0.2) ncdc 0.02 w2 0.6 tcp,t lc,dc Uniform (0.1, 0.2) ncds 0.01 w3 0.3 tcp,t dc,rm Uniform (0.1, 0.2) ecp m Uniform (15, 20) w4 0.3 tcp,t dc,rc Uniform (0.1, 0.2) ecp dc Uniform (15, 20) w5 0.4 tcp,t dc,ds Uniform (0.1, 0.2) ecp rm Uniform (15, 20) cop 2m Uniform (2, 5) tcp,t rm,m Uniform (0.1, 0.2) ecp ds Uniform (15, 20) cop 2rm Uniform (2, 5) tcp,t rm,s Uniform (0.1, 0.2) wvp m Uniform (5, 15) cop 2rc Uniform (5, 9) tcp,t rc,s Uniform (0.1, 0.2) wvp rc Uniform (5, 15) cop 2dc Uniform (7, 15) pcp,t m Uniform (0.7, 1.5) wvp rm Uniform (5, 15) co2v 2 vdc 4 wvp dc Uniform (5, 15) hp,t m Uniform (0.5, 1.5) vrm 3 pcp,t dc Uniform (0.7, 1.5) vrc 5 pcp,t rm Uniform (0.7, 1.5) It should be also pointed out that for implementing the model on the examples and extracting the Pareto solutions, the ε-constraint method is run on the GAMS software V.23.0 and on a computer with a Core i5 CPU and 4.0 GB RAM. p The number of embodied variables in the small-, medium-, and large-size problems are 1036, 8478, and 105,303, respectively. Also, it is mentioned that the CPU times for running p The number of embodied variables in the small-, medium-, and large-size problems are 1036, 8478, and 105,303, respectively. 4. The Numerical Results Also, it is mentioned that the CPU times for running Logistics 2024, 8, 29 18 of 25 18 of 25 the small-, medium-, and large-size problems were 160, 760, and 3500 s, respectively, on average. In the following, the results of the implementation are interpreted step by step. the small-, medium-, and large-size problems were 160, 760, and 3500 s, respectively, on average. In the following, the results of the implementation are interpreted step by step. 4.1. Objective Function Evaluations and Model Complexity As explained, to implement the ε-constraint method, the value of the objective func- tions for the ideal solution of each objective function should be determined first. Then, the values of Z∗ i and Z∗∗ i for all objective functions characterized by index i are calculated using Equations (57) and (58), respectively, and are shown in Table 5. It should also be noted that for each case problem, the model in (56) should be solved many times depending on the value of r in Equation (59). Hence, to stand for the model complexity, the average time of running the model for each case problem is taken into account. The last column of Table 5 shows the solving times of the case problems. Table 5. The values of Z∗ i and Z− i for the case problems. Table 5. The values of Z∗ i and Z− i for the case problems. Z* 1 Z− 1 Z* 2 Z− 2 Z* 3 Z− 3 Z* 4 Z− 4 Solving Time (s) Example 1 14,016 165,320 43,918 384,146 −3735 −112 10 16 135 Example 2 83,549 493,640 130,690 941,240 −10,490 −450 10 27 592 Example 3 506,381 1,630,951 639,500 2,013,510 −34,050 −1884 21 45 2623 Next, taking into account the values in Table 5 and choosing r = 10, Equation (59) is employed to derive the values of εi for each objective function. Doing so and using the ε-constraint model in Equation (57), the Pareto solutions are extracted, as shown in Table 6. It is notable that in this table, the duplicate values for different values of εi have been removed. Also, Figures 2–4 include triple combinations of the objective functions to make it possible to display them in 3D spaces. Table 6. The Pareto solutions for the case problems. 4.1. Objective Function Evaluations and Model Complexity The Small-Size Problem The Medium-Size Problem The Large-Size Problem Objective Function Pareto Solution Z1 Z2 Z3 Z4 Z1 Z2 Z3 Z4 Z1 Z2 Z3 Z4 1 165,320 43,918 3735 10 493,640 130,690 10,490 11 1,630,951 639,500 34,050 21 2 140,540 81,725 3333 10 418,140 220,750 9377 13 1,474,490 826,440 30,030 24 3 122,160 119,528 2930 11 367,310 310,810 8261 15 1,328,780 1,013,400 26,010 27 4 104,510 157,337 2529 12 281,765 490,919 6029 18 1,183,165 1,200,300 21,980 30 5 89,297 195,136 2125 11 240,850 580,990 4913 20 1,036,904 1,387,300 17,960 33 6 74,089 232,945 1723 13 83,549 941,240 450 27 897,926 1,574,200 13,930 36 7 58,902 270,749 1318 14 - - - - 762,711 1,761,210 9909 39 8 44,060 308,554 917 14 - - - - 506,381 2,013,510 1884 45 9 29,219 346,359 513 15 - - - - - - - - 10 14,016 384,146 112 16 - - - - - - - - Table 6. The Pareto solutions for the case problems. Logistics 2024, 8, 29 19 of 25 - s 2024, 8, x FOR PEER REVIEW Figure 2. The Pareto solutions for the small-size problem in 3D space. Figure 2. The Pareto solutions for the small-size problem in 3D space. Figure 2. The Pareto solutions for the small-size problem in 3D space. 024, 8, x FOR PEER REVIEW Figure 2. The Pareto solutions for the small-size problem in 3D space. Figure 2. The Pareto solutions for the small-size problem in 3D space. Figure 2. The Pareto solutions for the small-size problem in 3D space. Figure 3. The Pareto solutions for the medium-size problem in 3D space. Figure 3. The Pareto solutions for the medium-size problem in 3D space. Figure 3. The Pareto solutions for the medium-size problem in 3D space. the medium-size problem in 3D space. Figure 3. The Pareto solutions Figure 3. The Pareto solutions for the medium-size problem in 3D space. Figure 3. The Pareto solutions for the medium-size problem in 3D space. Logistics 2024, 8, 29 20 of 25 2024, 8, x FOR PEER REVIEW 22 Figure 4. The Pareto solutions for the large-size problem in 3D space. Figure 4. The Pareto solutions for the large-size problem in 3D space. 024, 8, x FOR PEER REVIEW Figure 4. The Pareto solutions for the large-size problem in 3D space. Figure 4. The Pareto solutions for the large-size problem in 3D space. 4.1. Objective Function Evaluations and Model Complexity Table 6 and Figures 2–4 clearly demonstrate the ability of the constraint method to extract various Pareto points and to properly explore the solution space and, therefore, the validity of the method. Another point is the conﬂict between the objective functions, as indicated by the trend of Pareto solutions in Table 6 and Figures 2–4. To this end, the minimization of cost is in contrast with the minimization of the environmental goals. The cost and the social objective functions of the supply chain are also in opposite directions. This means that caring about the social aspects of the supply chain entails paying more, though the eﬀects of these measures on the supply chain proﬁtability may appear in the long term. Also, the Pareto solution that represents the ideal solution for the cost function is alone evident for claiming the supply chain seeking solely the minimization of costs has disappointing performances in the environmental and social dimensions. Thus, a sustain- able approach could provide a beAer alignment of the supply chain with social and na- tional concerns, as observed in other Pareto solutions. The ﬁnal note in this regard and in line with the model assumptions is that minimizing the costs does not necessarily mean using fewer facilities. That is, sometimes increasing the facilities reduces the transporta- tion and material ﬂow costs on the planned horizon, which in turn can oﬀset the invest- ment costs of the facilities. However, as this cost has to be paid at the beginning of the supply chain, sometimes it is not desirable for the stakeholders or even aﬀordable for them. Overall, the results of this section show that the objective functions are in conﬂict ith ea h othe a d thi ho the eed to u e a ulti obje ti e odel fo de i i a Table 6 and Figures 2–4 clearly demonstrate the ability of the ε-constraint method to extract various Pareto points and to properly explore the solution space and, therefore, the validity of the method. Another point is the conflict between the objective functions, as indi- cated by the trend of Pareto solutions in Table 6 and Figures 2–4. To this end, the minimization of cost is in contrast with the minimization of the environmental goals. The cost and the social objective functions of the supply chain are also in opposite directions. 4.1. Objective Function Evaluations and Model Complexity This means that caring about the social aspects of the supply chain entails paying more, though the effects of these measures on the supply chain profitability may appear in the long term. Also, the Pareto solution that represents the ideal solution for the cost function is alone evident for claiming the supply chain seeking solely the minimization of costs has disappointing performances in the environmental and social dimensions. Thus, a sustainable approach could provide a better alignment of the supply chain with social and national concerns, as observed in other Pareto solutions. The final note in this regard and in line with the model assumptions is that minimiz- ing the costs does not necessarily mean using fewer facilities. That is, sometimes increasing the facilities reduces the transportation and material flow costs on the planned horizon, which in turn can offset the investment costs of the facilities. However, as this cost has to be paid at the beginning of the supply chain, sometimes it is not desirable for the stakeholders or even affordable for them. Overall, the results of this section show that the objective functions are in conflict with each other, and this shows the need to use a multi-objective model for designing a closed-loop supply chain. with each other, and this show closed-loop supply chain. 4.2. Trend in Aggregate Variables p pp y 4.2. Trend in Aggregate Variables Another remarkable result of this section is the trends observed in the aggregate var- iables instead of reporting all variables separately. To do this, variables are categorized into three groups representing, respectively, the total number of established facilities (TF), the total number of processed products (produced, remanufactured, recycled, and dis- Another remarkable result of this section is the trends observed in the aggregate variables instead of reporting all variables separately. To do this, variables are categorized into three groups representing, respectively, the total number of established facilities (TF), the total number of processed products (produced, remanufactured, recycled, and disposed) in the planning horizon (TP), and the total transportation in the planning horizon (TV). Table 7 shows the results of case problems with respect to the mentioned variables. Logistics 2024, 8, 29 21 of 25 Table 7. The aggregate variables of Pareto solutions in the case problems. The Small-Size Problem The Medium-Size Problem The Large-Size Problem Aggregate Variable Pareto Solution TF TP TV TF TP TV TF TP TV 1 3 4216 2326 3 19,846 9364 5 69,902 37,772 2 3 3763 2077 3 17,763 8683 5 63,605 36,327 3 3 3310 1851 3 16,074 8603 6 57,225 34,794 4 3 2857 1624 3 12,485 7763 6 50,957 32,438 5 3 2404 1398 3 10,824 6903 7 44,603 29,667 6 3 1951 1171 8 7157 4136 8 38,430 26,879 7 3 1497 945 - - - 8 32,211 25,384 8 3 1044 718 - - - 11 25,994 23,066 9 3 591 491 - - - - - - 10 4 166 210 - - - - - - Logistics 2024, 8, x FOR PEER REVIEW 23 of 27 4 3 2857 1624 3 12,485 7763 6 50,957 32,438 5 3 2404 1398 3 10,824 6903 7 44,603 29,667 6 3 1951 1171 8 7157 4136 8 38,430 26,879 7 3 1497 945 - - - 8 32,211 25,384 8 3 1044 718 11 25 994 23 066 Table 7. The aggregate variables of Pareto solutions in the case problems. The Small-Size Problem The results in Table 7 indicate that by reducing the supply chain costs, the level of operation is also reduced. with each other, and this show closed-loop supply chain. 4.2. Trend in Aggregate Variables However, sometimes increasing the facilities of the supply chain decreases the supply chain’s overall costs and this will be due to the reduction in other supply chain costs, such as transportation between facilities. , , 9 3 591 491 - - - - - - 10 4 166 210 - - - - - - The results in Table 7 indicate that by reducing the supply chain costs, the level of operation is also reduced. However, sometimes increasing the facilities of the supply chain 4.3. Sensitivity Analysis decreases the s supply chain co We also carry out a sensitivity analysis on the demand and the number of returned products. For this purpose, three levels are considered; (1) the parameters in the ini- tial values, (2) the parameters multiplied by 1.5, and (3) the parameters multiplied by 2. Figure 5 shows the effects of objective functions versus the changes in the parameters rep- resented by the demand and returned products of the supply chain. The trends of changes indicate the positive and direct effects of demand on all objective functions. However, the effects are more severe on the cost objective function than others. Nevertheless, n an uncertain business environment, the effects of changes in the demand and returned products could be of practical value. That is, if the supply chain and the business context do not have the necessary flexibility to change facilities when these parameters fluctuate, other sustainability goals are diminished inevitably. Therefore, agility and flexibility are strategies that strengthen the supply chain toward sustainability. 4.3. Sensitivity Analysis We also carry out a sensitivity analysis on the demand and the number of returned products. For this purpose, three levels are considered; (1) the parameters in the initial values, (2) the parameters multiplied by 1.5, and (3) the parameters multiplied by 2. Figure 5 shows the eﬀects of objective functions versus the changes in the parameters represented by the demand and returned products of the supply chain. The trends of changes indicate the positive and direct eﬀects of demand on all objective functions. However, the eﬀects are more severe on the cost objective function than others. Nevertheless, n an uncertain business environment, the eﬀects of changes in the demand and returned products could be of practical value. That is, if the supply chain and the business context do not have the necessary ﬂexibility to change facilities when these parameters ﬂuctuate, other sustaina- bility goals are diminished inevitably. Therefore, agility and ﬂexibility are strategies that strengthen the supply chain toward sustainability. 0 200,000 400,000 600,000 800,000 0 2 4 6 8 10 12 Z1 PARETO SOLUTION The parameters in the initial values The parameters multiplied by 1.5 The parameters multiplied by 2 a) 0 200,000 400,000 600,000 800,000 1,000,000 0 2 4 6 8 10 12 Z2 PARETO SOLUTION The parameters in the initial values The parameters multiplied by 1.5 The parameters multiplied by 2 b) Figure 5. Cont. 5. 5. Discussion tegic and operational decisions. Each of these goals addresses a particular aspect of inter- est to managers, although they can be contradictory to each other. Also, it should balance between long-term and short-term goals because short-term goals target the use of limited resources in order to gain proﬁt in the short term, while long-term goals aim at the possi- bility of developing the supply chain in the future and ﬂexibility against uncertain events along with equipping the supply chain to earn stable and continuous proﬁts. In this re- gard, a sustainable perspective provides such a viewpoint to all short- and long-term ob- jectives as well as protecting the rights of future generations with an emphasis on the en- vironment and the development of human societies. In this paper, such a perspective was studied in the design of a closed-loop supply chain, and in this context, new goals were introduced against the conventional goals in supply chain design issues. As discussed, social and environmental goals, although they have general ac- ceptance, require paying more costs in the supply chain. Nevertheless, paying aAention to these dimensions of the supply chain will increase the credibility of the business in the future and somehow guarantee its survival in the future. This is in line with other supply In designing a closed-supply chain, there are various objectives that influence its strategic and operational decisions. Each of these goals addresses a particular aspect of interest to managers, although they can be contradictory to each other. Also, it should balance between long-term and short-term goals because short-term goals target the use of limited resources in order to gain profit in the short term, while long-term goals aim at the possibility of developing the supply chain in the future and flexibility against uncertain events along with equipping the supply chain to earn stable and continuous profits. In this regard, a sustainable perspective provides such a viewpoint to all short- and long-term objectives as well as protecting the rights of future generations with an emphasis on the environment and the development of human societies. In this paper, such a perspective was studied in the design of a closed-loop supply chain, and in this context, new goals were introduced against the conventional goals in supply chain design issues. future and somehow guarantee its survival in the future. 4.3. Sensitivity Analysis decreases the s supply chain co 0 200,000 400,000 600,000 800,000 0 2 4 6 8 10 12 Z1 PARETO SOLUTION The parameters in the initial values The parameters multiplied by 1.5 The parameters multiplied by 2 a) 0 200,000 400,000 600,000 800,000 1,000,000 0 2 4 6 8 10 12 Z2 PARETO SOLUTION The parameters in the initial values The parameters multiplied by 1.5 The parameters multiplied by 2 b) The parameters in the initial values The parameters multiplied by 1.5 The parameters multiplied by 2 Figure 5. Cont. Figure 5. Cont. Logistics 2024, 8, 29 22 of 25 27 Figure 5. The eﬀects of demand changes on the objective functions, where sub-ﬁgures (a–d) show those eﬀects on the objective functions Z1, Z2, Z3, and Z4, respectively. 5 Discussion 0 1500 3000 4500 6000 7500 9000 0 2 4 6 8 10 12 Z3 PARETO SOLUTION The parameters in the initial values The parameters multiplied by 1.5 The parameters multiplied by 2 c) 0 10 20 30 0 2 4 6 8 10 12 Z4 PARETO SOLUTION The parameters in the initial values The parameters multiplied by 1.5 The parameters multiplied by 2 d) Figure 5. The effects of demand changes on the objective functions, where sub-figures (a–d) show those effects on the objective functions Z1, Z2, Z3, and Z4, respectively. 0 1500 3000 4500 6000 7500 9000 0 2 4 6 8 10 12 Z3 PARETO SOLUTION The parameters in the initial values The parameters multiplied by 1.5 The parameters multiplied by 2 c) Figure 5. The eﬀects of demand changes on the objective functions, where sub-ﬁgures (a–d) show those eﬀects on the objective functions Z1, Z2, Z3, and Z4, respectively. 5 Di i Figure 5. The effects of demand changes on the objective functions, where sub-figures (a–d) show those effects on the objective functions Z1, Z2, Z3, and Z4, respectively. 6. Conclusions In this paper, a multi-period, multi-product, and multi-objective function model was presented that aimed to design and optimize a closed-loop supply chain. To do so, two economic objective functions of logistic costs and the number of facilities as well as one environmental and one social objective function were considered. The cost ob- jective functions intended to be minimized included the establishment cost of facilities, the transportation costs between facilities, the processing cost of products, the holding costs of products, and the cost associated with the employee support schemes. In the environmental objective function, the minimization of CO2 emissions, water usage, and energy consumption was investigated. Moreover, the enhancement of job creation and self-sufficiency throughout the activities of the supply chain were pursued by the social objective function of the paper. Indeed, the main goal of sustainable development is to consider as much as possible objectives that affect human life on a long-term horizon and in coordination with the concerns of future generations. Thus, the proposed paper made contributions to introducing new objective functions in the mentioned dimensions of a sustainable design of the supply chain. To extract the Pareto solutions of the model, the ε-constrained method was utilized. The results of the paper in this regard illustrated the conflicts between the objective functions and more emphasis on one of the objective functions in each of the Pareto solutions. We also interpreted the different trends in the solutions, such as the fact that we could not address all design concerns of the supply chain by just emphasizing the total cost of the supply chain. It was shown that this objective function is unable to even address the economic objectives, such as the number of facilities employed in the supply chain. Furthermore, the sensitivity analysis of the demands of the supply chain revealed that these parameters mainly affect the supply chain cost and their effects on the objective functions are slight. For future research, it is suggested to propose the coordination mechanism within the studied supply chain to provide necessary incentives for cooperation. Also, the use of other multi-objective methods such as compromise and goal programming are suggested. Considering the uncertainty in each of the mentioned parameters of the model could make the model more realistic and increase the capability of the supply chain in response to different changes in the real world and is left as another suggestion for future studies. 5. 5. Discussion This is in line with other supply chain design studies that aAempt to beneﬁt the sustainable perspective [15,18,20]. Another point is the number of facilities in the supply chain. In a short-term perspective, a smaller number of facilities will cost the investors less and may be favorable for them at the start of their business, but in a long-term perspective, more facilities will reduce costs and re- lease of pollutants in the entire supply chain. Therefore, considering this objective along with other sustainability goals provides a beAer balance in long-term and short-term de- cisions and makes sustainability research in the ﬁeld of the supply chain more aligned with the long-term goals of managers. As discussed, social and environmental goals, although they have general acceptance, require paying more costs in the supply chain. Nevertheless, paying attention to these dimensions of the supply chain will increase the credibility of the business in the future and somehow guarantee its survival in the future. This is in line with other supply chain design studies that attempt to benefit the sustainable perspective [15,18,20]. Another point is the number of facilities in the supply chain. In a short-term perspective, a smaller number of facilities will cost the investors less and may be favorable for them at the start of their business, but in a long-term perspective, more facilities will reduce costs and release of pollutants in the entire supply chain. Therefore, considering this objective along with other sustainability goals provides a better balance in long-term and short-term decisions and makes sustainability research in the field of the supply chain more aligned with the long-term goals of managers. g g g The current research could guide managers in policy making and strategic planning in several directions. In line with many scholars, the environmental and social dimensions of the supply chain affect the configuration of the closed-loop supply chain [22]. Improving the distribution planning by including the sustainable goals stemming from the proposed model is also cited as a strategic decision for enhancing the corporate social responsibility of the supply chain [24]. Moreover, the results of the research showed that in strategic Logistics 2024, 8, 29 23 of 25 thinking, a proper balance between long-term and short-term goals should be created. 5. 5. Discussion In this regard, if managers are able to finance more in the initial stages of the business to extend the supply chain facilities, they exhibit high performance in all sustainability goals. Meanwhile, if the supply chain is equipped with appropriate flexibility and agility strategies, it will have a better response to business changes such as demand fluctuations, but otherwise, it will be forced to sacrifice some sustainability indicators for others. The results of this research clearly showed that the objectives studied in the supply chain design are in contradiction with each other. On the other hand, the ε-constraint method extracted Pareto points with high ability, and each point paid more attention to one of the goals. The nature of the contradiction in supply chain design goals was evident even in the sensitivity analysis of the parameters, in that changing the parameters that had a positive effect on one objective function had an adverse effect on some other objective functions. But in any case, one should choose a solution from the Pareto points that reflects the subjective preferences of the decision makers well. In this regard, the fuzzy analytic hierarchy process proposed in [28,29] is beneficial for weighting the objective functions and choosing the most desired solution from the Pareto solution set. The proposed method in this paper only extracted Pareto’s dominant points but did not provide a framework for trade-offs between different solutions that were ultimately preferred by decision makers. Therefore, a supplementary study that can help to choose the final answer correctly and in accordance with the assumptions of the research community is necessary. 6. Conclusions The closed-loop supply chain is undoubtedly affected by the policies and laws of social and national institutions. Therefore, in line with many emerging investigations, it is worthwhile Logistics 2024, 8, 29 24 of 25 to study the effect of these measures on supply chain design issues and scrutinize the interventions of the government and other external institutions in future research. Author Contributions: Conceptualization, M.A.M., V.J. and M.B.; methodology, M.A.M.; software, M.A.M.; validation, M.A.M., V.J. and M.B.; formal analysis, M.A.M. and V.J.; investigation, M.A.M. and M.B.; resources, M.A.M. and M.B.; data curation, M.A.M. and V.J.; writing—original draft preparation, M.A.M. and M.B.; writing—review and editing, M.A.M. and V.J.; visualization, M.A.M.; supervision, M.A.M. and V.J.; project administration, V.J.; funding acquisition, V.J. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Data Availability Statement: The authors confirm that all data generated during this study are included in this published paper. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References Analysis of electronic waste reverse logistics decisions using Strategic Options Develop- A l i h d l A B ili J Cl P d 2016 133 1105 1117 [C R f] 5. Giri, B.C.; Chakraborty, A.; Maiti, T. Pricing and return product collection decisions in a closed loop supply chain with dual channel in both forward and reverse logistics. J. Manuf. Syst. 2017, 42, 104–123. [CrossRef] 6. Guarnieri, P.; e Silva, L.C.; Levino, N.A. Analysis of electronic waste reverse logistics decisions using Strategic Options Develop- ment Analysis methodology: A Brazilian case. J. Clean. Prod. 2016, 133, 1105–1117. [CrossRef] g J f y 6. Guarnieri, P.; e Silva, L.C.; Levino, N.A. 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https://openalex.org/W3122065532	https://rua.ua.es/dspace/bitstream/10045/112446/1/Peinado_etal_2021_IntJMolSci.pdf	English	null	Impact of Maturation and Vitrification Time of Human GV Oocytes on the Metaphase Plate Configuration	International journal of molecular sciences	2,021	cc-by	13,803	Article Impact of Maturation and Vitriﬁcation Time of Human GV Oocytes on the Metaphase Plate Conﬁguration o 1 , Isabel Moya 1 , Paula Sáez-Espinosa 2, Macarena Barrera 1,2 , Laura García-Valverde 1 cés 1,3 , Patricia Torres 1 and María José Gómez-Torres 2,4,* Irene Peinado 1 , Isabel Moya 1 , Paula Sáez-Espinosa 2, Macarena Barrera 1,2 , Laura García-Valverde 1,2, Raquel Francés 1,3 , Patricia Torres 1 and María José Gómez-Torres 2,4,* 1 Assisted Human Reproduction Unit, La Fe University and Polytechnic Hospital, 46026 Valencia, Spain; peinado_ire@gva.es (I.P.); imoyamarin@gmail.com (I.M.); macarenabarreragallardo@gmail.com (M.B.); lmgv25@gmail.com (L.G.-V.); raquelfrances1@gmail.com (R.F.); patriblanc81@hotmail.com (P.T.) 2 Biotechnology Department, Alicante University, 03690 Alicante, Spain; paula.saez@ua.es 3 Energy and Memory, Brain Plasticity Unit, CNRS, ESPCI Paris, PSL Research University, 75005 Paris, France 4 Cátedra Human Fertility, Universidad de Alicante, 03690 Alicante, Spain * Correspondence: mjose.gomez@ua.es 1 Assisted Human Reproduction Unit, La Fe University and Polytechnic Hospital, 46026 Valencia, Spain; peinado_ire@gva.es (I.P.); imoyamarin@gmail.com (I.M.); macarenabarreragallardo@gmail.com (M.B.); lmgv25@gmail.com (L.G.-V.); raquelfrances1@gmail.com (R.F.); patriblanc81@hotmail.com (P.T.) 2 Biotechnology Department, Alicante University, 03690 Alicante, Spain; paula.saez@ua.es 3 Energy and Memory, Brain Plasticity Unit, CNRS, ESPCI Paris, PSL Research University, 75005 Paris, France 4 Cátedra Human Fertility, Universidad de Alicante, 03690 Alicante, Spain 1 Assisted Human Reproduction Unit, La Fe University and Polytechnic Hospital, 46026 Valencia, Spain; peinado_ire@gva.es (I.P.); imoyamarin@gmail.com (I.M.); macarenabarreragallardo@gmail.com (M.B.); lmgv25@gmail.com (L.G.-V.); raquelfrances1@gmail.com (R.F.); patriblanc81@hotmail.com (P.T.) gy y y 4 Cátedra Human Fertility, Universidad de Alicante, 03690 Alicante, Spain * Correspondence: mjose.gomez@ua.es Abstract: The combination of in vitro maturation (IVM) techniques and oocyte vitriﬁcation (OV) could increase the number of useful oocytes in different types of patients. IVM and subsequent OV is the most widely used clinical strategy. Would the results improve if we reverse the order of the techniques? Here, we evaluated survival, in vitro maturation, time to extrude the ﬁrst polar body (PB), and the metaphase plate conﬁguration of human prophase I (GV) oocytes before or after their vitriﬁcation. Speciﬁc, 195 GV oocytes from 104 patients subjected to controlled ovarian stimulation cycles were included. We stablished three experimental groups: GV oocytes vitriﬁed and IVM (Group GV-Vit), GV oocytes IVM and vitriﬁed at MII stage (Group MII-Vit), and GV oocytes IVM (Group not-Vit). All of them were in vitro matured for a maximum of 48 h and ﬁxed to study the metaphase plate by confocal microscopy. Citation: Peinado, I.; Moya, I.; Sáez-Espinosa, P.; Barrera, M.; García-Valverde, L.; Francés, R.; Torres, P.; Gómez-Torres, M.J. Impact of Maturation and Vitriﬁcation Time of Human GV Oocytes on the Metaphase Plate Conﬁguration. Int. J. Mol. Sci. 2021, 22, 1125. https:// doi.org/10.3390/ijms22031125 Keywords: in vitro maturation; cryopreservation; spindle conﬁguration Academic Editor: Marc Yeste Received: 23 December 2020 Accepted: 21 January 2021 Published: 23 January 2021 Academic Editor: Marc Yeste Received: 23 December 2020 Accepted: 21 January 2021 Published: 23 January 2021 Citation: Peinado, I.; Moya, I.; Sáez-Espinosa, P.; Barrera, M.; García-Valverde, L.; Francés, R.; Torres, P.; Gómez-Torres, M.J. Impact of Maturation and Vitriﬁcation Time of Human GV Oocytes on the Metaphase Plate Conﬁguration. Int. J. Mol. Sci. 2021, 22, 1125. https:// doi.org/10.3390/ijms22031125 International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences Article Impact of Maturation and Vitriﬁcation Time of Human GV Oocytes on the Metaphase Plate Conﬁguration According to our results, the vitriﬁcation of immature oocytes and their subsequent maturation presented similar survival, maturation, and metaphase plate conformation rates, but a signiﬁcantly higher percentage of normal spindle than the standard strategy. Additionally, the extension of IVM time to 48 h did not seem to negatively affect the oocyte metaphase plate conﬁguration. 1. Introduction Oocytes vitriﬁcation (OV) protocols developed during the last decade have allowed the introduction of this technique in all assisted reproduction (AR) laboratories. This is based on the quick freezing of the oocytes in a medium with high cryoprotectants (CRP) concentrations, preventing ice crystals formation [1]. Previous studies have focused on avoiding the toxic effects of the CRP, reducing the exposure and freezing time [2–4]. OV has required a lengthier optimization and implantation period than embryo vitriﬁcation (EV). One of the greatest challenges of OV is the disruption of the meiotic spindle [5–9]. The spindle, which is formed mainly by microtubules, is responsible of chromosome segregation [10]. The microtubules are very sensitive to temperature so, therefore, oocyte preservation is associated with an increase in embryonic aneuploidies [11–13]. However, it is also known that this structure is dynamic and can repolymerize when the physiological conditions are recovered [14–19]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Oocyte preservation can be performed in immature oocytes or after their in vitro maturation (IVM). Conventionally, prophase I (GV) oocytes have been considered more suitable for preservation than metaphase II (MII) oocytes, since during meiosis arresting at GV, chromatin is protected in the germinal vesicle and the spindle is not formed yet [20–22]. Nevertheless, even if this problem was hypothetically avoided with GV oocytes, the https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 1125. https://doi.org/10.3390/ijms22031125 Int. J. Mol. Sci. 2021, 22, 1125 (http://creativecommons.org/licens 2 of 16 ed yet possible alteration in the cortical granules distribution, cytoplasmic organelles, RNA, and proteins may compromise their maturation [8,23–25]. and proteins may compromise their maturation [8,23–25]. Immature oocytes collection and in vitro maturation are techniques with multiple t ti l i di ti P ti t t i k f i h ti l ti (OHSS) ith possible alteration in the cortical granules distribution, cytoplasmic organelles, RNA, and proteins may compromise their maturation [8,23–25]. and proteins may compromise their maturation [8,23–25]. 1. Introduction Immature oocytes collection and in vitro maturation are techniques with multiple t ti l i di ti P ti t t i k f i h ti l ti (OHSS) ith Immature oocytes collection and in vitro maturation are techniques with multiple potential indications: Patients at risk of ovarian hyperstimulation (OHSS) or with Polycystic Ovary Syndrome (PCOS); women that underwent invasive surgery; patients at risk of exposure to estrogens; oocyte donation program; optimization of stimulated cycles; classic in vitro fertilization (IVF) or assisted by intracytoplasmic sperm microinjection (ICSI), and fertility preservation for social reasons or cancer patients [26–28]. In this last group, patients for oocyte vitriﬁcation and ovarian cortex preservation are included. However, this technique is still under development, and is not a clear alternative to other AR techniques. IVM involves nuclear and cytoplasmic changes that, as previously mentioned, are necessary for the subsequent proper embryonic development. The understanding of the molecular basis of the chromosome segregation process during IVM would reveal the full potential of this technique. Consequently, the quality of the collected MII oocytes will depend not only on the intrinsic characteristics of the original GV oocyte, but also on the vitriﬁcation protocol chosen and the conditions of the IVM process. Both processes can contribute to a deﬁcient embryonic development or to the start of apoptotic processes [29]. potential indications: Patients at risk of ovarian hyperstimulation (OHSS) or with Polycystic Ovary Syndrome (PCOS); women that underwent invasive surgery; patients at risk of exposure to estrogens; oocyte donation program; optimization of stimulated cycles; classic in vitro fertilization (IVF) or assisted by intracytoplasmic sperm microinjection (ICSI), and fertility preservation for social reasons or cancer patients [26–28]. In this last group, patients for oocyte vitrification and ovarian cortex preservation are included. However, this technique is still under development, and is not a clear alternative to other AR techniques. IVM involves nuclear and cytoplasmic changes that, as previously mentioned, are necessary for the subsequent proper embryonic development. The understanding of the molecular basis of the chromosome segregation process during IVM would reveal the full potential of this technique. Consequently, the quality of the collected MII oocytes will depend not only on the intrinsic characteristics of the original GV oocyte, but also on the vitrification protocol chosen and the conditions of the IVM process. Both processes can contribute to a deficient embryonic development or to the start of apoptotic processes [29]. 1. Introduction In the recent years, many children were born after OV [30–33], IVM [34–39], or a combination of both [40,41]. However, this combination remains controversial [42,43]. In previous studies performed by our group, the vitriﬁcation before or after the IVM for 48 h of GV did not show signiﬁcant differences in some constituents of their ultrastructure [25]. In the present work, we aimed to evaluate the impact of IVM time and/or vitriﬁcation on the following rates: Survival, maturation, spindle conﬁguration, and chromosomes distribution. To that end, we distributed the oocytes in three experimental groups: Group GV-Vit (frozen GV oocytes and then matured in vitro), Group MII-Vit (frozen MII oocytes after being matured in vitro), and Group not-Vit (GV oocytes matured in vitro and not vitriﬁed) (see Figure 1). p In the recent years, many children were born after OV [30–33], IVM [34–39], or a combination of both [40,41]. However, this combination remains controversial [42,43]. In previous studies performed by our group, the vitrification before or after the IVM for 48 h of GV did not show significant differences in some constituents of their ultrastructure [25]. In the present work, we aimed to evaluate the impact of IVM time and/or vitrification on the following rates: Survival, maturation, spindle configuration, and chromosomes distribution. To that end, we distributed the oocytes in three experimental groups: Group GV-Vit (frozen GV oocytes and then matured in vitro), Group MII-Vit (frozen MII oocytes after being matured in vitro), and Group not-Vit (GV oocytes matured in vitro and not vitrified) (see Figure 1). Figure 1. Experimental design that describes in detail the different study groups and the methodology used at the different in vitro maturation times (24 and 48 h). GV, prophase I; MI, metaphase I; MII, metaphase II, OV, oocytes vitrification; IVM, in vitro maturation; FIX, fixation; Vit, vitrification. Figure 1. Experimental design that describes in detail the different study groups and the methodology used at the different in vitro maturation times (24 and 48 h). GV, prophase I; MI, metaphase I; MII, metaphase II, OV, oocytes vitriﬁcation; IVM, in vitro maturation; FIX, ﬁxation; Vit, vitriﬁcation. Figure 1. Experimental design that describes in detail the different study groups and the methodology used at the different in vitro maturation times (24 and 48 h). GV, prophase I; MI, metaphase I; MII, metaphase II, OV, oocytes vitrification; IVM, in vitro maturation; FIX, fixation; Vit, vitrification. Figure 1. 2.2. Maturation Rate (MR) Regardless of the study groups or prior state of the oocyte, fresh (Group MII-Vit + Group not-Vit) or warmed (Group GV-Vit), no signiﬁcant differences were observed in maturation rates (Table 1). However, the germinal vesicle breakdown (GVBD) was signiﬁcantly higher in the Group GV-Vit vs. Group MII-Vit [88.9% (40/45) vs. 71.8% (61/85); p-value < 0.05] (see Table 1). The percentage of oocytes that matured during the ﬁrst 24 h of culture was 62.6% (114/182), with an increase of 11% (20/182) during 25–48 h. Thus, 73.6% (134/182) of the initial GV oocytes matured after 48 h. 2.1. Survival Rate (SR) 2.1. Survival Rate (SR) Depending on the maturation stage on which the oocytes were vitrified, the survival rate (SR) after their warming did not show significant differences in GV (Group GV-Vit) and MII (Group MII-Vit) [Group GV-Vit 77.6% (45/58) vs. Group MII-Vit 68.2% (30/44)] (Table 1). Table 1. Rates calculated according to the groups of study. Survival rate (SR), maturation rates (MR): MR by germinal vesicle breakdown (GVBD); after 24 h and 48 h. Average percentage by group and p-value. () p-value < 0.05 Group GV-Vit vs. Group MII-Vit. (–) data not possible. Group GV-Vit Group MII-Vit Group Not-Vit SR 45/58 (77.6%) 30/44 (68.2%) – MR GVBD 40/45 (88.9%) 61/85 (71.8%) * 43/52 (82.7%) 24 h 27/45 (60%) 56/85 (65.9%) 31/52 (59.6%) 48 h 33/45 (73.3%) 65/85 (76.5%) 36/52 (69.2%) Similarly, the SR was calculated according to the maturation time (24 or 48 h). For this analysis, only IVM oocytes from Group MII-Vit (n = 44) were selected, since Group GV-Vit maturation could be conditioned by the previous vitriﬁcation of the oocytes. This study did not show differences depending on the maturation time [24 h 65% (26/40) vs. 48 h 100% (4/4)]. 1. Introduction Experimental design that describes in detail the different study groups and the methodology used at the different in vitro maturation times (24 and 48 h). GV, prophase I; MI, metaphase I; MII, metaphase II, OV, oocytes vitriﬁcation; IVM, in vitro maturation; FIX, ﬁxation; Vit, vitriﬁcation. Int. J. Mol. Sci. 2021, 22, 1125 3 of 16 3 of 16 Our results showed comparable vitriﬁcation survival, maturation, and metaphase plate conﬁguration rates whether oocytes were cryopreserved before or after IVM, sug- gesting a possible beneﬁt if oocytes were vitriﬁed at GV. Furthermore, we showed that extending IVM could increase the number of available oocytes without altering their quality, at least regarding the metaphase plate conﬁguration. 2.3. Spindle Characteristics and Chromosome Arrangement (C) Normal spindle complex oriented perpendicular to the focal plane and chromosomes arranged in a circular way. (D) Spindle showing completely disorganized appearance with abnormal chromosomes configuration. (E) Spindle complex displaying reduction in the longitudinal dimension of the spindle and compact metaphase plate. (F) Normal spindle conformation with some chromosomes slightly displaced from the plane of the metaphase plate (arrows). Figure 2. Confocal micrographs of spindle chromosome complexes. Anti-α/β-tubulin antibody and Alexa Fluor 488 to visualize the spindle (green) and DAPI to stain the chromosomes (blue). (A,B) Normal barrel-shaped spindle conﬁguration with chromosomes arranged at the equator of the structure. (C) Normal spindle complex oriented perpendicular to the focal plane and chromosomes arranged in a circular way. (D) Spindle showing completely disorganized appearance with abnormal chromosomes conﬁguration. (E) Spindle complex displaying reduction in the longitudinal dimension of the spindle and compact metaphase plate. (F) Normal spindle conformation with some chromosomes slightly displaced from the plane of the metaphase plate (arrows). ult of these observations, four categories regarding spindle and chromosome stablished. The first category involved oocytes with normal configuration of es (N/N, Figure 3A). More in detail, a barrel-polymerized spindle positioned r to the first PB and aligned chromosomes at the equator of the meiotic econd included oocytes containing a normal spindle and partially abnormal arrangement (N/PA, Figure 3A). In this case, some chromosomes are de of the spindle equatorial plane (Figure 3). The third category included indle configuration (depolymerized spindle) and partially abnormal distribution (A/PA, Figure 3A). The last category corresponded to oocytes al spindle and chromosome alignment (A/A Figure 3A) As a result of these observations, four categories regarding spindle and chromosome status were established. The ﬁrst category involved oocytes with normal conﬁguration of both structures (N/N, Figure 3A). More in detail, a barrel-polymerized spindle positioned perpendicular to the ﬁrst PB and aligned chromosomes at the equator of the meiotic spindle. The second included oocytes containing a normal spindle and partially abnormal chromo- somal arrangement (N/PA, Figure 3A). In this case, some chromosomes are located outside of the spindle equatorial plane (Figure 3). The third category included abnormal spindle conﬁguration (depolymerized spindle) and partially abnormal chromosome distribution (A/PA, Figure 3A). The last category corresponded to oocytes with abnormal spindle and chromosome alignment (A/A, Figure 3A). al spindle and chromosome alignment (A/A, Figure 3A). 2.3. Spindle Characteristics and Chromosome Arrangement The reconstruction of Z-stacks sections allowed us to observe spindle conﬁguration and chromosome alignment in each oocyte. Regarding the normal spindle conformation, if the spindle was oriented parallel to the focal plane, the complex looked as a typical barrel-shaped (Figure 2A,B). On the contrary, if the spindle was oriented perpendicular to the focal plane the ﬁbers became circular in shape (Figure 2C). Abnormal spindles had a disorganized appearance (Figure 2D) or were shorter than usual (Figure 2E). Normal chromosomes arrangement showed the genetic material aligned in a compact metaphase plate at the spindle equator (Figure 2A,B,E) or in a perfect circle depending on the focal plane (Figure 2C). If some chromosomes were found to be slightly displaced from the metaphase plate, this conﬁguration was classiﬁed as partially abnormal (Figure 2F) and, in the case of being completely disorganized, as abnormal (Figure 2D). 4 of 16 Int. J. Mol. Sci. 2021, 22, 1125 21, 22, x FOR PEER REVIEW Figure 2. Confocal micrographs of spindle chromosome complexes. Anti-α/β-tubulin antibody and Alexa Fluor 488 to visualize the spindle (green) and DAPI to stain the chromosomes (blue). (A,B) Normal barrel-shaped spindle configuration with chromosomes arranged at the equator of the structure. (C) Normal spindle complex oriented perpendicular to the focal plane and chromosomes arranged in a circular way. (D) Spindle showing completely disorganized appearance with abnormal chromosomes configuration. (E) Spindle complex displaying reduction in the longitudinal dimension of the spindle and compact metaphase plate. (F) Normal spindle conformation with some chromosomes slightly displaced from the plane of the metaphase plate (arrows). Figure 2. Confocal micrographs of spindle chromosome complexes. Anti-α/β-tubulin antibody and Alexa Fluor 488 to visualize the spindle (green) and DAPI to stain the chromosomes (blue). (A,B) Normal barrel-shaped spindle conﬁguration with chromosomes arranged at the equator of the structure. (C) Normal spindle complex oriented perpendicular to the focal plane and chromosomes arranged in a circular way. (D) Spindle showing completely disorganized appearance with abnormal chromosomes conﬁguration. (E) Spindle complex displaying reduction in the longitudinal dimension of the spindle and compact metaphase plate. (F) Normal spindle conformation with some chromosomes slightly displaced from the plane of the metaphase plate (arrows). Figure 2. Confocal micrographs of spindle chromosome complexes. Anti-α/β-tubulin antibody and Alexa Fluor 488 to visualize the spindle (green) and DAPI to stain the chromosomes (blue). (A,B) Normal barrel-shaped spindle configuration with chromosomes arranged at the equator of the structure. 2.3. Spindle Characteristics and Chromosome Arrangement N/N: Polymerized spindle and normal chromosome distribution; N/PA: Polymerized spindle and partially abnormal chromosome distribution; A/PA: Depolymerized spindle and partially abnormal chromosome distribution; A/A: Depolymerized spindle and abnormal chromosome distribution. (A) Oocyte morphology by bright field microscopy and confocal micrographs of spindle chromosomes complexes. Anti-α/β-tubulin antibody and Alexa Fluor 488 to visualize the spindle Figure 3. Oocyte categories according to the whole spindle chromosomes complex conﬁguration. N/N: Polymerized spindle and normal chromosome distribution; N/PA: Polymerized spindle and partially abnormal chromosome distribution; A/PA: Depolymerized spindle and partially abnormal chromosome distribution; A/A: Depolymerized spindle and abnormal chromosome distribution. (A) Oocyte morphology by bright ﬁeld microscopy and confocal micrographs of spindle chromosomes complexes. Anti-α/β-tubulin antibody and Alexa Fluor 488 to visualize the spindle conﬁguration (green) and DAPI to stain the chromosomes arrangement (blue). Chromosomes slightly displaced from the plane of the metaphase plate (write arrow). In the dotted box, the mitotic spindle and chromosomes were ampliﬁed for easy viewing. (B) Proportion of oocytes according to their Spindle chromosomes complex conﬁguration in the three groups of study (Group GV-Vit, Group MII-Vit, and Group not-Vit), p-value > 0.05. This graph also contains, inside the bar, information on the number of oocytes included in each group. Figure 3. Oocyte categories according to the whole spindle chromosomes complex configuration. N/N: Polymerized spindle and normal chromosome distribution; N/PA: Polymerized spindle and partially abnormal chromosome distribution; A/PA: Depolymerized spindle and partially abnormal chromosome distribution; A/A: Depolymerized spindle and abnormal chromosome distribution. (A) Oocyte morphology by bright field microscopy and confocal micrographs of spindle chromosomes complexes. Anti-α/β-tubulin antibody and Alexa Fluor 488 to visualize the spindle Figure 3. Oocyte categories according to the whole spindle chromosomes complex conﬁguration. N/N: Polymerized spindle and normal chromosome distribution; N/PA: Polymerized spindle and partially abnormal chromosome distribution; A/PA: Depolymerized spindle and partially abnormal chromosome distribution; A/A: Depolymerized spindle and abnormal chromosome distribution. (A) Oocyte morphology by bright ﬁeld microscopy and confocal micrographs of spindle chromosomes complexes. Anti-α/β-tubulin antibody and Alexa Fluor 488 to visualize the spindle conﬁguration (green) and DAPI to stain the chromosomes arrangement (blue). Chromosomes slightly displaced from the plane of the metaphase plate (write arrow). In the dotted box, the mitotic spindle and chromosomes were ampliﬁed for easy viewing. (B) Proportion of oocytes according to their Spindle chromosomes complex conﬁguration in the three groups of study (Group GV-Vit, Group MII-Vit, and Group not-Vit), p-value > 0.05. 2.3. Spindle Characteristics and Chromosome Arrangement g ( g ) The comparison of the mean percentage of oocytes with normal metaphase plate con- ﬁguration rate (N/N) between the three study groups did not show signiﬁcant differences (see Figure 3B). No differences were found once the rest of the categories were compared (N/PA; A/PA or A/A) in the three groups. It is worth mentioning that despite not reaching statistical signiﬁcance, oocytes frozen at GV (Group GV-Vit) or non-frozen (Group not-Vit) seemed to show a normal metaphase plate conﬁguration in a higher percentage than the one obtained when the oocytes were matured and subsequently vitriﬁed (Group MII-Vit). 5 of 16 6 Int. J. Mol. Sci. 2021, 22, 1125 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of Figure 3. Oocyte categories according to the whole spindle chromosomes complex configuration. N/N: Polymerized spindle and normal chromosome distribution; N/PA: Polymerized spindle and partially abnormal chromosome distribution; A/PA: Depolymerized spindle and partially abnormal chromosome distribution; A/A: Depolymerized spindle and abnormal chromosome distribution. (A) Oocyte morphology by bright field microscopy and confocal micrographs of spindle chromosomes complexes. Anti-α/β-tubulin antibody and Alexa Fluor 488 to visualize the spindle Figure 3. Oocyte categories according to the whole spindle chromosomes complex conﬁguration. N/N: Polymerized spin and normal chromosome distribution; N/PA: Polymerized spindle and partially abnormal chromosome distribution; A/ Depolymerized spindle and partially abnormal chromosome distribution; A/A: Depolymerized spindle and abnorm chromosome distribution. (A) Oocyte morphology by bright ﬁeld microscopy and confocal micrographs of spin chromosomes complexes. Anti-α/β-tubulin antibody and Alexa Fluor 488 to visualize the spindle conﬁguration (green) a DAPI to stain the chromosomes arrangement (blue). Chromosomes slightly displaced from the plane of the metaphase pl (write arrow). In the dotted box, the mitotic spindle and chromosomes were ampliﬁed for easy viewing. (B) Proportion oocytes according to their Spindle chromosomes complex conﬁguration in the three groups of study (Group GV-Vit, Gro MII-Vit, and Group not-Vit), p-value > 0.05. This graph also contains, inside the bar, information on the number of oocy included in each group. Oocyte categories according to the whole spindle chromosomes complex configuration. N/N: Polymerized d l h di t ib ti N/PA P l i d i dl d ti ll b l h categories according to the whole spindle chromosomes complex conﬁguration. N/N: Polymerized spindle Figure 3. Oocyte categories according to the whole spindle chromosomes complex configuration. 2.3. Spindle Characteristics and Chromosome Arrangement On the other hand, the evaluation of the spindle polymerization, regardless of the chromosomal distribution, showed a signiﬁcantly higher percentage of oocytes with an optimal spindle conﬁguration in the oocytes that matured at 48 h compared to those that did it during the ﬁrst 24 h of culture (Table 2). However, these results have to be interpreted very carefully since the number of oocytes that maturated after 48 h was fairly low (n = 13). Table 2. Normal spindle configuration and chromosomal position of oocytes depending on the in vitro maturation time (IVM), 24 or 48 h. * p-value < 0.05. IVM, in vitro maturation; CHR, chromosomes. IVM n CHR SPINDLE 24 h 61 24/61 (39.3%) 39/61 (63.9%) * 48 h 13 5/13 (38.5%) 12/13 (92.3%) * 2.4. Influence of In Vitro Maturation (IVM) Time on the Configuration Metaphase Plate 2.4. Inﬂuence of In Vitro Maturation (IVM) Time on the Conﬁguration Metaphase Plate Counting the 74 oocytes from the immunofluorescence study (Group GV-Vit = 26, Group MII-Vit = 27, and Group not-Vit = 21), regardless of the group that they belonged to and depending on the IVM time, 24 or 48 h, the analysis of the four different metaphase plate configurations showed significant differences between both study times [N/N: IVM24 h 39.3% (24/61) vs. IVM48 h: 38.5% (5/13); N/PA: IVM24 h 24.6% (15/61) vs. IVM48 h: 53.8% (7/13); A/PA IVM24 h 31.1% (19/61) vs. IVM48 h: 0% (0/13); A/A IVM24 h 4.9% (3/61) vs. IVM48 h: 7.7% (1/13); p-value < 0.05]. Counting the 74 oocytes from the immunoﬂuorescence study (Group GV-Vit = 26, Group MII-Vit = 27, and Group not-Vit = 21), regardless of the group that they belonged to and depending on the IVM time, 24 or 48 h, the analysis of the four different metaphase plate conﬁgurations showed signiﬁcant differences between both study times [N/N: IVM24 h 39.3% (24/61) vs. IVM48 h: 38.5% (5/13); N/PA: IVM24 h 24.6% (15/61) vs. IVM48 h: 53.8% (7/13); A/PA IVM24 h 31.1% (19/61) vs. IVM48 h: 0% (0/13); A/A IVM24 h 4.9% (3/61) vs. IVM48 h: 7.7% (1/13); p-value < 0.05]. p When the results were analyzed separately, the study of the chromosomal distribution, regardless of the spindle conformation, did not show significant differences between the groups (see Table 2). 2.3. Spindle Characteristics and Chromosome Arrangement This graph also contains, inside the bar, information on the number of oocytes included in each group. Int. J. Mol. Sci. 2021, 22, 1125 6 of 16 frozen higher The study of the chromosomal distribution, regardless of the spindle conformation, did not show signiﬁcant differences in any of the cases (Figure 4A). However, the exclusive evaluation of the normal spindle polymerization (barrel structure of the metaphase plate) showed signiﬁcant differences between the groups studied (see Figure 4B). This signiﬁcance was higher when we compared them one by one (Group GV-Vit vs. Group MII-Vit p-value = 0.038 and Group MII-Vit vs. Group not-Vit p-value = 0.030). Thus, the results exposed that MIIIVM oocytes that were vitriﬁed at GV (Group GV-Vit) were similar to than those MIIIVM that were not vitriﬁed (Group not-Vit), presenting a better polymerized spindle than the MIIIVM vitriﬁed at MII (Group MII-Vit). vitrified (Group MII Vit). The study of the chromosomal distribution, regardless of the spindle conformation, did not show significant differences in any of the cases (Figure 4A). However, the exclusive evaluation of the normal spindle polymerization (barrel structure of the metaphase plate) showed significant differences between the groups studied (see Figure 4B). This significance was higher when we compared them one by one (Group GV-Vit vs. Group MII-Vit p-value = 0.038 and Group MII-Vit vs. Group not-Vit p-value = 0.030). Thus, the results exposed that MIIIVM oocytes that were vitrified at GV (Group GV-Vit) were similar to than those MIIIVM that were not vitrified (Group not-Vit), presenting a better polymerized spindle than the MIIIVM vitrified at MII (Group MII-Vit). Figure 4. Chromosomes and spindle normal configuration of the oocytes in the three groups of study (Group GV-Vit, Group MII-Vit, and Group not-Vit). (A) Chromosomes normal configuration, p-value > 0.05. (B) Spindle normal configuration * p-value Group GV-Vit vs. Group MII-Vit = 0.038 and ** p-value Group MII-Vit vs. Group not-Vit = 0.03. Figure 4. Chromosomes and spindle normal conﬁguration of the oocytes in the three groups of study (Group GV-Vit, Group MII-Vit, and Group not-Vit). (A) Chromosomes normal conﬁguration, p-value > 0.05. (B) Spindle normal conﬁguration * p-value Group GV-Vit vs. Group MII-Vit = 0.038 and ** p-value Group MII-Vit vs. Group not-Vit = 0.03. Figure 4. Chromosomes and spindle normal configuration of the oocytes in the three groups of study (Group GV-Vit, Group MII-Vit, and Group not-Vit). (A) Chromosomes normal configuration, p-value > 0.05. 2.3. Spindle Characteristics and Chromosome Arrangement (B) Spindle normal configuration * p-value Group GV-Vit vs. Group MII-Vit = 0.038 and ** p-value Group MII-Vit vs. Group not-Vit = 0.03. Figure 4. Chromosomes and spindle normal conﬁguration of the oocytes in the three groups of study (Group GV-Vit, Group MII-Vit, and Group not-Vit). (A) Chromosomes normal conﬁguration, p-value > 0.05. (B) Spindle normal conﬁguration * p-value Group GV-Vit vs. Group MII-Vit = 0.038 and ** p-value Group MII-Vit vs. Group not-Vit = 0.03. 2.4. Influence of In Vitro Maturation (IVM) Time on the Configuration Metaphase Plate Counting the 74 oocytes from the immunofluorescence study (Group GV-Vit = 26, Group MII-Vit = 27, and Group not-Vit = 21), regardless of the group that they belonged to and depending on the IVM time, 24 or 48 h, the analysis of the four different metaphase plate configurations showed significant differences between both study times [N/N: IVM24 h 39.3% (24/61) vs. IVM48 h: 38.5% (5/13); N/PA: IVM24 h 24.6% (15/61) vs. IVM48 h: 53.8% (7/13); A/PA IVM24 h 31.1% (19/61) vs. IVM48 h: 0% (0/13); A/A IVM24 h 4.9% (3/61) vs. IVM48 h: 7.7% (1/13); p-value < 0.05]. When the results were analyzed separately, the study of the chromosomal distribution, regardless of the spindle conformation, did not show significant differences between the groups (see Table 2). On the other hand, the evaluation of the spindle polymerization, regardless of the chromosomal distribution, showed a significantly 2.4. Inﬂuence of In Vitro Maturation (IVM) Time on the Conﬁguration Metaphase Plate Counting the 74 oocytes from the immunoﬂuorescence study (Group GV-Vit = 26, Group MII-Vit = 27, and Group not-Vit = 21), regardless of the group that they belonged to and depending on the IVM time, 24 or 48 h, the analysis of the four different metaphase plate conﬁgurations showed signiﬁcant differences between both study times [N/N: IVM24 h 39.3% (24/61) vs. IVM48 h: 38.5% (5/13); N/PA: IVM24 h 24.6% (15/61) vs. IVM48 h: 53.8% (7/13); A/PA IVM24 h 31.1% (19/61) vs. IVM48 h: 0% (0/13); A/A IVM24 h 4.9% (3/61) vs. IVM48 h: 7.7% (1/13); p-value < 0.05]. When the results were analyzed separately, the study of the chromosomal distribution, regardless of the spindle conformation, did not show signiﬁcant differences between the groups (see Table 2). 3. Discussion The use of in vitro matured oocytes in reproductive cycles with or without stimulation, combined with the vitriﬁcation technique, involves novel ways of action and allows us to maximize the efﬁciency of the AR laboratories’ techniques. Their clinical use is conditioned to the collection of competent MII oocytes, which are able to sustain early embryonic development. Currently, the use of immature oocytes (rescue oocytes) is one of the research ﬁelds with the greatest potential to develop and optimize emerging AR techniques in humans. Therefore, there are several aspects to improve, specially IVM technique. However, there is high controversy in the literature regarding vitriﬁcation before or after IVM [42,44,45]. For that, this study was divided into different experimental groups depending on their maturation state prior to vitriﬁcation: Group GV-Vit (GV oocytes frozen and then matured in vitro), Group MII-Vit (frozen MII oocytes after being matured in vitro), and Group not-Vit (GV oocytes matured in vitro and not vitriﬁed). The surface/volume ratio is similar in immature (GV) oocytes or after their in vitro maturation (MII). However, the microstructure confers a priori advantages to GV oocytes over MII: (1) Chromatin protected by the nuclear membrane inside the GV and (2) absence of the microtubule assembly complex during meiosis. Nevertheless, there is no consensus in the literature about this topic [21,46–49]. Our results did not show statistical signiﬁcance when comparing the survival rate of GV oocytes vs. MIIIVM, which agreed with other studies published in humans, where survival after GV warming was comparable to the one showed by MIIIVM, either with oocytes from stimulated [44,49–51] or non-stimulated [21] cycles. Therefore, in stimulated or non-stimulated cycles, we assumed that the preservation stage did not affect oocyte survival. However, it is necessary to inquiry whether their competence might be compromised. In this study, progression to MII at 48 h did not show signiﬁcant differences depending on the previous state of the oocytes (fresh or warmed). Most of the published studies support this result, obtaining similar [52,53] or higher [44,54,55] maturation rates, if oocytes were cryopreserved at MII stage. However, a recent meta-analysis questions the fact that vitriﬁed GV oocytes show worse results [42]. In fact, our results suggested a higher GVBD in those cases where oocytes were vitriﬁed prior to their maturation. This agrees with previous studies, which showed that the reagents used for vitriﬁcation induce cytoplasmic changes that favor GVBD. 2.3. Spindle Characteristics and Chromosome Arrangement On the other hand, the evaluation of the spindle polymerization, regardless of the chromosomal distribution, showed a significantly When the results were analyzed separately, the study of the chromosomal distribution, regardless of the spindle conformation, did not show signiﬁcant differences between the groups (see Table 2). On the other hand, the evaluation of the spindle polymerization, regardless of the chromosomal distribution, showed a signiﬁcantly higher percentage of oocytes with an optimal spindle conﬁguration in the oocytes that matured at 48 h compared to those that did it during the ﬁrst 24 h of culture (Table 2). However, these results have to be interpreted very carefully since the number of oocytes that maturated after 48 h was fairly low (n = 13). Table 2. Normal spindle configuration and chromosomal position of oocytes depending on the in vitro maturation time (IVM), 24 or 48 h. * p-value < 0.05. IVM, in vitro maturation; CHR, chromosomes. IVM n CHR SPINDLE 24 h 61 24/61 (39.3%) 39/61 (63.9%) * 48 h 13 5/13 (38.5%) 12/13 (92.3%) * Table 2. Normal spindle configuration and chromosomal position of oocytes depending on the in vitro maturation time (IVM), 24 or 48 h. * p-value < 0.05. IVM, in vitro maturation; CHR, chromosomes. 7 of 16 Int. J. Mol. Sci. 2021, 22, 1125 3. Discussion Speciﬁcally, they indicate an increase in the intracellular levels of Ca2+ and intra-oocyte cAMP degradation, favoring the activation of the maturation promoting factor (MPF) and meiosis resumption [54,56]. Following this line, other research has shown that the short-term presence of the cryoprotectants dimethyl sulfoxide (DMSO) and ethylene glycol (EG) facilitate Ca2+ trafﬁc, having an impact on the increase of the MR [57,58]. DMSO would mobilize the intra-oocyte Ca2+ deposits, especially the endoplasmic reticulum, while EG would favor the entry of extracellular Ca2+ inside the oocyte [58]. y y In this work, we stablished a time for IVM of 48 h, generally longer than the one recom- mended by most of the published literature, which is usually stablished at a maximum of 24/32 h [59,60]. Under these conditions, maturation of both vitriﬁed and fresh GV oocytes was similar at 24 h and 48 h. This result contradicts the hypothesis that vitriﬁed GV oocytes before their IVM need more time to extrude the ﬁrst polar body [44,49]. Furthermore, in opposition to this hypothesis, the results published by Lowther and colleagues showed differences in time regarding the GVBD, but only during the ﬁrst 2 h of culture, which is the time of adaptation to the physiological culture conditions re-established after the warming. However, 6.5 h later, the rates were similar, even slightly higher than the group with frozen oocytes [53]. Meanwhile, after studying if culture time affects survival rate, our results did not show signiﬁcant differences when comparing Group MII-Vit MIIIVM oocytes at 24 vs. 48 h. Hence, contrary to what is indicated by the studies mentioned above, our results suggest that extending the culture time does not alter the structures more sensitive to cryogenic damage. Once the maturation obstacle is overcome and in order to continue evaluating the competence of the collected oocytes, it is necessary to evaluate the possible deﬁciencies Int. J. Mol. Sci. 2021, 22, 1125 8 of 16 that vitriﬁcation and IVM may have caused in the oocytes, and observe whether altering the order of these leads to better results. Understanding how human oocytes perform chromosomal segregation is greatly important, since errors during this process explain most of the human aneuploidies [61]. Microtubular depolymerization caused by tem- perature changes has been described both in mature and immature oocytes [52,62–65]. However, repolymerization of the spindle once physiological conditions are recovered has also been reported [5,66,67]. 3. Discussion Despite this, previous studies describe that frozen oocytes, both GV and MIIIVM, show abnormal spindles and an altered chromosomal distribution when compared to non-frozen oocytes [44,52,63]. Therefore, temporary depolymerization would be recoverable, but its functionality could be affected, resulting in an abnormal chromosomal segregation, maturation arrest, and aneuploidy formation [5]. The Conﬁguration rate metaphase plate obtained in this study did not show signiﬁ- cant differences between none of the groups of study when the chromosomal and spindle conﬁguration were evaluated together. When we evaluated the spindle conﬁguration, regardless of the chromosomal distribution, we observed signiﬁcant differences in favor of non-vitriﬁed MIIIVM or vitriﬁed at GV stage and then matured, compared to oocytes that were vitriﬁed after maturation. The time required to recover physiological conditions after warming in this study was 2 h, which is recommended by different authors [68–70], although others recommend a slightly higher interval [7,47,52]. Likely, the low spindle conﬁguration rates in Group MII-Vit were due to an insufﬁcient recovery time after warm- ing. These results support that, after warming, the spindle could be reassembled, but in an abnormal way. Moreover, it conﬁrms that GV vitriﬁcation does not prevent the correct formation of the spindle after their vitriﬁcation. p In addition to cryopreservation, suboptimal culture conditions during IVM could increase alterations in the meiotic apparatus [62,71]. However, an exhaustive observation of this between MII oocytes matured in vivo and in vitro did not show variations in the actin intensity pattern in the proximal and distal domains of the metaphase spindle, but showed an increase in the cytoplasmic actin in MIIIVM oocytes [72]. It is possible that this reﬂects an adaptative response to IVM conditions to increase the cytoplasmic actin ﬂuxes, previously described in rodent oocytes [73]. Our study did not include MII oocytes matured in vivo as a second control group for IVM because human MII oocytes matured in vivo are highly valuable in the clinic and are used exclusively for the patient. However, there are other studies that point to a worse spindle conﬁguration and chromosome distribution in oocytes matured in vitro vs. in vivo [74–76]. 3. Discussion Depending on the maturation time, Escrich and colleagues concluded to not exceed the culture more than 24 h, since oocytes that matured between the ﬁrst 18.4 ± 2.7 h showed better activation and division rates and a lower rate of meiotic errors in comparison with the ones that did it later [59]. Nevertheless, in this work, regarding the metaphase plate conﬁguration, we observed signiﬁcant differences in favor to the oocytes that matured between 25–48 vs. 0–24 h. Therefore, according to our results, the time of culture of oocytes up to 48 h did not imply higher meiotic error rates. These would suggest, again, that extending the time of culture would be beneﬁcial, favoring the synchronization between nuclear and cytoplasmic maturation, allowing better oocyte meiotic development. As already mentioned, there are contradictory results in the literature, mainly because it is a procedure that combines several techniques, each one of them with variable results depending on the conditions in which they have been performed [7,67,77–80]. In addition, oocytes generated in vivo may also present low competence due to the characteristics of the donor or the patient [81,82]. Therefore, at this point it is worthwhile to highlight that most of the published studies randomly assign the oocytes to the groups of study, but do not describe the characteristics of the women that participate in the study or the homogeneity of the groups. In the literature, we found some studies that relate vitriﬁcation, IVM, or the metaphase plate conﬁguration with some of the clinical characteristics of the patients. For instance, they point that in vitro maturation rate is not affected by the age [83], except prepubescent or elderly women who show lower maturation rates [84,85]. However, Int. J. Mol. Sci. 2021, 22, 1125 9 of 16 9 of 16 survival to vitriﬁcation is diminished by age increase [7,81]. The increase of the age also inﬂuences on the correct organization of the metaphase plate in human oocytes. It has been reported a higher percentage of chromosome misalignment in those cases [86], due to a lack of adhesion [87] caused by a reduction of the assembly points between kinetochores and microtubules [88,89]. 4.1. Oocyte Collection The patients were subjected to controlled ovarian stimulation following a short antago- nist protocol. Pituitary suppression was performed by administration of (150–300 IU/day) rec-FSH (Gonal F 1050; Merck and Co, Madrid, Spain) and GnRH (Orgalutran®; MSD and Co., Hoddesdon, UK). When the growth of at least three follicles >16 mm was ob- served, ovulation was induced by the administration of 250 µg of rec-hCG (Ovitrelle, Merck, London, UK). Oocyte retrieval was performed 36 h after hCG administration via ultrasound-guided transvaginal puncture-aspiration. Cumulus-corona-oocyte (CCO) complexes denudation was carried out using hyaluronidase SynVitro® Hyadase; Origio® Solution, Màlov, Denmark) for a maximum of 30 sec with a denudation pipette (Denudation pipette Flexipet®, Cook® Medical, Bloomington, IN, USA). Removal of cumulus-corona cells is required to evaluate and classify the oocyte nuclear maturation state. Despite com- ing from stimulated cycles, a total of 199 GV oocytes showed an intracytoplasmic nucleus called the germinal vesicle, characteristic of the prophase of the ﬁrst meiotic division. After the IVM rescue, we included in this study all the oocytes that showed an approximately circular shape and a variable size between 120–140 µm; homogeneous or slightly heteroge- neous cytoplasm with no granularity such as inclusions or refractile bodies. Four oocytes (2%) were excluded from the study for being giant, presenting dimorphisms in their zona pellucida, or showing large vacuoles or signs of atresia/degeneration in their ooplasm. 4. Materials and Methods This study was approved by the Institutional Review Board of the Hospital Universitario y Politécnico La Fe, Valencia, Spain. All 104 patients included in the study were fully informed and gave their signed consent to donate the 195 GV oocytes collected from their ICSI cycles carried out in the Human Reproductive Unit of the previously mentioned Hospital. 3. Discussion There is also evidence in the literature regarding the diagnosis of infertility [32,37]; for example, in women with endometriosis, a lower IVM rate, higher zona pellucida hardening, premature exocytosis of the GC, and a high disruption of the chromosomes in the metaphase plate have been described [90]. All these factors should be taken into account when conducting further studies in humans. g Overall, vitriﬁcation and IVM techniques can alter essential structures for the proper development of the embryo. These alterations could impede meiosis, fertilization, or early embryonic development, preventing the activation of the embryonic genome and therefore, avoiding the formation of a blastocyst with implantation capacity [91,92]. Further studies in humans that allow us to deﬁne the moment and the conditions in which to perform these techniques are required to consolidate their combination and increase their range of indications. It should not be forgotten that rescue IVM may be the only gestation opportunity, without resorting to egg donation, for certain types of patients as a oncological or low response, among others. 4.3. In Vitro Maturation 4.3. In Vitro Maturation GV oocytes were i GV oocytes were individually cultured in micro-drops of 25 µL of culture medium cov- ered by mineral oil OVOILTM on 1008 plates at 37 ◦C and 5% CO2. We used SAGE 1-StepTM (Origio®, Màlov, Denmark) culture medium, supplemented with human menopausal gonadotropin (hMG, Menopur® 75 UI, Ferring®, Madrid, Spain) and synthetic serum substitute (SSS IrvineScientiﬁc®, Santa Ana, CA, USA). Oocytes were serially observed under the inverted microscope (Olympus, IX70, Tokio, Japan) 20, 24, 44, and 48 h after the IVM. Mature oocyte (MII) were considered to be those in which a rupture of the GV was observed, and the presence of a ﬁrst polar corpuscle in the perivitelline space during the ﬁrst 48 h of culture was observed under inverted optical microscope (Figure 5). y y p μ covered by mineral oil OVOILTM on 1008 plates at 37 °C and 5% CO2. We used SAGE 1- StepTM (Origio®, Màlov, Denmark) culture medium, supplemented with human menopausal gonadotropin (hMG, Menopur® 75 UI, Ferring®, Madrid, Spain) and synthetic serum substitute (SSS IrvineScientific®, Santa Ana, CA, USA). Oocytes were serially observed under the inverted microscope (Olympus, IX70, Tokio, Japan) 20, 24, 44, and 48 h after the IVM. Mature oocyte (MII) were considered to be those in which a rupture of the GV was observed, and the presence of a first polar corpuscle in the perivitelline space during the first 48 h of culture was observed under inverted optical microscope (Figure 5). Figure 5. Sequence at different stages of meiosis of the human oocyte. Oocyte with an intact nucleus (prophase I, GV), late metaphase of meiosis I, onset of anaphase I, onset of polar body extrusion and meiosis II, total polar body extrusion. Images were obtained in Primo Vision Time-Lapse System (Vitrolife® Göteborg, Sweden) in the Human Reproduction Unit of Hospital Universitario y Politécnico, La Fe. Figure 5. Sequence at different stages of meiosis of the human oocyte. Oocyte with an intact nucleus (prophase I, GV), late metaphase of meiosis I, onset of anaphase I, onset of polar body extrusion and meiosis II, total polar body extrusion. Images were obtained in Primo Vision Time-Lapse System (Vitrolife® Göteborg, Sweden) in the Human Reproduction Unit of Hospital Universitario y Politécnico, La Fe. Figure 5. Sequence at different stages of meiosis of the human oocyte. 4.3. In Vitro Maturation 4.3. In Vitro Maturation GV oocytes were i Oocyte with an intact nucleus (prophase I, GV), late metaphase of meiosis I, onset of anaphase I, onset of polar body extrusion and meiosis II, total polar body extrusion. Images were obtained in Primo Vision Time-Lapse System (Vitrolife® Göteborg, Sweden) in the Human Reproduction Unit of Hospital Universitario y Politécnico, La Fe. Figure 5. Sequence at different stages of meiosis of the human oocyte. Oocyte with an intact nucleus (prophase I, GV), late metaphase of meiosis I, onset of anaphase I, onset of polar body extrusion and meiosis II, total polar body extrusion. Images were obtained in Primo Vision Time-Lapse System (Vitrolife® Göteborg, Sweden) in the Human Reproduction Unit of Hospital Universitario y Politécnico, La Fe. 4.4. Oocyte Vitrification and Warming 4.4. Oocyte Vitriﬁcation and Warming We used vitrification/warming medium and the Cryotop® open system device commercialized by Kitazato® (BioPharma Co, Shizuoka, Japan). The vitrification/warming protocol used was the one recommended by the commercial company developed by We used vitriﬁcation/warming medium and the Cryotop® open system device com- mercialized by Kitazato® (BioPharma Co, Shizuoka, Japan). The vitriﬁcation/warming protocol used was the one recommended by the commercial company developed by Kuwayama [4]. This protocol was used in all the oocytes involved in this study, regard- less of their maturation stage (GV or MII). Survival rate was evaluated microscopically with Hoffman contrast 2 h after warming and was based on morphology and integrity observations of the oocyte membrane. Prospective, randomized cohort study. Prospective, randomized cohort study. To evaluate the effects of cryopreservation on oocytes in vitro maturation, the study was divided in different experimental groups depending on their maturation state prior to vitriﬁcation: Group GV-Vit (GV oocytes vitriﬁed and then matured in vitro), Group MII-Vit (vitriﬁed MII oocytes after being matured in vitro), and Group not-Vit (GV oocytes matured in vitro and not vitriﬁed) (Figure 1). Int. J. Mol. Sci. 2021, 22, 1125 10 of 16 Vit (GV 10 of 16 Vit (GV 4.3. In Vitro Maturation 4.3. In Vitro Maturation GV oocytes were i 4.6. Tubulin and Chromatin Staining 4.6. Tubulin and Chromatin Staining In order to observe the spindle conformation we performed the next methodology based on previous reports (7, 54, 79). All ﬁxed oocytes were incubated with a mixture of two primary tubulin antibodies produced in mouse, anti-β-tubulin, and anti-α-tubulin (1:100) (Sigma-Aldrich) for 90 min. After that, the cells were washed three times for 10 min in PBS and incubated with the secondary antibody anti-mouse conjugated with Alexa Fluor 488 (1:500) (Jackson ImmmunoResearch, Ely, UK) for 1 h in the dark. Then, the oocytes were washed again three times for 10 min in PBS. For chromatin staining, we added 5µL of mounting medium with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Vector Laboratories, Burlingame, CA, USA) and left for incubation for 5 min in the dark. All of the process was performed at room temperature. This part was performed at the Department of Biotechnology of the University of Alicante, Alicante, Spain. 4.5. Oocyte Fixation For all groups (Group GV-Vit, Group MII-Vit, and Group not-Vit, see experimental design in Figure 1), the MII oocytes obtained by in vitro maturation [20–48 h] were ﬁxed with 2% (w/v) paraformaldehyde solution (Electron Microscopy Sciences, Hatﬁeld, PA USA), 0.5% (w/v) Tritón X-100 (Sigma-Aldrich®, Saint Louis, MO, USA), and 1µmol/L of Taxol® (Paclitaxel, Sigma-Aldrich) in phoshate-buffered saline without calcium or magnesium, pH 7.4 (PBS, Biowest, Nuaillé, France) for 30 min at 37 ◦C and 5% (v/v) CO2. Oocytes where then washed three times for 15 min in PBS and stored in 2% (w/v) bovine serum albumin (BSA, Sigma-Aldrich), 0.1 mol/L of glycine (Sigma-Aldrich), 0.01% (w/v) Triton X-100, and PBS until they were immunolabeled (see Figure 1). All reagents were aliquoted in PBS except of Taxol, that as recommended by the commercial company was aliquoted in DMSO. Int. J. Mol. Sci. 2021, 22, 1125 11 of 16 11 of 16 4.7. Image Acquisition A total of 199 oocytes were mounted using cavity slides, and the spindle characteristics and chromosome arrangement were evaluated by Confocal Laser Scanning Zeiss LSM 800 Microscope (Zeiss, Oberkochen, Germany) and Zeiss Imaging Software at the technical services of the University of Alicante. Z-stacks sections (1040 × 1040 pixels) of the entire spindle of each oocyte were obtained using an oil 40x objective and 408 nm and 561 nm lasers. Then, the sections were reconstructed using ZEN 2.5 lite software (Zeiss). 4.8. Statistical Analysis 2021, 22, 1125 Abbreviations A Abnormal AR Assisted reproduction cAMP Cyclic adenosine monophosphate CHR Chromosome CRP Cryoprotectants DMSO Dimethylsulfoxide EG Ethylene Glycol EV Embryo vitriﬁcation GC Granulosa cells GV Germinal vesicle (prophase I) GVBD Germinal vesicle breakdown h Hours ICSI Intracytoplasmic sperm microinjection IVF In vitro fertilization IVM In vitro maturation MII Metaphase II MPF Maturation promoting factor MR Maturation rate N Normal OHSS Ovarian hyperstimulation OV Oocyte vitriﬁcation PA Partially abnormal PB Polar body PCOS Polycystic Ovary Syndrome SR Survival rate Abbreviations A Abnormal AR Assisted reproduction cAMP Cyclic adenosine monophosphate CHR Chromosome CRP Cryoprotectants DMSO Dimethylsulfoxide EG Ethylene Glycol EV Embryo vitriﬁcation GC Granulosa cells GV Germinal vesicle (prophase I) GVBD Germinal vesicle breakdown h Hours ICSI Intracytoplasmic sperm microinjection IVF In vitro fertilization IVM In vitro maturation MII Metaphase II MPF Maturation promoting factor MR Maturation rate N Normal OHSS Ovarian hyperstimulation OV Oocyte vitriﬁcation PA Partially abnormal PB Polar body PCOS Polycystic Ovary Syndrome SR Survival rate Abbreviations A Abnormal AR Assisted reproduction cAMP Cyclic adenosine monophosphate CHR Chromosome CRP Cryoprotectants DMSO Dimethylsulfoxide EG Ethylene Glycol EV Embryo vitriﬁcation GC Granulosa cells GV Germinal vesicle (prophase I) GVBD Germinal vesicle breakdown h Hours ICSI Intracytoplasmic sperm microinjection IVF In vitro fertilization IVM In vitro maturation MII Metaphase II MPF Maturation promoting factor MR Maturation rate N Normal OHSS Ovarian hyperstimulation OV Oocyte vitriﬁcation PA Partially abnormal PB Polar body PCOS Polycystic Ovary Syndrome SR Survival rate References 1. Cobo, A.; Garcia-Velasco, J.A.; Domingo, J.; Remohí, J.; Pellicer, A. Is vitriﬁcation of oocytes useful for age-related fertility decline and in cáncer patients? Fertil. Steril. 2013, 99, 1485–1495. [CrossRef] 1. Cobo, A.; Garcia Velasco, J.A.; Domingo, J.; Remohí, J.; Pellicer, A. Is vitriﬁcation of oocytes useful for fertility preservation for age-related fertility decline and in cáncer patients? Fertil. Steril. 2013, 99, 1485–1495. [CrossRef] 2 Chian R C ; Son W Y; Huang J Y; Cui S J ; Buckett W M ; Tan S L High survival rates and pregnancies of human oocytes g y p , , [ ] 2. Chian, R.C.; Son, W.Y.; Huang, J.Y.; Cui, S.J.; Buckett, W.M.; Tan, S.L. High survival rates and pregnancies of human oocytes g y p , , [ ] 2. Chian, R.C.; Son, W.Y.; Huang, J.Y.; Cui, S.J.; Buckett, W.M.; Tan, S.L. High survival rates and pregnancies of human oocytes g y p 2. Chian, R.C.; Son, W.Y.; Huang, J.Y.; Cui, S.J.; Buckett, W.M.; Tan, S.L. High survival rates and pregn following vitriﬁcation: Preliminary report. Fertil. Steril. 2005, 84, S36. [CrossRef] 3. Jain, J.K.; Paulson, R.J. Oocyte cryopreservation. Fertil. Steril. 2006, 86 (Suppl. 4), 1037–1046. [CrossRef] 3. Jain, J.K.; Paulson, R.J. Oocyte cryopreservation. Fertil. Steril. 2006, 86 (Suppl. 4), 1037–1046. [CrossRef] 4 Kuwayama M Highly efﬁcient vitriﬁcation for cryopreservation of human oocytes and embryos: The Cryotop method Theri- 4. Kuwayama, M. Highly efﬁcient vitriﬁcation for cryopreservation of human oocytes and embryos: The Cryotop method. Theri- ogenology 2007, 67, 73–80. [CrossRef] [PubMed] 4. Kuwayama, M. Highly efﬁcient vitriﬁcation for cryopreservation of human oocytes and embryos: The Cryotop method. Theri- ogenology 2007, 67, 73–80. [CrossRef] [PubMed] g gy 5. Rienzi, L.; Ubaldi, F.; Iacobelli, M.; Minasi, M.G.; Romano, S.; Greco, E. Meiotic spindle visualizat Reprod. Biomed. Online 2005, 10, 192–198. 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The homogeneity of the groups was evaluated with the Kolmogorov–Smirnov test. The differences between the quantitative variables were veriﬁed using T-Test or Mann–Whitney U test. For the qualitative variables, the X2 test or Fisher’s test was used if both variables to be compared were dichotomous or some cell contained an expected frequency percentage of less than 5%. Comparison of Maturation Rates [MR = n◦MII oocytes (24–48 h)/n◦GV oocytes × 100], Survival Rate [SR = (n◦2 h viable devitriﬁed oocytes/n◦vitriﬁed oocytes) × 100], and Normal Conformation Rate [NCR = n◦oocytes with normal-partially normal conformation/n◦oocytes evaluated) × 100], was performed using contingency tables, X2 test, with a level of α equal to 0.05, therefore, it was considered that there were signiﬁcant differences when p-value was < 0.05. Author Contributions: All authors contributed to this study and performed one or more experiments. Investigation, I.P., I.M., M.J.G.-T., P.S.-E., M.B., L.G.-V., R.F. and P.T.; Methodology, I.M., P.S.-E., L.G.-V. and P.T.; Supervision, M.J.G.-T.; Writing–original draft, I.P., M.J.G.-T and R.F.; Writing–review & editing, I.P., M.J.G.-T., P.S.-E. and R.F. All authors have read and agreed to the published version of the manuscript and funding acquisition M.J.G.-T. Funding: This research was funded by Department of Biotechnology of the University of Alicante (VIGROB-186). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of La Fe Health (protocol coe: 2018/0357 and date of approval: 19th of December, 2018). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available in the article. Data Availability Statement: The data presented in this study are available in the article. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 12 of 16 12 of 16 Int. J. Mol. Sci. 13. Franciosi, F.; Goudet, G.; Tessaro, I.; Papillier, P.; Dalbies-Tran, R.; Reigner, F.; Deleuze, S.; Douet, C.; Miclea, I.; Lodde, V.; et al. In vitro maturation affects chromosome segregation, spindle morphology and acetylation of lysine 16 on histone H4 in horse oocytes. Reprod. Fertil. Dev. 2017, 29, 721–730. [CrossRef] [PubMed] References Ambrosini, G.; Andrisani, A.; Porcu, E.; Rebellato, E.; Revelli, A.; Caserta, D.; Cosmi, E.; Marci, R.; Moscarini, M. Oocytes cryopreservation: State of art. Reprod. Toxicol. 2006, 22, 250–262. [CrossRef] [PubMed] y p p [ ] [ ] 7. 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https://openalex.org/W3202447791	https://zenodo.org/record/5771024/files/Authenticity%20Paradox%20an%20Examination%20on%20Local%20Gastronomy.pdf	English	null	Authenticity Paradox: an Examination on Local Gastronomy	Anais Brasileiros de Estudos Turísticos	2,021	cc-by	9,347	Resumo ___________________________________________________________________________________________ A gastronomia local oferece autenticidade, um elemento importante na experiência turística. No entanto, alguns turistas não conseguem experimentar os produtos gastronômicos em sua forma autêntica. Este problema se deve ao tipo de turista, ao nível de importância da gastronomia e à busca por autenticidade. Para atender a um variado número de turistas, os produtores gastronômicos locais fazem mudanças na essência dos produtos. Este estudo pretende investigar o processo de mudança de autenticidade de produtos gastronómicos, oferecidos no contexto do turismo, e desenvolver uma compreensão das causas e custos da deterioração. Metodologicamente, a literatura existente foi avaliada com inferências conceituais. Como os resultados sugerem, as mudanças começam com a exploração do turismo, seguida pela deterioração relacionada ao turismo. Após a deterioração, se o processo de recuperação for realizado, o retorno à forma exata de autenticidade parece não ser possível. Novas formas alternativas de autenticidade são mais prováveis de ocorrer. Além disso, perspectivas equivocadas, como considerar medidas preventivas de recuperação como ações de curto prazo, podem levar à deterioração novamente, como em uma espiral. Aumentar a conscientização para evitar essa espiral é a proposta crítica deste estudo para os destinos. Abrir novos horizontes para o fenômeno da autenticidade e derrubar a ideia de que os turistas são o único motivo de deterioração são contribuições à literatura. As mudanças que levam à deterioração também são de responsabilidade da população local. Palavras-chave: Autenticidade. Paradoxo. Gastronomia. Produtos de Turismo Gastronômico. Pesquisa Conceitual. _Abstract Local gastronomy offers authenticity, an important element of tourist experience. However, some tourists cannot experience gastronomic products in their exact authentic forms. This issue causes from tourists' typology, importance level of gastronomy, and quest for authenticity. For serving widely variated tourists, producers of the local gastronomic products make changes in the essence of products. Current study aims to investigate changing process of gastronomic products’ authenticity which are served in the context of tourism, and to develop an understanding on causes and costs of deterioration. Methodologically, existing literature had evaluated with conceptual inferences. As the findings propose, the changes start with touristic exploration which is followed by tourism-related deterioration. After deterioration, if process of recovery conducted, turning back to the exact form of authenticity does not seem possible. The alternative forms of authenticity are most likely to occur. Additionally, mistaken perspectives like considering recovery preventions as short-term actions can lead the deterioration again, like a loop. Raising awareness for prevent this loop is this study's critical proposition to destinations. Opening new horizons for the phenomenon of authenticity and overthrowing the idea that see tourists as the sole reason for deterioration are the contributions to the literature. The changes that lead deterioration are responsibility of locals, too. Keywords: Authenticity. Paradox. Gastronomy. Gastronomic Tourism Products. Conceptual Research. REVISTA ANAIS BRASILEIROS DE ESTUDOS TURÍSTICOS / ABET (e-ISSN 2238-2925) ARTIGO CONCEITUAL / CONCEPTUAL PAPER / ARTÍCULO CONCEPTUAL REVISTA ANAIS BRASILEIROS DE ESTUDOS TURÍSTICOS / ABET (e-ISSN 2238-2925) ARTIGO CONCEITUAL / CONCEPTUAL PAPER / ARTÍCULO CONCEPTUAL AUTHENTICITY PARADOX: AN EXAMINATION ON LOCAL GASTRONOMY Gü k A b k * & Ed Al h ** Gürkan Aybek * & Eda Alphan ** Licenciada por Creative Commons 4.0 / Internacional CC BY 4.0 * Master in Gastronomy and Culinary Arts/Akdeniz University (2020). Degree in Gastronomy and Culinary Arts/Akdeniz University (2017). Research Assistant of Gastronomy and Culinary Arts Department at Tourism Faculty/Akdeniz University. ORCID: https://orcid.org/0000-0002-5536-5440 [ gurkanaybek@akdeniz.edu.tr ] *Master in Gastronomy and Culinary Arts/Gazi University (2017). Degree in Gastronomy and Culinary Arts/Gazi University (2015). Research Assistant of Gastronomy and Culinary Arts Department at Tourism Faculty/Akdeniz University. ORCID: https://orcid.org/0000-0001-8294-5718 [ edaalphan@akdeniz.edu.tr ] 1 INTRODUCTION Even there are many studies point out the negative effects of tourism on authenticity of gastronomy (e.g., Cohen & Avieli, 2004; Archer et al., 2005; Kırlar Can et al., 2017), there is no study that tries to explain these effects as a holistic process which contains stages of exploration, deterioration, and recovery. It is believed that understanding this process step by step can ease the prevention from authentic deterioration for destinations. Authenticity is seen as a contributor of tourism experience (Wang, 1999; Özdemir & Seyitoğlu, 2017; Zhang et al., 2019; Tiberghien et al., 2020). In tourism literature, many researchers proposed that the idea of authenticity, which associates “traditionality” and “genuinity”, is one of the main motivations of some tourists (Cohen, 1988; Wang, 1999; Stankova & Vassenska, 2015; Özdemir & Seyitoğlu, 2017; Guerreiro & Marques, 2017; Zhang et al., 2019). Experiencing the values which are not produced specifically for mass market (Cohen, 1988) creates an opportunity to feel unique themselves (Tiberghien et al., 2020). Moreover, in this paper, it is proposed that the deterioration and recovery processes might turn into an authenticity paradox. Deterioration that takes place after exploration triggers the concerns about the danger to lose authenticity in the mind of local stakeholders. Then, the recovery actions may be developed as a reflection of these concerns. However, considering the actions as temporary and short-term preventions can lead to observe the same deterioration process. Thus, a loop named as authenticity paradox takes place in the destination. Although there are tourists who travels with authenticity seeking behavior actively, the fact that, there are also the ones who experience authentic features just because of their spontaneous encounter at the destination (Özdemir & Seyitoğlu, 2017). Furthermore, for some tourists, authenticity may be something to be avoided according to their personal traits and perceptions. This paper is a conceptual research as it evaluated the current literature on gastronomy, tourism, and authenticity topics. Even there is a lack of empirical data in this study, it can open new horizons for future empiric and conceptual research. In this manner, this study will contribute to the field with new research possibilities. Also, developing a model to demonstrate authenticity paradox and its stages is tidying up to general debates about tourism and authenticity. 2 LITERATURE REVIEW One of the main concerns of the destinations that are popular among mass of tourists is protecting authentic features from tourism-related deterioration (Cohen, 1988; Getz, 1995; Archer et al., 2004; Butler, 2017; Kırlar Can et al., 2017). More tourists mean more consumption and, for locals, more income expectation consequently (Teixeira & Ribeiro, 2013). Resumen La gastronomía local ofrece autenticidad, un elemento importante en la experiencia turística. Sin embargo, algunos turistas no pueden experimentar los productos gastronómicos en su forma auténtica. Este problema se debe al tipo de turista, el nivel de importancia de la gastronomía y su búsqueda por autenticidad. Para atender a una gran variedad de turistas, los productores gastronómicos locales realizan cambios en la esencia de sus productos. Este estudio tiene como objetivo investigar el proceso cambiante de la autenticidad de los productos gastronómicos, ofertados en el contexto del turismo y desarrollar una comprensión de las causas y los costos de su deterioro. Metodológicamente, la literatura existente se ha evaluado con inferencias conceptuales. Como los resultados demuentran, los cambios comienzan con la exploración turística, seguida de un deterioro relacionado con el turismo. Después del deterioro, aún si se lleva a cabo el proceso para la recuperación de la autenticidad del producto, no es posible volver a su forma exacta. Es más probable que ocurran nuevas formas alternativas de autenticidad. Además, perspectivas erróneas como considerar medidas preventivas de recuperación como acciones a corto plazo, pueden conducir nuevamente al deterioro, como una espiral. Concientizar para prevenir esa espiral, es la propuesta crítica de este estudio para los destinos. Abrir nuevos horizontes para el fenómeno de la autenticidad y derribar la idea de que son los turistas el único motivo de deterioro, son las contribuciones a la literatura. Los cambios que provocan el deterioro también son responsabilidad de los lugareños. Palabras clave: Autenticidad. Paradoja. Gastronomía. Productos del Turismo Gastronómico. Investigación Conceptual. Palabras clave: Autenticidad. Paradoja. Gastronomía. Productos del Turismo Gastronómico. Investigación Concep Licenciada por Creative Commons 4.0 / Internacional CC BY 4.0 Master in Gastronomy and Culinary Arts/Akdeniz University (2020). Degree in Gastronomy and Culinary Arts/Akdeniz University (2017). Research Assistant of Gastronomy and Culinary Arts Department at Tourism Faculty/Akdeniz University. ORCID: https://orcid.org/0000-0002-5536-5440 [ gurkanaybek@akdeniz.edu.tr ] **Master in Gastronomy and Culinary Arts/Gazi University (2017). Degree in Gastronomy and Culinary Arts/Gazi University (2015). Research Assistant of Gastronomy and Culinary Arts Department at Tourism Faculty/Akdeniz University. ORCID: https://orcid.org/0000-0001-8294-5718 [ edaalphan@akdeniz.edu.tr ] Rev. Anais Bras. de Est. Tur./ ABET, Juiz de Fora (Brasil), e-ISSN 2238-2925, v.11, n. único, pp.1 – 12, Jan./ Dez., 2021 1 Palabras clave: Autenticidad. Paradoja. Gastronomía. Productos del Turismo Gastronómico. Investigación Conceptual. 1 1 INTRODUCTION But regardless from the typology of tourists; the objects, locations, and products, which are called as “authentic” in the context of tourism, already exist in a destination as a part of the everyday life of local people. For residents, these are routine and ordinary “localities” which reflects their way of living. And offering these localities for tourist consumption may cause some problems like deterioration accordingly (Kırlar Can et al., 2017). 2.1 Tourism & Authenticity Authenticity is a significant provider of tourism experience (Butler, 2017; Guerreiro & Marques, 2017; Corpas & Castillo, 2019). However, tourists’ authenticity-seeking behavior during their tourism experience differentiates from each other according to their personal characteristics (Özdemir & Seyitoğlu, 2017). Relevant to this differentiation, authenticity context in a destination also has different approaches. And meeting with higher amount of demand with different wants and preferences requires to leave the traditional ways and become more tourist oriented (Kırlar Can et al., 2017). Thus, local producers may change the essence of the products and deteriorate them with their own hands to get a bigger slice of the cake (Wirth & Freestone, 2003; Ekin, 2018). In this sense, tourism may damage the authenticity inevitably through both tourists and locals. Wang (1999) proposes authenticity has 4 basic approaches in the context of tourism as objectivism, constructivism, existential, and post-modernism. Objectivism approach insists authenticity is the genuine form of an object without any touches on it. However, authenticity has to be reconstructed for harmonization to the tourism spectrum (Cohen & Avieli, 2004). According to what has been conveyed so far, this study examines the authentic deterioration and ensuing recovery processes of local gastronomic products. Local gastronomy is chosen as the authentic object that reflects the stages due to its link to the culture (Bogataj, 2019), changeable nature with dependency to human (Martin, 2014), and importance in tourism as a tourism product (Björk & Kauppinen-Räisänen, 2017). In constructivist view, tourists will not be able to experience objects or events in their authentic forms. So, alterations, which are referred by Maccannell (1973) as staged authenticity, are inevitable in tourism settings. 2 Rev. Anais Bras. de Est. Tur./ ABET, Juiz de Fora (Brasil), e-ISSN 2238-2925, v.11, n. único, pp.1 – 12, Jan./ Dez., 2021 AUTHENTICITY PARADOX: AN EXAMINATION ON LOCAL GASTRONOMY Gürkan Aybek & Eda Alphan Wang’s (1999) existential authenticity concept connects authenticity to tourist’s perception about the activity. Chang et al. (2011) explain this through dining experience during travel. If tourists’ participation level to local culture could be endured by dining experience, then positive perceptions about authenticity can be developed (Özdemir & Seyitoğlu, 2017). For post- modernist, the arguments on authenticity are pointless since there is no way to know what is authentic actually (Wang, 1999). gastronomy (existential and experimental), especially local food experience satisfies this desire (Björk & Kauppinen-Räisänen, 2017; Bogataj, 2019; Zhang et al., 2019). 2.1 Tourism & Authenticity And the authenticity is one of the key parts of the local food experience (Chang et al., 2011; Timothy & Ron, 2013; Zhang et al., 2019; Skinner et al., 2020) Components of local gastronomic identity are the sources of authenticity (Sims, 2009; Timothy & Ron, 2013; Zhang et al., 2019). So, food-related typologies of tourists also determine their quest for authenticity. In tourism literature, it is widely assumed authenticity will appear in the context of constructivist and existential approaches (e.g., Wang, 1999; Cohen & Avieli, 2004; Timothy & Ron, 2013; Özdemir & Seyitoğlu, 2017). Therefore, studies mostly focus on possible effects of tourism (e.g., Wirth & Freestone, 2003; Timothy & Ron, 2013) and tourist’s authenticity- related experience (e.g., Tasci & Knutson, 2004; Özdemir & Seyitoğlu, 2017). The study conducted by Özdemir & Seyitoğlu (2017) revealed that tourists can be divided into three categories (which are authenticity seekers, moderates, and comfort seekers) according to their quest for authenticity. Authenticity seekers are in an active search for food that reflects genuine authenticity despite the possible risks. The tourists who are defined as moderates also looking for authentic and novel food but in acceptable level of risk. So, reducing risk to an acceptable level through changing some ingredients is one way to ensure their consumption. But in the third context, the authors conceptualized them as comfort seekers who intend to maintain their comfort and safety and willing to stay in their “environmental bubble”, there is an avoidance habit from local authentic foods and search for familiar ones. But yet, there is no study focusing on how tourism-related changes can be observed and, if the changes are negative, how after-deterioration process of authentic objects develops in a destination. The current study aims to examine and explain the process in a holistic view with the objectivist approach to fill this gap. Since the gastronomic tourism products are chosen as “the object”, the relationship with gastronomy, tourism, and authenticity should be explained. It is thinkable that if tourists describe themselves as authenticity seekers, they will hanker to consume local food products that reflect the gastronomic identity of the destination. On the other hand, tourists, who have concerns about food with high-risk perceptions and avoidance habit, are expected to choose restaurants which not reflecting local texture. of Gastronomic Tourism 3.1 Authenticity Paradox and Dilemma Concepts in the Existing Tourism Literature 3.1 Authenticity Paradox and Dilemma Concepts in the Existing Tourism Literature The relationship between tourism and authenticity of destinations already examined under the name of “dilemma” or “paradox” in several studies (Table 1) but there is not any common definition of the theme. It is possible to say that the authenticity in a destination can be shaped by tourism. Some of the researchers have been thinking this “shaping” process creates a dilemma according to coexistence of positive and negative effects of tourism (Getz, 1995; Archer et al., 2004; Teixeira & Ribeiro, 2013; Cobb, 2014). Additionally, sometimes adding more “authentic- like” features can increase tourist attention and financial income (Kırlar Can et al., 2017). Depending on these, authentic products can become too mainstream or, as opposite, too exaggerated. In both cases, deterioration of authenticity occurs. Cohen (1988) mentioned that if there is a deterioration of authenticity, it will not solely be perceived by locals who knows the “real” form of the authenticity, tourists will also recognize it. Wirth and Freestone (2003) tie this situation to another paradox that is about homogenization of a culture’s heterogeneous values according to commodification of the culture for tourist consumption, but the heterogeneity was the attraction foremost. ) On one hand, tourism increases awareness and familiarity of the authentic local products which lead to protection and regeneration of them (Getz, 1995; Grünewald, 2002; Teixeira & Ribeiro, 2013). On the other hand, increasing awareness and demand creates a pressure on the products along with the desire of locals earning more money through meeting the higher amount of demand than before and preferring tourists rather than locals to serve (Getz, 1995; Grünewald, 2002; Wirth & Freestone, 2003; Rickly, 2019). Efforts for supply to this demand and meet with the different preferences cause lower quality and uniformization in products (Cobb, 2014). Losing this heterogeneity facilitates tourist consumption at first, but with time that pass, tourists will recognize the commoditization and not get attracted by destination anymore. However, Martin (2014) thinks differently about losing tourist attraction. According to him, it does not that matter for tourists if it is real or not unless it satisfies the search for authenticity. Tourists cannot detect the decreased authenticity and, probably, would believe what they experienced is the real. Table 1: Authenticity Paradox & Dilemma in the Literature Paper Mention Context Focus of Conflict Cohen (1988) Paradox Tourism in general Locals vs. 3.1 Authenticity Paradox and Dilemma Concepts in the Existing Tourism Literature Tourist Getz (1995) Dilemma Event tourism Destroying vs. Regenerating Wirth & Freestone (2003) Paradox Cultural heritage Heterogeneity vs. homogeneity Cohen (2007) Dilemma Tourism in general Before vs. After Popularization Ashworth (2009) Dilemma Cultural heritage Economic Contribution vs. Locals’ Disrelish Timothy & Ron (2013) Not specified Tourism in general Preserving vs. Commoditization Cobb (2014) Paradox Tourism and digitalization Preserving vs. Uniformization and Commodification Martin (2014) Paradox Cajun cuisine Reaching vs. Searching to Authenticity Ekin (2018) Dilemma Emerging destinations Authenticity Seeking vs. Local Deceptive Source: Evaluated from the literature Table 1: Authenticity Paradox & Dilemma in the Literature Nevertheless, unlike Martin (2014), Leong (2016) points out that, Hong Kong has lost her authenticity according to rapid development in tourism which homogenizes uniqueness and distinctiveness of the city’s authentic values and it causes reduction in the number of travelers. All these research handle authenticity as “paradox” or “dilemma” with somehow close but conceptually different meanings in many different contexts. However, even the local gastronomic products are contributors of authenticity (Sims, 2009; Martin, 2014), no study held the structure of local gastronomic products as the main object. The current study tries to fill this gap, too by theoretically investigating how these products as contributors of authenticity (Sims, 2009; Martin, 2014) can be shaped and changed according to tourism development. It is thought that the nature of these products may cause sudden changes according to dominant tourist profile in the destination (Martin, 2014). Source: Evaluated from the literature Also, this pressure may force producers and servers in a destination to create “deceptive” authenticity (Timothy & Ron, 2013; Ekin, 2018). As can be remembered from the MacCannel’s (1973) “staged 3 CONCEPTUAL FRAMEWORK authenticity” concept, tourists may never reach a real and genuine authenticity in a destination. Because of the pure authenticity may be too much for tourists, little changes have made to streamline the products to tourist consumption (Cohen & Avieli, 2004). 2.2 Authenticity Consumption According to Quan & Wang (2004), tourists’ food consumption behavior can be observed in two contexts: peak experience or supporting experience. For tourists, who want to experience food in a destination as the contrast to their everyday life, local gastronomy can be the peak experience. On the other hand, for tourists that refer food as “supporting experience” local food is not the main search and can be perceived as an element to be avoided. Mkono et al. (2013) conducted a netnographic research which supports the propositions of Quan and Wang’s (2004) model. It is understood that the literature on gastronomic consumption in tourism and authenticity or locality is mostly focused on the tourist side of the concept. There is a variety of tourists from authenticity-seekers to authenticity-avoiders. A destination can be visited by tourists who are not interested in gastronomy and its authenticity (McKercher et al., 2008; Özdemir & Seyitoğlu, 2017). As a response to this, there are different types of food service businesses which target different segments of the tourist market (Yılmaz & Özdemir, 2017). While Quan and Wang (2004) ponder about the context of tourist’s food consumption, Hjalager (2004) divides tourists into four typologies in terms of their food quest. These are existential, recreational, diversionary, and experimental. The existential and experimental tourists are willing to consume local gastronomy, however, diversionary and recreational tourists are assumed to not have a desire for gastronomy, and sometimes they even try to stay away (Cohen & Avieli, 2004). For the ones who desire to consume local However, no study which proposes a theoretical view on the local gastronomic products’ alteration as an authentic object. Further, authenticity and tourism relation is a multifaceted and paradoxical subject since there are negative and positive observations on it (e.g., Wirth & Freestone, 2003; Archer et al., 2005; Teixeira & Ribeiro, 2013; Zhang et al., 2019; Cobb, 2014). Thus, there is a paradox can emerge in the changing process of local gastronomic products. 3 3 Rev. Anais Bras. de Est. Tur./ ABET, Juiz de Fora (Brasil), e-ISSN 2238-2925, v.11, n. único, pp.1 – 12, Jan./ Dez., 2021 AUTHENTICITY PARADOX: AN EXAMINATION ON LOCAL GASTRONOMY Gürkan Aybek & Eda Alphan AUTHENTICITY PARADOX: AN EXAMINATION ON LOCAL GASTRONOMY Gürkan Aybek & Eda Alphan AUTHENTICITY PARADOX: AN EXAMINATION ON LOCAL GASTRONOMY Gürkan Aybek & Eda Alphan experience desire (Quan & Wang, 2004; Hjalager, 2004) and authenticity-seeking behavior (Özdemir & Seyitoğlu, 2017) are somehow related (Skinner et al., 2020). This study based on these relationships’ effects on the context of the authenticity in a destination (MacCannel, 1973; Wang, 1999). Thus, one of the main goals and contributions of this study to this debate is to concretize how local producers change the structure of their gastronomic products’ authenticity to gain all possible income through them. Table 2 summarized the relationships. For remembrance, since there are tourists visit a destination with the desire to consume local and authentic gastronomic products, there are also the ones who search for other attractiveness and sometimes avoids (Cohen & Avieli, 2004; McKercher et al., 2008; Özdemir & Seyitoğlu, 2017). Source: own elaboration. Table 2: Theoretical relations of gastronomy, authenticity, and product structure Food as an Experience Tourist Types Quest for Authenticity Authenticity Context Products’ Authentic Structure Peak Experience Existential High Existential Genuine local products Experimental Moderate High Commodification, Staged Authenticity, Existential Local products with shows and modified ingredients, techniques etc. Supporting Experience Diversionary Moderate Low Staged Authenticity, Existential Local products that streamlined to tourists’ desire and needs Recreational Low Staged Authenticity, Inauthenticity Products that satisfy tourists without concerning authenticity Quan & Wang (2004) Hjalager (2004) Özdemir & Seyitoğlu (2017) MacCannell (1973); Wang (1999) The current study’s theoretical contribution Table 2: Theoretical relations of gastronomy, authenticity, and product structure Q t f According to Quan & Wang (2004), tourists’ food consumption behavior can be observed in two contexts: peak experience or supporting experience. For tourists, who want to experience food in a destination as oppose to their everyday life, local gastronomy can be the peak experience. On the other hand, for tourists that refers food as supporting experience local food is not the main search and can be perceived as an element to be avoided. and identity indicator (Steiner & Reisinger, 2006). So, it is possible to say that experimental tourists are also the moderate authenticity-seekers (Özdemir & Seyitoğlu). Exaggerated presentations and shows with plentiful ingredients than usual are always on display in social media which attracts this kind of tourists to a destination. Hence, offering exaggeratedly commodified food as authentic can also be found in destinations. AUTHENTICITY PARADOX: AN EXAMINATION ON LOCAL GASTRONOMY Gürkan Aybek & Eda Alphan The following idea may come to mind according to what are told so far; if tourists in search for authenticity, like existential tourists that see food as peak experience, then they choose a destination that has strong reflection of local gastronomic identity and authenticity. And the others, the ones who can be called as recreational or comfort seekers, can travel to more suitable destinations for them. Similarly, Hjalager (2004) divides tourists into four typologies in terms of their food quest. These are existential, recreational, diversionary, and experimental. While existential and experimental tourists are willing to consume local gastronomy, diversionary and recreational tourists are assumed to not have a desire for gastronomy, and sometimes they even try to stay away. Also, there is not just one type of food or restaurant in a destination (Yılmaz & Özdemir, 2017), they can visit the ones which serve what they want. All these doubts are welcomed and somehow true. But it is also known that a destination cannot be chosen solely by gastronomic tourists (McKercher et al., 2008). There are the ones with expectations and searches other than local gastronomy and authenticity. Especially in emerging destinations (Ekin, 2018), producers and service providers may try to appeal to every types of tourists with a specific product to earn more money with minimum effort. So, some modifications can be observed that aim to streamline and ease the consumption of moderate authenticity- seekers and keep attracted the main authenticity- seekers at the same time (Cohen & Avieli, 2004). Also, there is not just one type of food or restaurant in a destination (Yılmaz & Özdemir, 2017), they can visit the ones which serve what they want. All these doubts are welcomed and somehow true. But it is also known that a destination cannot be chosen solely by gastronomic tourists (McKercher et al., 2008). Our main question is what happens to structure of local gastronomic products as authentic objects when different types of tourists visit a destination. For instance, diversionary tourists see food as a supporting experience element, and not in an active search for local gastronomy. It can be thought that they are moderates in authenticity-seeking behavior. There are the ones with expectations and searches other than local gastronomy and authenticity. 3.2 Authentic Structure of Gastronomic Tourism Products As mentioned above, tourists’ gastronomic As mentioned above, tourists’ gastronomic 4 Rev. Anais Bras. de Est. Tur./ ABET, Juiz de Fora (Brasil), e-ISSN 2238-2925, v.11, n. único, pp.1 – 12, Jan./ Dez., 2021 AUTHENTICITY PARADOX: AN EXAMINATION ON LOCAL GASTRONOMY Gürkan Aybek & Eda Alphan Especially in emerging destinations (Ekin, 2018), producers and service providers may try to appeal to every types of tourists with a specific product to earn more money with minimum effort. So, some modifications can be observed that aim to streamline and ease the consumption of moderate authenticity- seekers and keep attracted the main authenticity- seekers at the same time (Cohen & Avieli, 2004). So, to attract them, local producers streamline their products for reducing the risks when they encounter. Changing some of the ingredients which are too “extreme” for tourists to more familiar ones is an example to it (Cohen, 2007). The other side of the coin, experimental tourists desire to experience the show and they are in search for popularity more than everything (Çalışkan, 2013). From a point of view, authenticity is a popularity 5 5 Rev. Anais Bras. de Est. Tur./ ABET, Juiz de Fora (Brasil), e-ISSN 2238-2925, v.11, n. único, pp.1 – 12, Jan./ Dez., 2021 AUTHENTICITY PARADOX: AN EXAMINATION ON LOCAL GASTRONOMY Gürkan Aybek & Eda Alphan display the stages to help destinations with similar struggles so they can determine if they are in this loop or in a stage of it. It is hoped that this will at least raise awareness of authenticity for practitioners and create new ideas for future research. This study fills gaps in the literature which are mentioned above. According to these, we aim to understand that how this streamlining process, which includes staged authenticity and commodification, deteriorate the authenticity of gastronomy. These will also be investigated that how the recognition of locals on the negative effects of deterioration takes place and which precautions could be taken for not live a situation like this again. Withal, the point of the paradox that in which circumstances a destination lives these processes as a loop continuously will be discussed. The paradox or loop contains three main stages as exploration, deterioration, and recovery. The exploration refers the gaining popularity as a tourist destination through locals’ marketing efforts and pioneer tourist attention. The service approach of destination turns tourist-oriented with this stage. After exploration, if tourists divided variously and local gastronomy producers try to target almost all of them, deterioration of authenticity can be observed. 5.1 Exploration to Deterioration Thanks to the development of communication technologies, a destination which is preferred by a few tourists in a period can become the focus of the tourist masses (Miguéns et al., 2008; Jalilvand et al., 2012; Cobb, 2014). Especially with social media, a small piece of information about authenticity may grow among many users who are also potential tourists (Xiang & Gretzel, 2010). Oliveira and Baracho (2018) show that in Brazil case, it is possible to assess tourism structure through social media. Since this study is a theoretical essay, the biggest limitation of the study is the lack of empirical data. Also, the authors might be overlooked some of the papers in the literature. However, it is believed this study creates a remarkable basis for future research as it will be discussed in the conclusion. Authentic experience indicators can flow between the experiencer and potential experiencer faster. It is even possible to say that there are “social media tourists” who do not care about authenticity but search for popularity and identity through it. For example, “The Eastern Express” in Turkey become popular among mass of tourists thanks to social media with its authentic and social media related shareable features (Çakmak & Altaş, 2018; Doğan et al., 2018; Eryılmaz & Yücetürk, 2018). AUTHENTICITY PARADOX: AN EXAMINATION ON LOCAL GASTRONOMY Gürkan Aybek & Eda Alphan Furthermore, another struggle that is not a stable concept, in fact, it is a changeable and living phenomena which can affected by factors like tourism (Martin, 2014; Timothy & Ron, 2013; Tiberghien et al., 2020). If the recovery process applied successfully after deterioration, will the authentic products be the same as they were will also be mentioned. In the framework of all these, this study examines authenticity from an objectivist view by treating local gastronomic products as an authentic object in tourism spectrum (Wang, 1999). Deterioration will bring recovery actions thanks to local concerns about authenticity. Recovered authenticity is expected to gain tourist attention back. However, if the recovery actions got stuck with short- term understatement and the same problems observed after re-exploration, a paradox of authenticity occurs. Every loop brings more casualty in the object’s authenticity and alternative forms (O1 and O2) emerge. Stages will be explained deeper in following sections. 4 METHODOLOGY & LIMITATIONS This study is a conceptual research that evaluates existing literature on authenticity, tourism, and gastronomy. The authors collected the studies in the literature and linked them to create a theoretical framework for the goals. Changes in the product structure and stages in the model (Figure 1) first discussed separately and individually to prevent orientation to each other. Then the authors met and mutually discussed the subject ones again. The formation of the model was decided with consultation by a professor in the gastronomy field. 5 A CONCEPTUAL MODEL PROPOSAL OF “AUTHENTICITY PARADOX” Figure 1 shows authenticity paradox as a loop that intends to explain how gastronomic products of a destination may lose their authentic features and gain them back in the context of tourism in the light of the literature. If this loop can be considered as a learning process by destinations, there is no need to worry about a paradox. But if the post-recovery process cannot well managed and similar problems are observed, then a vicious circle may begin, and it becomes a causal paradox. This research aims to Social media also creates marketing options for locals (Katsikari et al., 2020). Social media’s structure which removes time, location, and cultural barriers between societies creates faster, cheaper, and wider marketing opportunities (Oliveira & Baracho, 2018). As Perinotto et al. (2020) verified for Brazil, it is important to have a social media profile for tourism organizations which affects the successful development of 6 Rev. Anais Bras. de Est. Tur./ ABET, Juiz de Fora (Brasil), e-ISSN 2238-2925, v.11, n. único, pp.1 – 12, Jan./ Dez., 2021 AUTHENTICITY PARADOX: AN EXAMINATION ON LOCAL GASTRONOMY Gürkan Aybek & Eda Alphan - Local Demand-oriented Service Approach Exploration of Gastronomic Identity as an Authentic Object in Destination - Staged Authenticity - Commodification & Commercialization - Miracle of Consensus on “Inauthenticity” in the Destination - Public Interventions (Quality Standarts, Quota Production, Geographical Indications) - Pioneer Tourist Attention & Locals’ Marketing Efforts Deterioration Recovery O1 O2 . . . - Tourist-oriented Service Approach - Miracle of Consensus on “Authenticity” in the Destination Possible Loss in Authenticity Possible Loss in Authenticity Possible Loss in Authenticity O1 & O2: New possible forms of object authencity of local gastronomic products Figure 1: Conceptual Proposal of Gastronomic Products' Authenticity Paradox Source: own elaboration. Figure 1: Conceptual Proposal of Gastronomic Products' Authenticity Paradox - Tourist-oriented Service Approach - Miracle of Consensus on “Authenticity” in the Destination Po in Possible Loss in Authenticity Possible Loss in Authenticity - Pioneer Tourist Attention & Locals’ Marketing Efforts - Commodification & Commercialization Deterioration Recovery - Miracle of Consensus on “Inauthenticity” in the Destination - Local Demand-oriented Service Approach O1 & O2: New possible forms of object authencity of local gastronomic products Source: own elaboration. O1 & O2: New possible forms of object authencity of local gastronomic products Source: own elaboration. Source: own elaboration. destinations. 5.3 After the Recovery: Paradox or not? In this paper, we thought that the authenticity paradox takes place after the gastronomic products of a destination regain their popularity. Same steps can be observed after the recovery in some circumstances. Pioneer tourists might visit destination again, and their shares and locals’ marketing efforts can make authenticity features visible anew. Another concern is that the tourism product is a product that need brand, package, and shelf life. Depending on these conditions, some changes in ingredients and preparations are inevitable. These commercialization and commodification processes lead loss in the sense of authenticity between before and after the rapid development of tourism in the area (Martin, 2014), and it most likely ends up with cultural deterioration. Furthermore, recovery process may be interesting and even attractive for tourists, and destinations can build their marketing strategy on this base (Fox, 2007). But when this paradox occurs? In fact, it is not easy to say in one breath, but it takes place if the locals have their greed for earning back without learning from the past. As we mentioned before, if these losing and recovering stages of authenticity seen as a learning process then there is no need to discuss a paradox or loop. But, if locals see tourism as exploitable opportunity, then living this loop again is almost inevitable. AUTHENTICITY PARADOX: AN EXAMINATION ON LOCAL GASTRONOMY Gürkan Aybek & Eda Alphan AUTHENTICITY PARADOX: AN EXAMINATION ON LOCAL GASTRONOMY Gürkan Aybek & Eda Alphan expectations, may become tourist-oriented and focus the wants and needs of tourists rather than locals (Grünewald, 2002; Wirth & Freestone, 2003; Martin, 2014). A staged authenticity is possible to emerge after it. If tourists do not volunteer to consume local gastronomic products as they are, changes and rearrangements occur to ease consumption (Cohen & Avieli, 2004; Cohen, 2007). Although it is not obviously revealed by evidence in any research, it is most likely to think as local consumers are already willing to eat local foods as they are naturally. So, serving and producing towards local demand may cause to reuse authentic recipes with original ingredients and techniques. Also, the cultural concerns of locals will develop a need of action among local decisionmakers and administrators of destination. Setting rules that contain quality standards, limited production, and geographical indicators is an example of ways to preserving and recovering local gastronomy (Uslu & Kiper, 2006; Çalışkan, 2013; Ceccarini, 2014). As mentioned before, adding some “fancy” and “authentic-like” features like shows and ingredients is a way to create extra attractiveness, also. These two ways are the examples of locals’ trial to addressing more types of tourists with same group of products. Increased volume should be examined also to assess deterioration process. In such a case, amount of production that increases directly proportional to demand pushes locals to find alternative ways to produce more products in a shorter time easily (Wirth & Freestone, 2003; Martin, 2014). It is probably caused by the economic instinct that is about not to lose the opportunity to earn more money in a shorter period. 5 A CONCEPTUAL MODEL PROPOSAL OF “AUTHENTICITY PARADOX” Because the information in social media shapes expectations and perceptions of tourists about the service providers in a destination (Lima et al., 2020). Thus, authentic tourism experiences a destination offers can be reflected by locals through social media as a marketing tool. Tourists with desire to experience authenticity can reach these marketing efforts as information at the same time thanks to social media. Higher amount of information flows through internet than before and this situation causes a faster development of tourism (Xiang & Gretzel, 2010). We called this awareness and popularization stage as exploration, but it is not just about exploring the destination, it also means exploration of more demand and income chances by locals. In addition to pioneer tourists, local people who want to promote their everyday life as an attraction or just have desire to show their life contribute this consensus also (Tiberghien et al., 2020). Farmers to restaurateurs, many different producers and service providers use media channels to promote the authenticity of their products (Cobb, 2014; Pilař et al., 2016). All these efforts occur because of the awareness among residents that there is an income possibility through localities, and this awareness developed thanks to pioneer tourists. The paradox starts with when a destination which was preferred by tourists, who can be called as authenticity-seeker pioneer tourists, becomes popular and sit on the focus of tourist masses. There is a need of awareness among tourists about authenticity for creating an attraction through it. Media and electronic-word-of-mouth are playing enormous role to widen the information about the local gastronomy and create awareness on it (Cohen, 2007; Miguéns et al., 2008; Andilolo & Ranteallo, 2017). Rickly (2019) calls it as “miracle of consensus” which means collective awareness and knowledge on a specific attraction of the destination. With social media, sharing process became easier and faster recently, and popularization of destinations as well. Of course, exploration cannot happen at once, it is a process that has cumulative sharing and promoting background and expands towards a saturation point. For a while, this expanding leads to increasing number of tourists which causes higher demand and income. In this course, locals, that have higher income 7 Rev. Anais Bras. de Est. Tur./ ABET, Juiz de Fora (Brasil), e-ISSN 2238-2925, v.11, n. único, pp.1 – 12, Jan./ Dez., 2021 6 DISCUSSION, CONCLUSION, & FUTURE RESEARCH IDEAS This paper aims to reveal the tourism-related structural changing process of local gastronomic products’ authenticity. Even there are studies indicate the effects of tourism on these products, no study offers conceptual understanding on the topic. For this reason, this study evaluates current literature to concretize deterioration of the gastronomic products’ authenticity and demonstrates this changing process on the theoretical model of the authenticity paradox. The study also contributes a conceptual model proposition of authenticity paradox. The authors consider authenticity paradox as a loop, contrary to general understanding. In a scenario which recovery actions cannot be maintained or implemented only in short-term, same struggles can emerge in destination again. And every loop causes some irreversible damages in authenticity. However, for some destinations, observing this paradox at least once might be a positive thing in an aspect. This kind of “calamity” may cause a development of an action plan for preserving authenticity of local food products. But these plans may change the products to “tourism-resistant” or “tourism-related”. Thus, determining if the recovered products are the real form of the gastronomy is almost impossible. Alternative versions of authenticity will most likely replace the “old” one. The first gap this study tries to fill is about the focus of the study related to authenticity approaches. In the existing literature, authenticity has mostly been explained with constructivist or existential approaches, and it discussed the effects of authenticity on the tourist’s experience. Unlike previous research, this study adopted an objectivist approach and chose the gastronomic tourism products as the “authentic object”. Second, this research demonstrates the paradox that it generates during the changing process of local gastronomic products. And also, it provides suggestions about after deterioration, how the recovery process works and what can happen as a result of this process. y p Furthermore, the authors do not see tourists or locals as the main culprit. Even though a perception that sees tourists as “the guilty” exists in the literature (Ashworth, 2009), this problem also about the respond of the residents to tourism demand. Tourists that are not exact authenticity or comfort-seekers always look food alternatives in the destination (Özdemir & Seyitoğlu, 2017). Almost every destination offers gastronomic products that can appeal different types of tourists like ethnic, global, or tourist-oriented foods (Yılmaz & Özdemir, 2018). 5.2 Deterioration to Recovery Deterioration is not noticeable at first by local businesses. But residents who do not expect money from tourism start to develop an idea on “inauthenticity” in the homeland. Therefore, it is possible to say that prejudice against tourism and cultural concerns of locals will eventually rise (Kırlar Can et al., 2017), and it is possibly will turn to a conflict between residents and other stakeholders. But deterioration of authentic features also will be noticed by tourists eventually (Cohen, 1988; Leong, 2016). Especially the existential and authenticity-seeker tourists would likely to be the first ones. However, it is worth to note that, some destinations can be owed their existence to commodification (Çokişler, 2018). It is possible to see that commodify the authenticity may regenerate them and make them possible to live longer (Cohen, 1988). Even some researchers have seen authenticity as a boundary (Cobb, 2014) that restricting cultures. But can a destination, which does not see authenticity as a boundary and try to recover their corrupt culture, gain the original form of authenticity back? It is another dilemma that must be investigated with a case study, but for us, it is not seeming possible. Then, almost the same process that leads to exploration with eWoM and media channels starts to work opposite way. The miracle of consensus transforms into perception of “inauthenticity” and it brings diminishing in the number of the tourists demand which leads declining of income. At this point, locals who wait for income realize the problem finally. The recovery process can include the regenerating the products in a way that can withstand the negative effects of tourism. Because putting some standards also mean taking out naturality from the producing process and determining the attitude and creativity of locals (Ceccarini, 2014). It is even thinkable that, the genuine form of authenticity can be already From another perspective, output of these negative effects causes local producers and businesses focus back to local demand rather than tourists. 8 Rev. Anais Bras. de Est. Tur./ ABET, Juiz de Fora (Brasil), e-ISSN 2238-2925, v.11, n. único, pp.1 – 12, Jan./ Dez., 2021 AUTHENTICITY PARADOX: AN EXAMINATION ON LOCAL GASTRONOMY Gürkan Aybek & Eda Alphan that streamlined to tourists’ desire and needs”, and “products that satisfy tourists without concerning authenticity” to the literature. changed by locals before tourism development for easy using and producing for their everyday life. 5.2 Deterioration to Recovery Thus, from our point of view, some potential losses in authenticity are inevitable after a recovery process. The proposed forms also show authenticity is a changeable phenomenon and controlling this changing process in an acceptable level is a struggle for destinations (Butler, 2017). If the changing expands through deterioration, then the destination’s gastronomy will lose its attractiveness (Cohen, 1988; Leong, 2016). After that, with cultural and economic concerns the recovery of local gastronomy will put in operation (Fox, 2007; Ceccarini, 2014). 6 DISCUSSION, CONCLUSION, & FUTURE RESEARCH IDEAS The point is, sometimes, the locals’ desire to earn more income and their respond to demand takes place as changing their cultural values for tourist consumption. Although this paper has a certain contribution to the literature, it also has limitations. Due to the conceptual structure of the current research as theoretical essay, this study reflects the authors’ view on the literature. So, it lacks empirical data. Another limitation of the study originated from that when comparing the research results of the previous studies. Empirical data cannot be used due to the lack of studies in these fields. But still, this study can offer theoretical and practical implications for the field. As Cohen and Avieli (2004) already indicated, authenticity of gastronomy will eventually lose some of its aspects according to tourism development. The variation in gastronomic (Quan & Wang, 2004; Hjalager, 2004) and authentic (Özdemir & Seyitoğlu, 2017) experience seeking behaviors is the key in this loss. The study can open new doors for future research. As it is not easy to say all destinations get in this paradox, destinations which emerge with a well- planned destination management strategy must be staying out. Examining the product structure and the paradox with different destinations empirically is needed to confirm or disproof the suggestion of this research. The gastronomic tourism products’ structure in a destination has different forms according to encounter with the types of tourists. According to this, the current study offers four basic structural forms as “genuine local products”, “local products with shows and modified ingredients, techniques etc.”, “local products Otherwise, a destination can continue its tourism attractiveness even if it lost the authenticity, even some of them can be chosen by tourists thanks to their staged and/or exaggerated authentic features. These kinds of 9 9 Rev. Anais Bras. de Est. Tur./ ABET, Juiz de Fora (Brasil), e-ISSN 2238-2925, v.11, n. único, pp.1 – 12, Jan./ Dez., 2021 AUTHENTICITY PARADOX: AN EXAMINATION ON LOCAL GASTRONOMY Gürkan Aybek & Eda Alphan AUTHENTICITY PARADOX: AN EXAMINATION ON LOCAL GASTRONOMY Gürkan Aybek & Eda Alphan cases are also worth researching to reveal other sides of authenticity in the gastronomy and tourism literature. Butler, R. (2017). The tourist experience: can destinations maintain authenticity?. Worldwide Hospitality and Tourism Themes, 9(6), 617-626. doi:10.1108/WHATT- 09-2017-0044 Examining this paradox in a context of a specific destination or a gastronomic product is needed to ensure of applicability of the model. 6 DISCUSSION, CONCLUSION, & FUTURE RESEARCH IDEAS Also, previously mentioned alternative forms of authenticity after recovery process are another valuable research idea to investigate. Nonetheless, this paradox can be observed in any other types of authentic tourism products in a destination also. Other researchers who interested in this subject can adapt and advance this model according to their purpose. Ceccarini, R. (2014). Food Bureaucracy and the Making of Authentic Pizza. In R. Cobb (Ed.), The Paradox of Authenticity in a Globalized World (pp. 23-34). New York: Palgrave Macmillan. Chang, R. C., Kivela, J., & Mak, A. H. (2011). Attributes that influence the evaluation of travel dining experience: When East meets West. Tourism Management, 32, 307-316. doi:10.1016/j.tourman.2010.02.009 Cobb, R. (2014). Introduction: The Artifice of Authenticity in the Age of Digital Reproduction. In R. Cobb (Ed.), The Paradox of Authenticity in a Globalized World (1. ed., pp. 1-9). New York: Palgrave Macmillan. Practically, the study aims to create awareness among locals and decision-makers of destinations. Even local gastronomic products create present income possibilities with its authentic features, alterations that made to ensure production may cause losing the future income. Maintain balance between being tourist-oriented and local-oriented is a critic effort in this manner. Cohen, E. (1988). Authenticity and commoditization in tourism. Annals of Tourism Research, 15(3), 371-386. doi:10.1016/0160-7383(88)90028-X Cohen, E. (2007). 'Authenticity' in Tourism Studies: Aprés la Lutte. Tourism Recreation Research, 32(2), 75-82. doi:10.1080/02508281.2007.11081279 Cohen, E., & Avieli, N. (2004). 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Towards a structural model of the tourist experience: an illustration from food Çakmak, V., & Altaş, A. (2018). Sosyal Medya Etkileşiminde Tren Yolculukları: DOĞU EKSPRESİ İle İlgili Youtube 11 Rev. Anais Bras. de Est. Tur./ ABET, Juiz de Fora (Brasil), e-ISSN 2238-2925, v.11, n. único, pp.1 – 12, Jan./ Dez., 2021 Rev. Anais Bras. de Est. Tur./ ABET, Juiz de Fora (Brasil), e-ISSN 2238-2925, v.11, n. único, pp.1 – 12, Jan./ Dez., 2021 Acknowledgements To Prof. Bahattin Özdemir (PhD) for his indispensable contribution and mentorship to the realization of this research. Özdemir, B., & Seyitoğlu, F. (2017). A conceptual study of gastronomical quest of tourists: Authenticity or safety 12 12 Rev. Anais Bras. de Est. Tur./ ABET, Juiz de Fora (Brasil), e-ISSN 2238-2925, v.11, n. único, pp.1 – 12, Jan./ Dez., 2021
https://openalex.org/W4287281635	https://zenodo.org/records/4586823/files/Apriyanto%20et%20al.pdf	English	null	Water Sources on Organoleptics and Characteristics of Sago Flour	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	5,424	INTRODUCTION in the processing (Singhal et al., Sago into Sago flour is a viable alternative as reported (Ehara 2009; Jong and Widjono, Singhal et al., 2008). Metroxylon palms are distributed in not only freshwater areas but also in brackish water areas near the coast and so are considered to be salt tolerant. Moniharapon (2018) reported that saline water treatment up to EC 6 to 7 mmho/cm did not affect lea emergence in Sago palm. (Singhal et al., 2008). Processing of flour is a viable alternative as reported (Ehara 2009; Jong and Widjono, 2015; Palm et al., 2003; Metroxylon palms are distributed in not only freshwater areas but also in brackish water and so are considered to be salt tolerant. Moniharapon (2018) reported that saline water treatment up to EC 6 to 7 mmho/cm did not affect leaf in the processing (Singhal et al., Sago into Sago flour is a viable alternative as reported (Ehara 2009; Jong and Widjono, Singhal et al., 2008). Metroxylon palms are distributed in not only freshwater areas but also in brackish water areas near the coast and so are considered to be salt tolerant. Moniharapon (2018) reported that saline water treatment up to EC 6 to 7 mmho/cm did not affect lea emergence in Sago palm. (Singhal et al., 2008). Processing of flour is a viable alternative as reported (Ehara 2009; Jong and Widjono, 2015; Palm et al., 2003; Metroxylon palms are distributed in not only freshwater areas but also in brackish water and so are considered to be salt tolerant. Moniharapon (2018) reported that saline water treatment up to EC 6 to 7 mmho/cm did not affect leaf Potential Indonesia has an area of about 1,128 million hectares or 51.3% of the 2,201 million hectares of world Sago. Potential productivity of ± 30 tonnes/ha/year, far exceeding the productivity of rice 16 tonnes/ha/ (Habilla and Cheng, 2015; Jong and Widjono According to (Heryani and Silitonga, 2018; Palm et al. 2003), the largest content in Sago is carbohydrates, in 100 g of dry Sago, there is 94 to 96 g of carbohydrate that is higher than 80.4 g of rice, 71.7 potatoes 16.3 g. The total amount of starch storage in a trunk is approximately 300 kg (dry wt.) in case of palm, the elite species among the starch producing palms (Ehara, 2006). Merit Research Journal of Food Science and Technology (ISSN: 2354 Available online http://www.meritresearchjournals.org/fst/index.htm Copyright © 2021 Merit Research Journals Merit Research Journal of Food Science and Technology (ISSN: 2354-2527) Vol. 6(1) pp. 001-007, February, 2021 Available online http://www.meritresearchjournals.org/fst/index.htm , February, 2021 Merit Research Journal of Food Science and Technology (ISSN: 2354 Available online http://www.meritresearchjournals.org/fst/index.htm Copyright © 2021 Merit Research Journals Merit Research Journal of Food Science and Technology (ISSN: 2354-2527) Vol. 6(1) pp. 001-007, February, 2021 Available online http://www.meritresearchjournals.org/fst/index.htm , February, 2021 Merit Research Journal of Food Science and Technology (ISSN: 2354 Available online http://www.meritresearchjournals.org/fst/index.htm Copyright © 2021 Merit Research Journals Merit Research Journal of Food Science and Technology (ISSN: 2354-2527) Vol. 6(1) pp. 001-007, February, 2021 Available online http://www.meritresearchjournals.org/fst/index.htm , February, 2021 Copyright © 2021 Merit Research Journals Copyright © 2021 Merit Research Journals Copyright © 2021 Merit Research Journals Mulono Apriyanto1, Latarus Fangohoi Latarus Fangohoi2, Zulharman3, Dewi Farah Diba Abstract Dewi Farah Diba4, Yulianti1 2Politeknik Pembangunan Pertanian Manokwari Papua Barat 3Program Studi PGSD, STKIP Taman Siswa Bima 4Sekolah Tinggi Teknologi Kelautan, Makasar Sulawesi Selatan 2Politeknik Pembangunan Pertanian Manokwari Papua Barat 3Program Studi PGSD, STKIP Taman Siswa Bima 4Sekolah Tinggi Teknologi Kelautan, Makasar Sulawesi Selatan Corresponding Author Email: mulonoapriyanto71@gmail.com Corresponding Author Email: mulonoapriyanto71@gmail.com Keyword Sago Keyword: Aroma, Indonesian national standards (SNI), Sago Flavour Aroma, Indonesian national standards (SNI), Rainwater, River Water, Mulono Apriyanto1, Latarus Fangohoi Latarus Fangohoi2, Zulharman3, Dewi Farah Diba Abstract Dewi Farah Diba4, Yulianti1 Mulono Apriyanto1, Latarus Fangohoi Latarus Fangohoi2, Zulharman3, Dewi Farah Diba Abstract Dewi Farah Diba4, Yulianti1 1Departement Food Science, Isamic University of Indragiri, Tembilahan Hulu Indonesia The purpose of this study was to determine the best various water sources for the manufacturing process. The variable observed was sensory analysis which included organoleptic observations which included observations of color chemical properties includes exam calculation of yield. The research design used was factorial Completely Randomized Design (CRD) with 2 factors and 3 replications. The first factor is river water (S), rainwater (H) and factor is heating temperature with 3 levels, namely 130 so that they are combined into 27 experimental units. The results showed that oC produced the best treatm in accordance with SNI standards (Indonesian national standards). The purpose of this study was to determine the best various water sources for the manufacturing process. The variable observed was sensory analysis which included organoleptic observations which included observations of color and aroma. Analysis of physical and chemical properties includes examination of nutritional content and calculation of yield. The research design used was factorial Completely Randomized Design (CRD) with 2 factors and 3 replications. The first factor is river water (S), rainwater (H) and combination of S and H factor is heating temperature with 3 levels, namely 130 so that they are combined into 27 experimental units. The results showed that Sago flour using rainwater combined with a heating temperature of 140 C produced the best treatment. Treatment H140 produces in accordance with SNI standards (Indonesian national standards). The purpose of this study was to determine the best Sago starch from various water sources for the manufacturing process. The variable observed was sensory analysis which included organoleptic observations which Analysis of physical and ination of nutritional content and calculation of yield. The research design used was factorial Completely Randomized Design (CRD) with 2 factors and 3 replications. The first factor combination of S and H (Y), the second factor is heating temperature with 3 levels, namely 130oC, 140oC, and 150oC, so that they are combined into 27 experimental units. The results showed flour using rainwater combined with a heating temperature of 140 ent. Treatment H140 produces Sago flour that is in accordance with SNI standards (Indonesian national standards). INTRODUCTION Potential Indonesia has an area of about 1,128 million hectares or 51.3% of the 2,201 million hectares of world . Potential productivity of ± 30 tonnes/ha/year, far ductivity of rice 16 tonnes/ha/year Widjono, 2015). 2018; Palm et al., is carbohydrates, in 94 to 96 g of carbohydrate igher than 80.4 g of rice, 71.7g of corn and 16.3 g. The total amount of starch storage in a trunk is approximately 300 kg (dry wt.) in case of Sago palm, the elite species among the starch producing palms The resulting Sago flour has a fiber content ranging from 1% to 2%. As a requirement for industrial 1% (MS468,1976), starch granules according to SNI 01 3729-1995 standards, have a pH that meets the standards for food SIRIM MS 470: 1992 amounted to 4.5 to 6.5 and the highest yield was at 2 days immersion 26.40% and 3 days immersion time of 26.50% in the use of Bimo CF starter and the highest yield for the use of tape yeast starter was 24.75% at 1 day imm time. flour has a fiber content ranging from 1% to 2%. As a requirement for industrial Sago of 1% (MS468,1976), starch granules according to SNI 01- 1995 standards, have a pH that meets the M MS 470: 1992 amounted to 4.5 to 6.5 and the highest yield was at 2 days immersion 26.40% and 3 days immersion time of 26.50% in the use of Bimo CF starter and the highest yield for the use of tape yeast starter was 24.75% at 1 day immersion Processing of Sago by the community is still in the traditional way so that it is less hygienic when viewed from its sanitation and high water content causes its shelf life to be relatively short. Processing of Sago flour is one of the semi-finished alternatives w to extend the shelf life and reduce the use of clean water by the community is still in the traditional way so that it is less hygienic when viewed from its sanitation and high water content causes its shelf Sago into Sago finished alternatives which aims to extend the shelf life and reduce the use of clean water 002 Merit Res. J. Food Sci. Technol. 002 Merit Res. J. Food Sci. Technol. MATERIALS AND METHODS liked the most, this test was based on the panelists' scoring of color and aroma. The test used 25 untrained panelists. The assessment is given based on sensory criteria (Apriyanto et al. 2016, 2017). Physicochemical Analysis The physicochemical analysis carried out in this study included physical analysis (viscosity) using the Ostwald method (Ehara, 2009). Chemical analysis includes the calculation of yield (Ariyani et al., 2020), moisture content in the thermogravimetric method (Horwitz and George, 2005), protein content in the Biuret method (Horwitz and George W. Latimer 2005), fat content in the Soxhlet extraction method (Horwitz and George W. Latimer 2005), ash content in the thermogravimetric method (Horwitz and George W. Latimer 2005), reflux method fiber content (Horwitz and George W. Latimer 2005) and carbohydrate content by difference method (Horwitz and George W. Latimer 2005), pH (Horwitz and George W. Latimer 2005). Methods Research The raw material for Sago which is still in the form of logs of 50 to 60 cm is characterized by a proximate content analysis which aims to determine the proximate content or chemical components of the Sago material used, besides that, the proximate content analysis is also carried out on raw materials that are pressed directly without fermentation using a blender. as a comparison with the fineness of 100 mesh. The analysis carried out includes analysis of the moisture content of the material, the ash content, the protein content, the fat content, the fiber content, the starch content and the whiteness. After the raw material has been characterized, then the size is reduced by grading Organoleptic Test Recapitulation of the results of analysis of variance (ANOVA) of Sago starch products using the Heat Moisture Treatment (HMT) method. Combination of three different types of water sources on the organoleptic assessment which includes color, aroma, and texture as (100% - KA1) × Bp1 = (100% - KA2) × Bp2 (100% - KA1) × Bp1 = (100% - KA2) × Bp2 Materials The materials used in this study include wet Sago starch from Sago processing in the Gaung Anak Serka area. BatangTuaka river water, rainwater and combination of S and H. Analysis materials include biuret reagent, NaOH (technical), distilled water, H2SO4 (technical), and n- Hexan. Data analysis The amount of water the spray uses= BP2-BP1. KA1: Moisture content of starch in initial conditions, KA2: Water content of starch desired, BP1: Weight of starch in initial conditions, BP2: Weight of starch after reaching KA2. Data analysis in this study was obtained from organoleptic variables, degree of acidity (pH), degree of whiteness, water content analysis, protein content analysis, fat content analysis, and ash content analysis, fiber content analysis. Data were analyzed using analysis of variance (Analysis of Variants). The results which significantly affected the observed variables were continued with Duncan's Multiple Range Test (DMRT) at the 95% confidence level (α = 0.05). The starch which has been adjusted to 28% moisture content is then placed in a petri dish then stirred and closed. The starch is allowed to stand in the refrigerator for one night to uniform the moisture content. The pan filled with wet starch is heated in an oven with a heating time of 3 hours at a temperature of 130oC, 140oC, and 150oC. The starch is stirred every hour to uniform the heat distribution. After cooling, the modified starch was dried for 4 hours at 50oC. Research design The research design used was factorial Completely Randomized Design (CRD) with 2 factors and 3 replications. The first factor is river water (S), rainwater (H) and combination of S and H(Y), the second factor is heating temperature with 3 levels, namely 130oC, 140oC, and 150oC, so that they are combined into 27 experimental units. A total of 200 g of Sago flour which water content is adjusted to 28% by spraying the three types of water sources. For the amount of water added to achieve a water content of 28% starch, the following formula is used: (100% - KA1) × Bp1 = (100% - KA2) × Bp2 Color Based on the analysis of variance in Table 1, it shows that the organoleptic assessment of the color variable, the aroma variable, has a very significant effect on the treatment of various water sources. The texture variable only had a significant effect on treatment water sources. and HMT, while fermentation. Each pH has a very significant effect on the treatment of water sources. viscosity and yield did not significantly affect all treatments. Analysis of variance on HMT treatment of all variables had no significant effect. In the combination treatment of water sources and forages, the organoleptic assessment had a significant effect on the variable color and aroma variables. Based on the results of the assessment of the organoleptic test on Sago flour with the heat moisture treatment method combined with different water sources, it is known that the treatment of forage temperature variations showed a very significant effect on the color organoleptic assessment of the treatment of water sources and forages. The average results of the panelists' assessment of the organoleptic color of Sago flour with the heat moisture treatment method combined with different water sources and the results of the DMRT test are presented in Table 2. p Based on the data above, information is obtained that the treatment of different types of microbes has significant impact on the color or whiteness of Sago flour. The variation of heating temperature in Sago flour that has been washed with various water sources at the highest organoleptic assessment is H130, namely with water sources using rainwater and combination of S and H and heating forages with a temperature of 130oC of 3.60 and the lowest organic assessment is S130 treatment, namely by river water with a heating temperature of 130oC HMT of 3.10. Apriyanto et al. 003 Treatment codes: S (river water), H (rainwater), Y (mixture of H + S), 130 (temperature 130oC), 140 (temperature 140oC) and 150 (temperature 150oC). Note: The numbers followed by different letter notations show significant differences based on the DMRT test of 0.05 at the 95% confidence level. Treatment codes: S (river water), H (rainwater), Y (mixture of H + S), 130 (temperature 130oC), 140 (temperature 140oC) and 150 (temperature 150oC). Note: The numbers followed by different letter notations show significant differences based on the DMRT test of 0.05 at the 95% confidence level. Treatment codes: S (river water), H (rainwater), Y (mixture of H + S), 130 (temperature 130oC), 140 (temperature 140oC) and 150 (temperature 150oC). shown in Table 1. shown in Table 1. Organoleptic Tests The observation variables for the organoleptic test analysis included texture, aroma, color, and taste of Sago flour for each treatment, to determine what the panelists Apriyanto et al. 003 Apriyanto et al. 003 Apriyanto et al. 003 Table 1. Recapitulation analysis of various Sago starches using the Heat Moisture Treatment method (Forages) The combination of three different types of water sources for organoleptic assessment(color, aroma, and texture), viscosity, yield, and pH. Table 1. Recapitulation analysis of various Sago starches using the Heat Moisture Treatment method (Forages) The combination of three different types of water sources for organoleptic assessment(color, aroma, and texture), viscosity, yield, and pH. aroma, and texture), viscosity, yield, and pH. No Observation Variable Variety analysis Water sources HMT Water sources + HMT 1 Organoleptic color * tn * 2 Organoleptic aroma ** tn * 3 Organoleptic texture * tn tn 4 Viscosity tn tn tn 5 Rendement tn tn tn 6 pH tn tn Note: = Very significant effect, tn = not significant effect, * = Has significant effect, HMT = Heat Moisture Treatment Table 2. Average results of panelists' assessment of the color organoleptic Sago flour combination of water sources and HMT Treatment Average Appearance Y130 3,60a±0,17 white S130 3,50ab±0 slightly white H130 3,50ab±0 white H140 3,43ab±0,12 white S140 3,37bc±0,23 slightly white Y140 3,37bc±0,23 slightly white S150 3,30ab±0 slightly white H150 3,30bc±0 slightly white Y150 3,10c±0,17 slightly white Note: The numbers followed by different letter notations show significant differences based on the DMRT test of 0.05 at the 95% confidence level. Treatment codes: S (river water), H (rainwater), Y (mixture of H + S), 130 (temperature 130oC), 140 (temperature 140oC) and 150 (temperature 150oC). No Observation Variable Variety analysis Water sources HMT Water sources + HMT 1 Organoleptic color ** tn * 2 Organoleptic aroma ** tn * 3 Organoleptic texture * tn tn 4 Viscosity tn tn tn 5 Rendement tn tn tn 6 pH tn tn Note: = Very significant effect, tn = not significant effect, * = Has significant effect, HMT = Heat Moisture Treatment Table 2. Average results of panelists' assessment of the color organoleptic Sago flour combination of water sources and HMT Treatment Average Appearance Y130 3,60a±0,17 white S130 3,50ab±0 slightly white H130 3,50ab±0 white H140 3,43ab±0,12 white S140 3,37bc±0,23 slightly white Y140 3,37bc±0,23 slightly white S150 3,30ab±0 slightly white H150 3,30bc±0 slightly white Y150 3,10c±0,17 slightly white Note: The numbers followed by different letter notations show significant differences based on the DMRT test of 0.05 at the 95% confidence level. Rendement The results of the analysis of the yield of Sago starch resulting from several different treatments and variations in the temperature of the HMT based on the results of the analysis of variance showed that the water source treatment and heating temperature variations for HMT in Sago starch products did not significantly affect the yield in each treatment. The average number of yield of Sago flour through the washing method combined with the HMT method fermented with different water sources is presented in Table 4. The mean results of panelists assessment of the organoleptic aroma of Sago starch products combined with the HMT method are presented in Table 3. The results of the organoleptic assessment of the aroma of Sago flour which was a combination of water sources and HMT based on the results of the analysis of variance showed that the treatment of different types of water sources in the processing of Sago flour showed no significant effect on the organoleptic assessment, aroma in each treatment. Based on the data in Table 3, information was obtained that the variations in the type of water and temperature of forage in Sago starch showed significantly different results in the treatment of water sources and the combination of water sources and forages. The treatment of water sources and the combination of water sources and forages obtained Sago starch products on the organoleptic assessment of the highest aroma of the panelists' assessment in the Y130 treatment, namely a mixture of river water and rainwater and heating forages with a temperature of 130oC of 3.50 slightly smelly, the lowest was in the S130 treatment, namely river water and heating forages with a temperature of 130oC of 3.10. The change in the p Based on the data in Table 4, it is found that the highest average yield of Y 130 treatment is 66.79%, while the control is 58.94%. This shows that the highest yield in Y130 treatment is significantly different from the number of control yields, but not significantly different for each treatment. The decrease in yield value in all treatments was thought to be because the process of adding water increased the water content of Sago flour to 28% in the forage process. 004 Merit Res. J. Food Sci. Technol. 004 Merit Res. J. Food Sci. Technol. Table 3. Average results of panelists' assessment of the organoleptic aroma of combined Sago flour and forage sources. Table 3. Average results of panelists' assessment of the organoleptic aroma of combined Sago flour and forage sources. Treatment Average Appearance Y130 3,50a±0 smells S130 3,43ab±0,21 smells H130 3,40ab±0,17 It smells a little H140 3,37ab±0,06 It smells a little S140 3,37ab±0,06 smells Y140 3,33ab±0,06 smells S150 3,30bc±0,10 smells H150 3,23bc±0,15 It smells a little Y150 3,10c±0,17 smells Note: The numbers followed by different letter notations show significant differences based on the DMRT test of 0.05 at the 95% confidence level. Treatment codes: S (river water), H (rainwater), Y (mixture of H + S), 130 (temperature 130oC), 140 (temperature 140oC) and 150 (temperature 150oC). Note: The numbers followed by different letter notations show significant differences based on the DMRT test of 0.05 at the 95% confidence level. Treatment codes: S (river water), H (rainwater), Y (mixture of H + S), 130 (temperature 130oC), 140 (temperature 140oC) and 150 (temperature 150oC). Note: The numbers followed by different letter notations show significant differences based on the DMRT test of 0.05 at the 95% confidence level. Treatment codes: S (river water), H (rainwater), Y (mixture of H + S), 130 (temperature 130oC), 140 (temperature 140oC) and 150 (temperature 150oC). Decreasing preference of the panelists to the parameter of color or degree of whiteness of Sago flour and heating of the forage temperature occurred in all variations of the treatment. Reduced degree of Sago starch whiteness that occurs due to high heating temperatures in forage modification. This statement is in accordance with the research that has been done by (Ega and Lopulalan 2015), White degree is one of the physical properties that changes in the modification process, the results of observations made show that the higher the heating temperature is carried out, the white degree of the material food will decrease. It can be seen that heating the HMT Sago starch at a temperature of 150oC gives a lower whiteness value when compared to a heating temperature of 130oC. distinctive aroma of Sago flour is thought to be due to the washing process that is carried out several times, this statement is in accordance with the opinion of (Tuahta et al., 2014). 004 Merit Res. J. Food Sci. Technol. The cause of the loss of distinctive odor is influenced by clean rainwater on Sago starch where lactic acid bacteria hydrolyze starch granules to produce monosaccharides. as a raw material for producing organic acids, especially lactic acid, which will be contained in materials that cause odor changes in MOSAS. Color Based on the results of the assessment of the organoleptic test on Sago flour with the heat meanstre treatment method combined with different water sources, it is known that the treatment of HMT heating temperature variations shows the results have a very significant effect on the assessment. rit Res. J. Food Sci. Technol. Table 3. Average results of panelists' assessment of the organoleptic aroma of combined Sago flour and forage sources. Treatment Average Appearance Y130 3,50a±0 smells S130 3,43ab±0,21 smells H130 3,40ab±0,17 It smells a little H140 3,37ab±0,06 It smells a little S140 3,37ab±0,06 smells Y140 3,33ab±0,06 smells S150 3,30bc±0,10 smells H150 3,23bc±0,15 It smells a little Y150 3,10c±0,17 smells Note: The numbers followed by different letter notations show significant differences based on the DMRT test of 0.05 at the 95% confidence level. Treatment codes: S (river water), H (rainwater), Y (mixture of H + S), 130 (temperature 130oC), 140 (temperature 140oC) and 150 (temperature 150oC). 004 Merit Res. J. Food Sci. Technol. Rendement Treatment codes: S (river water), H (rainwater), Y (mixture of H + S), 130 (temperature 130oC), 140 (temperature 140oC) and 150 (temperature 150oC). . Average results of pH analysis of the combination of water and HMT Sago flour the flour becomes sticky and when dry the flour becomes hard and difficult to mash. acid in this substrate can be seen by decreasing the pH of the substrate (Ehara 2009; Ehara, MatsuI, and Naito 2006; Heryani and Silitonga, 2018). The average pH value of Sago flour that has been treated in various combination treatments with the HMT method is presented in Table 5. PH analysis The results of the analysis of the pH of Sago flour produced from several treatments and temperature variations for HMT based on the results of analysis of variance showed that the results had a very significant effect on the pH value in each treatment. Rendement In this process, it is suspected that the incubation process in the refrigerator has not been able to uniform the moisture content in the sample so that the ovening process at forage temperatures, namely 130oC, 140oC, and 150oC, gives gelatinization effect and the starch granules break. Cracked starch granules cause Apriyanto et al. 005 Apriyanto et al. 005 Table 4. Average yield of Sago flour combination of water sources and HMT Treatment Average (%) Y130 66,79±0,02 S130 66,75±0,02 H130 66,76±0,02 H140 66,77±0,02 S140 66,74±0,02 Y140 66,73±0,02 S150 66,76±0,02 H150 66,77±0,02 Y150 66,79±0,02 Note: The numbers followed by different letter notations show significant differences based on the DMRT test of 0.05 at the 95% confidence level. Treatment codes: S (river water), H (rainwater), Y (mixture of H + S), 130 (temperature 130oC), 140 (temperature 140oC) and 150 (temperature 150oC). Table 5. Average results of pH analysis of the combination of water and HMT Sago flour Treatment Average (%) Y130 7,51a±0,05 S130 7,50a±0,04 H130 7,47ab±0,06 H140 7,46ab±0.06 S140 7,43ab±0,03 Y140 7,33b±0,08 S150 6,32c±0,18 H150 6,28c±0,07 Y150 5,83d±0,07 Note: The numbers followed by different letter notations show significant differences based on the DMRT test of 0.05 at the 95% confidence level. Treatment codes: S (river water), H (rainwater), Y (mixture of H + S), 130 (temperature 130oC), 140 (temperature 140oC) and 150 (temperature 150oC). Table 4. Average yield of Sago flour combination of water sources and HMT Table 4. Average yield of Sago flour combination of water sources and HMT Table 4. Average yield of Sago flour combination of water sources and HMT Treatment Average (%) Y130 66,79±0,02 S130 66,75±0,02 H130 66,76±0,02 H140 66,77±0,02 S140 66,74±0,02 Y140 66,73±0,02 S150 66,76±0,02 H150 66,77±0,02 Y150 66,79±0,02 Note: The numbers followed by different letter notations show significant differences based on the DMRT test of 0.05 at the 95% confidence level. Treatment codes: S (river water), H (rainwater), Y (mixture of H + S), 130 (temperature 130oC), 140 (temperature 140oC) and 150 (temperature 150oC). Table 5. Average results of pH analysis of the combination of water and HMT Sago flour Treatment Average (%) Y130 7,51a±0,05 S130 7,50a±0,04 H130 7,47ab±0,06 H140 7,46ab±0.06 S140 7,43ab±0,03 Y140 7,33b±0,08 S150 6,32c±0,18 H150 6,28c±0,07 Y150 5,83d±0,07 Note: The numbers followed by different letter notations show significant differences based on the DMRT test of 0.05 at the 95% confidence level. REFERENCES 150oC for 3 hours and followed by drying for 5 hours. The decrease in protein content occurs in the foraging process, this is due to the presence of damaged protein during high heating, but the decrease in protein content is not too significant because the incubation process has occurred spontaneously fermentation, which is thought to increase the value of the protein content. Apriyanto M, Sutardi EH, Supriyanto (2016). “Fermentation Process Improvement of Cocoa Beans with Addition of Non Fermentation Inoculum of Saccharomyces Cerevisiae, Lactobacillus Lactis, and Acetobacter Aceti.” AGRITECH, 36(4): 410–15. Apriyanto M, Sutardi S, Supriyanto S, Eni H (2017). “Cocoa Beans Dry Fermentation Using Saccharomyces Cerevisiae, Lactobacillus Lactis and Acetobacter Aceti.” Agritech 37(3): 302. Lactis and Acetobacter Aceti.” Agritech 37(3): 302. The fat content that has decreased when compared to the control is also thought to be caused by high temperatures so that the fat in Sago flour is oxidized, while the decrease in ash and fiber content is thought to be due to the fermentation process carried out, according to Apriyanto et al. (2017) tape yeast consists of mold Rhizopus oryzae, Mucor), yeast (Sacharomyces cerevisiae, Sacharomyces verdomanni, Candida utilis) and bacteria (Pediococcus sp. and Bacillus sp.). from the results of research conducted by Tuahta et al. (2014) that Bacillus sp. Effective in reducing crude fiber content with an incubation time of 2 days. This is in line with the results of this study, the largest decrease in fiber content was up to 1.69% with 2 days of immersion time. And after 3 days the fiber content increases because the development of mold which consistently increases during the fermentation period can contribute crude fiber through the cell walls. And according to Moniharapon (2018) the generative time of mold is 3 to 6 days. Ariyani O, Fatma ZN, Mulono A, Veriani A (2020). “Total of Phenol and Antioxydant Activities of Rice Bran Yoghurt.” Jurnal Gizi dan Dietetik Indonesia (Indonesian J. Nutr. Dietetics) 7(2): 37–40. Ega, La, and Cynthia Gracia Christina Lopulalan. 2015. “Modifikasi Pati Sagu Dengan Metode Heat Moisture Treatment.” Agritekno: Jurnal Teknologi Pertanian 4(2): 33–40. Ehara, Hiroshi. 2009. “Potency of Sago Palm as Carbohydrate Resource for Strengthening Food Security Program.” Indonesian Journal of Agronomy 37(3): 209–19. Ehara, Hiroshi, Moe MatsuI, and Hitoshi Naito. 2006. “Avoidance Mechanism of Salt Stress in Sago Palm (Metroxylon Sagu Rottb.).” Japanese Journal of Tropical Agriculture 50(1): 36– 41. Habilla, C, and L H Cheng. 006 Merit Res. J. Food Sci. Technol. Table 6. Chemical composition of combined Sago starch and HMT No Composition Total (%) Control Y140 SNI 1 Water content 9.57 5.88 Max.13 2 Protein content 4.75 4.17 - 3 Ash content 0.09 0.12 Max.0,5 4 Fat level 0.9 0.55 - 5 Fiber content 1.5 1 0,1 6 Starch content 83.1 88.28 - Specification: Y140 (combined treatment of water source and drying temperature). Table 6. Chemical composition of combined Sago starch and HMT Specification: Y140 (combined treatment of water source and drying temperature). content, fiber content and carbohydrate content, as shown in Table 6. treatment and the HMT heating combination lowered the temperature viscosity of flour, reduced rancidity and can retain the color of Sago flour. the higher the heating temperature of the forage, the lower the viscosity value and the whiter the Sago starch produced. Based on the results of the analysis of variance in Table 6, it shows that the water content of the selected treatment is much lower than the control Sago flour, the main cause of a significant decrease in water content is due to heating the temperature of the forages using high temperatures, namely REFERENCES 2015. “Quality of Jelly Candy Made of Acid-Thinned Starch Added with Different Non-Starch Polysaccharides.” Journal of Food Research and Technology 3(1): 14–22. www.jakraya.com/journa/jfrt. Heryani, Susi, and Rhoito Frista Silitonga. 2018. “Penggunaan Tepung Sagu (Metroxylon Sp.) Asal Riau Sebagai Bahan Baku Kukis Cokelat.” Warta Industri Hasil Pertanian 34(2): 53. Horwitz, Wil liam, and Jr George W. Latimer. 2005. AOAC International Official Methods of Analysis of AOAC International. https://www.techstreet.com/standards/official-methods-of- analysis-of-aoac-international-20th-edition- 2016?product_id=1937367. Best Treatment Chemical Analysis The best treatment and control combination of modified Sago flour was based on chemical analysis, namely moisture content, protein content, ash content, fat content, fiber content, and carbohydrate content. The best treatment used in this research is Sago flour with various water sources and heating temperature of forage 140oC, while the control used is Sago flour with river water (traditionally). Recapitulation of the results of the chemical content analysis of Sago flour products with the best treatment and control of chemical properties including water content, protein content, ash content, fat p Based on the data in Table 5, it is found that the average pH of Sago flour for the best treatment is 7.51, while the control is 6.35. This shows that the pH of the best treated Sago starch is very significantly different from the control pH, by looking at the data that has been done, the results are very significant. The results of research by Majid (2015) show that with the increase in the amount of acid excreted by LAB due to the accumulation of acid in the substrate, it will increase the acidity of the substrate and increase the accumulation of 006 Merit Res. J. Food Sci. Technol. 006 Merit Res. J. Food Sci. Technol. CONCLUSION Jong, F S, Widjono, Adi. 2015. “Sagu: Potensi Besar Pertanian Indonesia.” Iptek Tanaman Pangan 2(1): 54–65. Sago flour which is produced from a combination of water and heating temperature of forage drying, namely 130oC, 140oC and 150oC, results in changes in the physical and chemical characteristics of Sago flour. The water source Moniharapon, Angcivioletta. 2018. “Pengaruh Konsentrasi Pati Sagu Termodifikasi Pada Pembuatan Permen.” Jurnal Penelitian Teknologi Industri 8(1): 51. Tuahta, Beltaser, Fajar Restuhad, and Usman Pato. 2014. “Studi Fermentasi untuk Modifikasi Pati Sagu oleh Bakteri Asam Laktat dengan Metode Perendaman.” Jom Faperta 1(2): 127–31. Apriyanto et al. 007 Palm, Sago, Cassava Starch, Corn Starch, and Ioannis S Arvanitoyannis. 2003. “Carbohydrates.” In Encyclopedia of Food Sciences and Nutrition (Second Edition). Singhal, Rekha S. et al. 2008. “Industrial Production, Processing, and Utilization of Sago Palm-Derived Products.” Carbohydrate Polymers 72(1): 1–20.
https://openalex.org/W2804584838	https://www.econstor.eu/bitstream/10419/195851/1/1025666399.pdf	English	null	On the Moments and the Distribution of Aggregate Discounted Claims in a Markovian Environment	Risks	2,018	cc-by	10,044	Article On the moments and the distribution of aggregate discounted claims in a Markovian environment Suggested Citation: Li, Shuanming; Lu, Yi (2018) : On the moments and the distribution of aggregate discounted claims in a Markovian environment, Risks, ISSN 2227-9091, MDPI, Basel, Vol. 6, Iss. 2, pp. 1-16, https://doi.org/10.3390/risks6020059 This Version is available at: https://hdl.handle.net/10419/195851 This Version is available at: https://hdl.handle.net/10419/195851 Terms of use: Die Dokumente auf EconStor dürfen zu eigenen wissenschaftlichen Zwecken und zum Privatgebrauch gespeichert und kopiert werden. Documents in EconStor may be saved and copied for your personal and scholarly purposes. Sie dürfen die Dokumente nicht für öffentliche oder kommerzielle Zwecke vervielfältigen, öffentlich ausstellen, öffentlich zugänglich machen, vertreiben oder anderweitig nutzen. You are not to copy documents for public or commercial purposes, to exhibit the documents publicly, to make them publicly available on the internet, or to distribute or otherwise use the documents in public. If the documents have been made available under an Open Content Licence (especially Creative Commons Licences), you may exercise further usage rights as specified in the indicated licence. Sofern die Verfasser die Dokumente unter Open-Content-Lizenzen (insbesondere CC-Lizenzen) zur Verfügung gestellt haben sollten, gelten abweichend von diesen Nutzungsbedingungen die in der dort genannten Lizenz gewährten Nutzungsrechte. https://creativecommons.org/licenses/by/4.0/ https://creativecommo Received: 7 May 2018; Accepted: 21 May 2018; Published: 23 May 2018 Received: 7 May 2018; Accepted: 21 May 2018; Published: 23 May 2018 Abstract: This paper studies the moments and the distribution of the aggregate discounted claims (ADCs) in a Markovian environment, where the claim arrivals, claim amounts, and forces of interest (for discounting) are inﬂuenced by an underlying Markov process. Speciﬁcally, we assume that claims occur according to a Markovian arrival process (MAP). The paper shows that the vector of joint Laplace transforms of the ADC occurring in each state of the environment process by any speciﬁc time satisﬁes a matrix-form ﬁrst-order partial differential equation, through which a recursive formula is derived for the moments of the ADC occurring in certain states (a subset). We also study two types of covariances of the ADC occurring in any two subsets of the state space and with two different time lengths. The distribution of the ADC occurring in certain states by any speciﬁc time is also investigated. Numerical results are also presented for a two-state Markov-modulated model case. Keywords: aggregate discounted claims; Markovian arrival process; partial integro-differential equation; covariance Shuanming Li 1,* and Yi Lu 2 1 Centre for Actuarial Studies, Department of Economics, The University of Melbourne, Melbourne 3010, Australia 2 Department of Statistics and Actuarial Science, Simon Fraser University, Burnaby, BC V5A 1S6, Canada; yilu@sfu.ca 1 Centre for Actuarial Studies, Department of Economics, The University of Melbourne, 1 Centre for Actuarial Studies, Department of Economics, The University of Melbourne, Melbourne 3010, Australia 2 Department of Statistics and Actuarial Science, Simon Fraser University, Burnaby, BC V5A 1S6, Canada; yilu@sfu.ca * Correspondence: shli@unimelb.edu.au risks risks 1. Introduction Consider a line of business or an insurance portfolio to be insured by a property and casualty insurance company. Suppose that random claims arrive in the future according to a counting process, denoted by {N(t)}t≥0, i.e., N(t) is the random number of claims up to time t. Assume that {Tn}n≥1 is a sequence of random claim occurrence times and {Xn}n≥1 is a sequence of corresponding random positive claim amounts (also called claim severities), and δ(t) is the force of interest at time t, which is modeled by a stochastic process. Then S(t) deﬁned by S(t) = N(t) ∑ n=1 Xne−R Tn 0 δ(s)ds , t ≥0 (1) (1) is the aggregate discounted claims (ADCs) up to certain time t, or the present value of the total amounts paid out by the company up to time t, which describes the random change over time of the insurer’s future liabilities at present time. Accordingly, {S(t)}t≥0 is the ADC process (compound discounted claims) for this business. At a ﬁxed time t, the randomness of S(t) comes from the number of claims up to time t, claim occurrence times, and corresponding sizes as well as the values of δ(s), 0 ≤s ≤t. It is an important quantity in the sense that, at the time of issue (t = 0), this quantity would help insurers set a premium for this particular line of business, and predict and manage their future liabilities. A simple case of model (1) is one in which the counting process {N(t)}t≥0 is a homogeneous Poisson process, independent of claim amounts, and the force of interest is deterministic. In this Risks 2018, 6, 59; doi:10.3390/risks6020059 www.mdpi.com/journal/risks www.mdpi.com/journal/risks 2 of 16 Risks 2018, 6, 59 paper, we assume that the counting process {N(t)}t≥0 is a Markovian arrival process (MAP) with representation (γ, D0, D1), introduced by Neuts (1979). That is, claim arrivals are inﬂuenced by an underlying continuous-time Markov process {J(t)}t≥0 on state space E = {1, 2, . . . , m} with an m × m intensity matrix D and initial distribution γ, where D = D0 + D1 = d0,ij + d1,ij , and is assumed to be irreducible. 1. Introduction Precisely, d0,ij represents the intensity of transitions from state i to state j without claim arrivals, while d1,ij(≥0) represents the intensity of transitions from state i to state j with an accompanying claim, having a cumulative distribution function Fi, density function fi, k-th moment µ(k) i , and Laplace transform ˆfi(s) = R ∞ 0 e−sx fi(x)dx. Here, the process {J(t)}t≥0 models the random environment, which affects the frequency and the severity of claims and thus the insurance business; for example, it is well known that the weather or climate conditions have impacts on automobile, property and casualty insurance claims. Moreover, we assume that the force of interest process {δ(t)}t≥0 in (1) is also governed by the same Markov process {J(t)}t≥0 and is assumed constant while staying at certain state, that is, when J(t) = i, δ(t) = δi(> 0), for all i ∈E. As the force of interest used for evaluation is mainly driven by the local or global economics conditions, we would reasonably model its random ﬂuctuations by a stochastic process that is different from {J(t)}t≥0. Technically, we can assume a two-dimensional Markov process as the environment or background process and other mathematical treatments would be the same as we do below. Hence, we make the above assumption in this paper to simplify notations and presentations. We note that studies of the inﬂuence of economic conditions such as interest and inﬂation on the classical risk theory can be found in papers by Taylor (1979), Delbaen and Haezendonck (1987), Willmot (1989), and Garrido and Léveill (2004). The MAP has received considerable attention in recent decades due to its versatility and feasibility in modeling stochastic insurance claims dynamics. MAPs include Poisson processes, renewal processes with the inter-arrival times following phase-type distributions, and Markov-modulated Poisson processes as special cases, which are intensively studied in actuarial science literature. Detailed characteristics and properties of MAPs can be found in papers by Neuts (1979) and Asmussen (2003). Below, we present a brief literature review on the related work based on models given by Equation (1) (including its special cases). g p Most of the studies on model (1) are under the assumption that {δ(t)}t≥0 is deterministic. 1. Introduction For the ADC, Léveillé and Garrido (2001a) give explicit expressions for its ﬁrst two moments in the compound renewal risk process by using renewal theory arguments, while Léveillé and Garrido (2001b) further derive a recursive formula for the moments calculation. Léveillé et al. (2010) study the moment generating function (mgf) of the ADC by ﬁnite and inﬁnite time under a renewal risk model or a delayed renewal risk model. Recently, Wang et al. (2018) studied the distribution of discounted compound phase-type renewal sums using the analytical results of their mgf obtained by Léveillé et al. (2010). Jang (2004) obtains the Laplace transform of the distribution of the ADC using a shot noise process. Woo and Cheung (2013) derive recursive formulas for the moments of the ADC using techniques used by Léveillé and Garrido (2001b), for a renewal risk process with certain dependence between the claim arrival and the amount caused. The impact of the dependency on the ADC are illustrated numerically. Kim and Kim (2007) derive simple expressions for the ﬁrst two moments of the ADC when the rates of claim arrivals and the claim sizes depend on the states of an underlying Markov process. Ren (2008) studies the Laplace transform and the ﬁrst two moments of the ADC following a MAP process, and Li (2008) further derives a recursive formula for the moments of the discounted claims for the same model. Barges et al. (2011) study the moments of the ADC in a compound Poisson model with dependence introduced by a Farlie–Gumbel–Morgenstern (FGM) copula; Mohd Ramli and Jang (2014) further derive Neumann series expression of the recursive moments by using the method of successive approximation. y g pp There are few papers that study models described by Equation (1) with a stochastic process {δ(t)}t≥0 in the literature of actuarial science. Leveille and Adekambi (2011, 2012) study the covariance and the joint moments of the discounted compound renewal sum at two different times Risks 2018, 6, 59 3 of 16 with a stochastic interest rate where the Ho–Lee–Merton and the Vasicek interest rate models are considered. Their idea of studying the covariance and the joint moments is adopted and extended in this paper. 1. Introduction Here, we assume that the components of the ADC process {S(t)}t≥0 described by Equation (1)—the number of claims, the size of the claims, and the force of interest for discounting—are all inﬂuenced by the same Markovian environment process, which enhances the ﬂexibility of the model parameter settings. It follows that S(t) depends on the trajectory of this underlying process whose states may represent different external conditions or circumstances that affect insurance claims. The main objective of this paper is to study the moments and the distribution of S(t) given in Equation (1), occurring in certain states (e.g., certain conditions) by time t. In general, while the expectation of S(t) at any given time t can be used as a reference for the insurer’s liability, the higher moments of S(t), describing further characteristics of the random variable such as the variability around the mean and how extreme outcomes could go, may be used to determine the marginals on reserves. Furthermore, the distributional results regarding S(t) would be useful for obtaining the risk measures such as the value at risk and the conditional tail expectation, which may help insurers prevent or minimize their losses from extreme cases. p p Our work is basically a generalization of some aforementioned studies. We ﬁrst obtain formulas for calculating mean, variance, and distribution of the ADC occurring in a subset of states at a certain time. The subset may represent a collection of similar conditions that the insurer would consider them as a whole. We then derive explicit matrix-analytic expressions for covariances of the ADC occurring in two subsets of the state space at a certain time and those occurring in a certain subset of states with two different time lengths. The motivation of studying these two types of covariance is that we believe they can reveal the correlation between the random discounted sums either between different underlying conditions or with different time lengths, and the information would be helpful for insurers to set their capital requirements for preventing future losses, and make strategic and contingency plans. Moreover, we obtain a matrix-form partial integro-differential equation satisﬁed by the distribution function of the ADC occurring in certain subset of states. The equation can be solved numerically to obtain the probability distribution function of the ADC, which again could be useful for measuring insurers’ risks of insolvency. The rest of the paper is organized as follows. 1. Introduction In Section 2, we study the joint Laplace transforms of the ADC occurring in each state by time t and pay attention to some special cases. Recursive formulas for calculating the moments of the ADC occurring in certain states are obtained. A formula for computing the covariance of the ADC occurring in two subsets of the state space is derived in Section 3, while the covariance of the ADC occurring in certain states with two different time lengths is studied in Section 4. The distribution of the ADC occurring in certain states is investigated in Section 5. Finally, some numerical illustrations are presented in Section 6. 2. The Laplace Transforms and Moments We ﬁrst decompose S(t) into m components as S(t) = m ∑ j=1 Sj(t) S(t) = m ∑ j=1 Sj(t) where where where Sj(t) = N(t) ∑ n=1 XnI(J(Tn) = j)e−R Tn 0 δ(s)ds Sj(t) = N(t) ∑ n=1 XnI(J(Tn) = j)e−R Tn 0 δ(s)ds is the ADC occurring in state j ∈E, with I(·) being the indicator function. For a given k(1 ≤k ≤m), 1 ≤l1 < l2 < . . . < lk ≤m denote Ek = {l1, l2, . . . , lk} ⊆E, a sub-state space of E. We then deﬁne is the ADC occurring in state j ∈E, with I(·) being the indicator function. For a given k(1 ≤k ≤m), 1 ≤l1 < l2 < . . . < lk ≤m denote Ek = {l1, l2, . . . , lk} ⊆E, a sub-state space of E. We then deﬁne SEk(t) = ∑ j∈Ek Sj(t) SEk(t) = ∑ j∈Ek Sj(t) 4 of 16 Risks 2018, 6, 59 to be the ADC occurring in the subset of state space Ek. In particular, SE(t) = S(t) and S{j}(t) = Sj(t). If δ(t) = 0 and Xi ≡1 for all i ∈N+, then SEk(t) = NEk(t), where NEk(t) is the number of claims occurring in the sub-state space Ek by time t. to be the ADC occurring in the subset of state space Ek. In particular, SE(t) = S(t) and S{j}(t) = Sj(t). If δ(t) = 0 and Xi ≡1 for all i ∈N+, then SEk(t) = NEk(t), where NEk(t) is the number of claims occurring in the sub-state space Ek by time t. g p y Let Pi and Ei denote conditional probability and conditional expectation given J(0) = i, respectively. Deﬁne iL(ξ1, ξ2, . . . , ξm; t) = Ei h e−∑m j=1 ξjSj(t)i , ξj ≥0, t ≥0, i ∈E (2) (2) to be the joint Laplace transform of S1(t), S2(t), . . . , Sm(t), given that the initial state is i. In particular, we have iL(ξ; t) = Ei h e−ξS(t)i = iL(ξ, ξ, . . . , ξ; t) iLEk(ξ; t) = Ei h e−ξSEk (t)i = iL(ξ1, ξ2, . . . , ξm; t) ξj=ξI(j=ln),n=1,2,...,k iLj(ξj; t) = Ei h e−ξjSj(t)i = iL(ξ1, ξ2, . . . , ξm; t) ξk=0,k̸=j . 2. The Laplace Transforms and Moments We deﬁne, for n ∈N+, the n-th moment of S(t), Sj(t), and SEk(t), respectively, as iV(n)(t) = Ei [Sn(t)] , i ∈E iV(n) j (t) = Ei h Sn j (t) i , i, j ∈E iV(n) Ek (t) = Ei h Sn Ek(t) i , 1 ≤k ≤m given that the initial state is i. g We write the following column vectors for the Laplace transforms We write the following column vectors for the Laplace transforms L(ξ1, ξ2, . . . , ξm; t) = 1L(ξ1, ξ2, . . . , ξm; t), . . . , mL(ξ1, ξ2, . . . , ξm; t) ⊤ L(ξ; t) = 1L(ξ; t), 2L(ξ; t), . . . , mL(ξ; t) ⊤ LEk(ξ; t) = 1LEk(ξ; t), 2LEk(ξ; t), . . . , mLEk(ξ; t) ⊤ Lj(ξj; t) = 1Lj(ξj; t), 2Lj(ξj; t), . . . , mLj(ξj; t) ⊤, with L(0; t) = LEk(0; t) = Lj(0; t) = 1 = (1, 1, . . . , 1)⊤. with L(0; t) = LEk(0; t) = Lj(0; t) = 1 = (1, 1, . . . , 1)⊤. j In this section, we ﬁrst show that L(ξ1, ξ2, . . . , ξm; t) satisﬁes a matrix-form ﬁrst-order partial differential equation, and derive recursive formulas for calculating the moments of various ADC depending on the initial state of the underlying Markovian process. We also consider some special cases. Theorem 1. L(ξ1, ξ2, . . . , ξm; t) satisﬁes ∂L(ξ1, ξ2, . . . , ξm; t) ∂t + δ m ∑ j=1 ξj ∂L(ξ1, ξ2, . . . , ξm; t) ∂ξj = D0L(ξ1, ξ2, . . . , ξm; t) + ˆf(ξ1, ξ2, . . . , ξm)D1 L(ξ1, ξ2, . . . , ξm; t) (3) where δ = diag(δ1, δ2, . . . , δm) and ˆf(ξ1, ξ2, . . . , ξm) = diag ˆf1(ξ1), ˆf2(ξ2), . . . , ˆfm(ξm) . ∂L(ξ1, ξ2, . . . , ξm; t) ∂t + δ m ∑ j=1 ξj ∂L(ξ1, ξ2, . . . , ξm; t) ∂ξj (3) (3) = D0L(ξ1, ξ2, . . . , ξm; t) + ˆf(ξ1, ξ2, . . . , ξm)D1 L(ξ1, ξ2, . . . , ξm; t) where δ = diag(δ1, δ2, . . . , δm) and ˆf(ξ1, ξ2, . . . 2. The Laplace Transforms and Moments , ξm) = diag ˆf1(ξ1), ˆf2(ξ2), . . . , ˆfm(ξm) . = D0L(ξ1, ξ2, . . . , ξm; t) + ˆf(ξ1, ξ2, . . . , ξm)D1 L(ξ1, ξ2, . . . , ξm; t) where δ = diag(δ1, δ2, . . . , δm) and ˆf(ξ1, ξ2, . . . , ξm) = diag ˆf1(ξ1), ˆf2(ξ2), . . . , ˆfm(ξm) . Risks 2018, 6, 59 5 of 16 Proof. For an inﬁnitesimal h > 0, conditioning on three possible events which can occur in [0, h]—no change in the MAP phase (state), a change in the MAP phase accompanied by no claims, and a change in the MAP phase accompanied by a claim—we have Proof. For an inﬁnitesimal h > 0, conditioning on three possible events which can occur in [0, h]—no change in the MAP phase (state), a change in the MAP phase accompanied by no claims, and a change in the MAP phase accompanied by a claim—we have iL(ξ1, ξ2, . . . , ξm; t) = [1 + d0,iih] iL ξ1e−δih, ξ2e−δih, . . . , ξme−δih ; t −h + m ∑ k=1,k̸=i d0,ikh kL ξ1e−δih, ξ2e−δih, . . . , ξme−δih ; t −h + m ∑ k=1 d1,ikh ˆfi ξie−δih kL ξ1e−δih, ξ2e−δih, . . . , ξme−δih ; t −h . (4) (4) As iL(ξ1, ξ2, . . . , ξm; t) is differentiable with respect to ξi (i ∈E) and t (the differentiability of iL with respect to t is justiﬁed in Appendix), we have As iL(ξ1, ξ2, . . . , ξm; t) is differentiable with respect to ξi (i ∈E) and t (the differentiability of iL with respect to t is justiﬁed in Appendix), we have iL ξ1e−δih, ξ2e−δih, . . . , ξme−δih ; t −h = iL(ξ1, ξ2, . . . , ξm; t) −h∂iL(ξ1, ξ2, . . . , ξm; t) ∂t −δih m ∑ l=1 ξl ∂iL(ξ1, ξ2, . . . , ξm; t) ∂ξl + o(h) (5) (5) where limh→0(o(h)/h) = 0. Substituting the expression above into Equation (4), dividing both sides by h, and letting h →0, we have where limh→0(o(h)/h) = 0. Substituting the expression above into Equation (4), dividing both sides by h, and letting h →0, we have δi m ∑ l=1 ξl ∂iL(ξ1, ξ2, . . . 2. The Laplace Transforms and Moments , ξm; t) ∂ξl + ∂iL(ξ1, ξ2, . . . , ξm; t) ∂t = m ∑ k=1 d0,ik kL(ξ1, ξ2, . . . , ξm; t) + m ∑ k=1 d1,ik ˆfi ξi kL(ξ1, ξ2, . . . , ξm; t) . (6) (6) Rewriting Equation (6) in matrix form gives Equation (3). Rewriting Equation (6) in matrix form gives Equation (3). Remark 1. Using the same argument, we have the follow results. Remark 1. Using the same argument, we have the follow results. Remark 1. Using the same argument, we have the follow results. (1) LEk(ξ; t) satisﬁes the following matrix-form ﬁrst-order partial differential equation: (1) LEk(ξ; t) satisﬁes the following matrix-form ﬁrst-order partial differential equation: ∂LEk(ξ; t) ∂t + δξ ∂LEk(ξ; t) ∂ξ = D0LEk(ξ; t) + ˆfEk(ξ)D1LEk(ξ; t) (7) (7) where ˆfEk(ξ) is an m × m diagonal matrix with the li-th entry being ˆfli(ξ), for i = 1, 2, . . . , k and all other entries being 1. where ˆfEk(ξ) is an m × m diagonal matrix with the li-th entry being ˆfli(ξ), for i = 1, 2, . . . , k and all other entries being 1. (2) L(ξ; t) satisﬁes (2) L(ξ; t) satisﬁes ∂L(ξ; t) ∂t + δξ ∂L(ξ; t) ∂ξ = D0L(ξ; t) + ˆf(ξ)D1L(ξ; t) where ˆf(ξ) = diag ˆf1(ξ), ˆf2(ξ), . . . , ˆfm(ξ) . ( ) g (3) Lj(ξj; t) satisﬁes g f f f (3) Lj(ξj; t) satisﬁes ∂Lj(ξj; t) ∂t + δξj ∂Lj(ξj; t) ∂ξj = D0Lj(ξj; t) + ˆfj(ξj)D1Lj(ξj; t) (3) Lj(ξj; t) satisﬁes ∂Lj(ξj; t) ∂t + δξj ∂Lj(ξj; t) ∂ξj = D0Lj(ξj; t) + ˆfj(ξj)D1Lj(ξj; t) where ˆfj(ξj) = diag 1, 1, . . . , ˆfj(ξj), 1, . . . , 1 . where ˆfj(ξj) = diag 1, 1, . . . , ˆfj(ξj), 1, . . . , 1 . Risks 2018, 6, 59 6 of 16 We now study the moments of the ADC considered in Theorem 1. Denote the vectors of the n-th moment of the corresponding ADC as Vn(t) = 1V(n)(t), 2V(n)(t), . . . , mV(n)(t) ⊤ Vn,Ek(t) = 1V(n) Ek (t), 2V(n) Ek (t), . . . , mV(n) Ek (t) ⊤ Vn,j(t) = 1V(n) j (t), 2V(n) j (t), . . . , mV(n) j (t) ⊤. 2. The Laplace Transforms and Moments From Equation (7), we obtain in Theorem 2 a matrix-form ﬁrst-order differential equation satisﬁed by the moments of SEk(t), Vn,Ek(t) and then, in Theorem 3, obtain recursive formulas for calculating them. From Equation (7), we obtain in Theorem 2 a matrix-form ﬁrst-order differential equation satisﬁed by the moments of SEk(t), Vn,Ek(t) and then, in Theorem 3, obtain recursive formulas for calculating them. Theorem 2. The moments of SEk(t) satisfy Theorem 2. The moments of SEk(t) satisfy V′ n,Ek(t) + nδ −D0 −D1 Vn,Ek(t) = n ∑ r=1 n r IEk µr D1 Vn−r,Ek(t) , n ∈N+, (8) (8) with initial conditions Vn,Ek(0) = 0 and V0,Ek(t) = 1. In particular, V′ 1,Ek(t) + δ −D0 −D1 V1,Ek(t) = IEkµ1D11 , t ≥0 V′ 1,Ek(t) + δ −D0 −D1 V1,Ek(t) = IEkµ1D11 , t ≥0 V′ 1,Ek(t) + δ −D0 −D1 V1,Ek(t) = IEkµ1D11 , t ≥0 where µr = diag µ(r) 1 , µ(r) 2 , . . . , µ(r) m , IEk is an m × m diagonal matrix with the li-th entry being 1, for i = 1, 2, . . . , k, and all other diagonal entries being 0. where µr = diag µ(r) 1 , µ(r) 2 , . . . , µ(r) m , IEk is an m × m diagonal matrix with the li-th entry being 1, for i = 1, 2, . . . , k, and all other diagonal entries being 0. Proof. By Taylor’s expansion (its existence is easily justiﬁed as we assume that fi has moment µ(n) i for any n ∈N+), we have Proof. By Taylor’s expansion (its existence is easily justiﬁed as we assume that fi has moment µ(n) i for any n ∈N+), we have ˆfi(ξ) = 1 + ∞ ∑ n=1 (−1)nξn n! µ(n) i . In matrix notation, In matrix notation, In matrix notation, In matrix notation, ˆfEk(ξ) = 1 + ∞ ∑ n=1 (−1)nξn n! IEkµn . (9) ˆfEk(ξ) = 1 + ∞ ∑ n=1 (−1)nξn n! IEkµn . (9) (9) Substituting Equation (9) together with Substituting Equation (9) together with Substituting Equation (9) together with LEk(ξ; t) = ∞ ∑ n=0 (−1)nξn n! Vn,Ek(t) LEk(ξ; t) = ∞ ∑ n=0 (−1)nξn n! Vn,Ek(t) into Equation (7) and equating the coefﬁcients of ξn give Equation (8). Corollary 1. 2. The Laplace Transforms and Moments If we set Ek = E and Ek = {j} in Theorem 3, we have the following recursive formulas for the moments of S(t) and Sj(t): Vn(t) = n ∑ k=1 n k Z t 0 e− n δ−(D0+D1) xµkD1Vn−k(t −x)dx Vn,j(t) = n ∑ k=1 n k Z t 0 e− n δ−(D0+D1) x IjµkD1Vn−k,j(t −x)dx . In particular, In particular, V1(t) = [δ −(D0 + D1)]−1 h I −e−[δ−(D0+D1)]ti µ1D11 V1,j(t) = [δ −(D0 + D1)]−1 h I −e−[δ−(D0+D1)]ti Ijµ1D11 . Remark 2. When t →∞, we have the following asymptotic results for the moments of the ADC for n ∈N+: Remark 2. When t →∞, we have the following asymptotic results for the moments of the ADC for n ∈N+: Vn,Ek(∞) = [nδ −(D0 + D1)]−1 n ∑ r=1 n r IEkµrD1Vn−r,Ek(∞) Vn(∞) = [nδ −(D0 + D1)]−1 n ∑ r=1 n r µr D1Vn−r(∞) Vn,j(∞) = [nδ −(D0 + D1)]−1 n ∑ r=1 n r Ij µr D1Vn−r,j(∞) where V0,Ek(∞) = V0(∞) = V0,j(∞) = 1. where V0,Ek(∞) = V0(∞) = V0,j(∞) = 1. 2. The Laplace Transforms and Moments We have the following results for the moments of S(t) and Sj(t). (i) Vn(t) satisﬁes the matrix-form ﬁrst-order differential equation: V′ n(t) + nδ −D0 −D1 Vn(t) = n ∑ r=1 n r µrD1 Vn−r(t) , n ∈N+ where Vn(0) = 0 and V0(t) = 1. In particular, V1(t) satisﬁes where Vn(0) = 0 and V0(t) = 1. In particular, V1(t) satisﬁes V′ 1(t) + δ −D0 −D1 V1(t) = µ1D11 , t ≥0. V′ 1(t) + δ −D0 −D1 V1(t) = µ1D11 , t ≥0. V′ 1(t) + δ −D0 −D1 V1(t) = µ1D11 , t ≥0. (ii) Vn,j(t) satisﬁes (ii) Vn,j(t) satisﬁes (ii) V′ n,j(t) + nδ −D0 −D1 Vn,j(t) = n ∑ r=1 n r IjµrD1 Vn−r,j(t) 7 of 16 Risks 2018, 6, 59 where Ij = I{j} is a diagonal matrix with the j-th entry being 1, and 0 otherwise, Vn,j(0) = 0 and V0,j(t) = 1. In particular, V1,j(t) satisﬁes where Ij = I{j} is a diagonal matrix with the j-th entry being 1, and 0 otherwise, Vn,j(0) = 0 and V0,j(t) = 1. In particular, V1,j(t) satisﬁes V′ 1,j(t) + δ −D0 −D1 V1,j(t) = Ijµ1D11 , t ≥0. Solving differential Equation (8) with Vn,Ek(0) = 0, we obtain the following recursive formulas for Vn,Ek(t). Solving differential Equation (8) with Vn,Ek(0) = 0, we obtain the following recursive formulas for Vn,Ek(t). Theorem 3. For t > 0 and n ∈N+, we have Vn,Ek(t) = n ∑ r=1 n r Z t 0 e− n δ−(D0+D1) x IEk µrD1Vn−r,Ek(t −x)dx . In particular, ar, V1,Ek(t) = Z t 0 e− δ−(D0+D1) xdx IEk µ1 D11 = [δ −(D0 + D1)]−1 h I −e−[δ−(D0+D1)]ti IEkµ1 D11 . (10) rly, we have V1,Ek(t) + V1,Ec k(t) = V1(t), where Ec k = E \ Ek. V1,Ek(t) = Z t 0 e− δ−(D0+D1) xdx IEk µ1 D11 = [δ −(D0 + D1)]−1 h I −e−[δ−(D0+D1)]ti IEkµ1 D11 . (10) (10) Clearly, we have V1,Ek(t) + V1,Ec k(t) = V1(t), where Ec k = E \ Ek. Corollary 2. If we set Ek = E and Ek = {j} in Theorem 3, we have the following recursive formulas for the moments of S(t) and Sj(t): Corollary 2. 3. The Covariance of ADC Occurring in Two Sub-State Spaces In this section, we ﬁrst calculate the joint moment of the ADC occurring in two subsets of the state space and then the covariance between them could be calculated. Risks 2018, 6, 59 Risks 2018, 6, 59 8 of 16 For 1 ≤l1 < l2 < . . . < lk ≤m and 1 ≤n1 < n2 < . . . < nj ≤m, where 2 ≤k + j ≤m, denote Ek = {l1, l2, . . . , lk} and Ej = {n1, n2, . . . , nj} to be two disjoint subsets of E, i.e., Ek ∩Ej = ∅. The aggregate discounted claim amounts occurring in Ek and Ej are SEk(t) = ∑ i∈Ek Si(t), SEj(t) = ∑ i∈Ej Si(t) . Deﬁne Deﬁne iLEk,Ej(ξk, ξj; t) = Ei h e −ξkSEk (t)−ξjSEj(t)i to be the joint Laplace transform of SEk(t) and SEj(t). Let LEk,Ej(ξk, ξj; t) be a column vector with the i-th entry being iLEk,Ej(ξk, ξj; t). Moreover, let to be the joint Laplace transform of SEk(t) and SEj(t). Let LEk,Ej(ξk, ξj; t) be a column vector with the i-th entry being iLEk,Ej(ξk, ξj; t). Moreover, let iVEk,Ej(t) = Ei h SEk(t)SEj(t) i be the joint moment of SEk(t) and SEj(t). Denote VEk,Ej(t) as an m × 1 column vector with the i-th entry being iVEk,Ej(t). A matrix-form integral expression of VEk,Ej(t) and its asymptotic formula when t →∞are presented in the theorem below. Theorem 4. For two disjoint subsets of E, Ek and Ej, the joint moment of SEk(t) and SEj(t) satisﬁes Theorem 4. For two disjoint subsets of E, Ek and Ej, the joint moment of SEk(t) and SEj(t) satisﬁes VEk,Ej(t) = Z t 0 e− 2δ−(D0+D1) x IEk µ1 D1V1,Ej(t −x)dx + Z t 0 e− 2δ−(D0+D1) x IEj µ1 D1V1,Ek(t −x)dx (11) (11) where V1,Ek(t) is given by Equation (10) in Theorem 3. When t →∞, we have VEk,Ej(∞) = 2δ −(D0 + D1) −1h IEk µ1D1V1,Ej(∞) + IEjµ1 D1V1,Ek(∞) i . (12) VEk,Ej(∞) = 2δ −(D0 + D1) −1h IEk µ1D1V1,Ej(∞) + IEjµ1 D1V1,Ek(∞) i . (12) (12) Proof. Following from Equation (3), we have Proof. 3. The Covariance of ADC Occurring in Two Sub-State Spaces Following from Equation (3), we have ∂LEk,Ej(ξk, ξj; t) ∂t + δξk ∂LEk,Ej(ξk, ξj; t) ∂ξk + δξj ∂LEk,Ej(ξk, ξj; t) ∂ξj = D0LEk,Ej(ξk, ξj; t) + ˆfEk,Ej(ξk, ξj)D1 LEk,Ej(ξk, ξj; t) (13) (13) where ˆfEk,Ej(ξk, ξj) is a diagonal matrix with the li-th entry being ˆfli(ξk), for i = 1, 2, . . . , k, with the ni-th entry being ˆfni(ξj), for i = 1, 2, . . . , j, and all other elements being 1. where ˆfEk,Ej(ξk, ξj) is a diagonal matrix with the li-th entry being ˆfli(ξk), for i = 1, 2, . . . , k, with the ni-th entry being ˆfni(ξj), for i = 1, 2, . . . , j, and all other elements being 1. j Taking partial derivatives with respect to ξk and ξj on both sides of Equation (13), setting ξk = 0 and ξj = 0, and noting that iVEk,Ej(t) = ∂2 iLEk,Ej(ξk, ξj; t) ∂ξk∂ξj ξk=ξj=0 , iVEk,Ej(t) = ∂2 iLEk,Ej(ξk, ξj; t) ∂ξk∂ξj ξk=ξj=0 , we obtain the following matrix-form ﬁrst-order differential equation for VEk,Ej(t): we obtain the following matrix-form ﬁrst-order differential equation for VEk,Ej(t): V ′ Ek,Ej(t) + [2δ −D0 −D1] VEk,Ej(t) = IEk µ1D1V1,Ej(t) + IEj µ1 D1V1,Ek(t) . Solving it gives Equation (11). Solving it gives Equation (11). Solving it gives Equation (11). Letting t →∞in Equation (11), we obtain expression (12) for the joint moment of SEk(∞) and SEj(∞). g g q Letting t →∞in Equation (11), we obtain expression (12) for the joint moment of SEk(∞) and SEj(∞). 9 of 16 Risks 2018, 6, 59 Remark 3. If Ek = {k} and Ej = {j}, and k ̸= j, we have Remark 3. If Ek = {k} and Ej = {j}, and k ̸= j, we have V{k},{j}(t) = Z t 0 e− 2δ−(D0+D1) x Ijµ1D1V1,k(t −x)dx + Z t 0 e− 2δ−(D0+D1) xIk µ1D1V1,j(t −x)dx . When t →∞, the joint moment of Sk(∞) and Sj(∞) can be expressed as When t →∞, the joint moment of Sk(∞) and Sj(∞) can be expressed as V{k},{j}(∞) = 2δ −(D0 + D1) −1 Ij µ1D1V1,k(∞) + Ik µ1 D1V1,j(∞) . Remark 4. 3. The Covariance of ADC Occurring in Two Sub-State Spaces If two subsets Ek and Ej are not disjoint, i.e., Ek ∩Ej = Ekj ̸= ∅, then Covi SEk(t), SEj(t) = Covi SEk\Ekj(t) + SEkj(t), SEj\Ekj(t) + SEkj(t) = Covi SEk\Ekj(t), SEkj(t) + Covi SEj\Ekj(t), SEkj(t) + Covi SEk\Ekj(t), SEj\Ekj(t) + Vari SEkj(t) . All the covariance terms in the expression above are for ADCs occurring in two disjoint sets. All the covariance terms in the expression above are for ADCs occurring in two disjoint sets. All the covariance terms in the expression above are for ADCs occurring in two disjoint sets. 4. The Covariance of the ADC with Two Different Time Lengths In this section, we investigate the covariance of the ADCs occurring in two (overlapped) time periods, i.e., we want to evaluate Covi(SEk(t), SEk(t + h)) ≜Cov(SEk(t), SEk(t + h) J(0) = i) = Ei SEk(t)SEk(t + h) −Ei[SEk(t)]Ei[SEk(t + h)] for t, h > 0 and Ek = {l1, l2, . . . , lk} with k ≤m. Denote REk(t, t + h) as a column vector with the i-th entry being Ei SEk(t)SEk(t + h) . In the following, we ﬁrst show in a lemma a result that is needed for deriving the expression for REk(t, t + h). We then present an explicit formula of REk(t, t + h) in a theorem below. As SEk(t + h) = SEk(t + h) −SEk(t) + SEk(t), we have Ei SEk(t)SEk(t + h) = Ei h S2 Ek(t) i + Ei SEk(t) SEk(t + h) −SEk(t) . (14) (14) Deﬁne Ft = σ(S(v); 0 ≤v ≤t) to be σ-algebra generated by the ADC process by time t. Using the law of iterated expectation, we have Deﬁne Ft = σ(S(v); 0 ≤v ≤t) to be σ-algebra generated by the ADC process by time t. Using the law of iterated expectation, we have Ei SEk(t) SEk(t + h) −SEk(t) = Ei E SEk(t) SEk(t + h) −SEk(t) Ft = Ei SEk(t)E SEk(t + h) −SEk(t) Ft = Ei n SEk(t)e−R t 0 δ(s)dsE SEk(t, t + h) Ft o = Ei n SEk(t)e−R t 0 δ(s)dsE SEk(t, t + h) J(t) o = m ∑ j=1 Ei n SEk(t)e−R t 0 δ(s)dsE SEk(t, t + h) J(t) = j o P(J(t) = j\|J(0) = i) = m ∑ j=1 Ei h SEk(t)e−R t 0 δ(s)dsI(J(t) = j) i Ej[SEk(h)]P(J(t) = j\|J(0) = i) (15) (15) = Ei n SEk(t)e−R t 0 δ(s)dsE SEk(t, t + h) J(t) o (15) = m ∑ j=1 Ei n SEk(t)e−R t 0 δ(s)dsE SEk(t, t + h) J(t) = j o P(J(t) = j\|J(0) = i) = m ∑ j=1 Ei h SEk(t)e−R t 0 δ(s)dsI(J(t) = j) i Ej[SEk(h)]P(J(t) = j\|J(0) = i) where SEk(t, t + h) is the present value, at time t, of the claims occurring in states within Ek over (t, t + h]. 4. The Covariance of the ADC with Two Different Time Lengths where SEk(t, t + h) is the present value, at time t, of the claims occurring in states within Ek over (t, t + h]. where SEk(t, t + h) is the present value, at time t, of the claims occurring in states within Ek over (t, t + h]. 10 of 16 Risks 2018, 6, 59 (t) = Mi,j,Ek(t) m×m, where Denote MEk(t) = Mi,j,Ek(t) m×m, where Denote MEk(t) = Mi,j,Ek(t) m×m, where Denote MEk(t) = Mi,j,Ek(t) m×m, where Mi,j,Ek(t) = Ei SEk(t)e−R t 0 δ(s)dsI(J(t) = j) . The following lemma gives a matrix-form integral expression for MEk(t). Lemma 1. MEk(t) is of the form The following lemma gives a matrix-form integral expression for MEk(t). Lemma 1. MEk(t) is of the form The following lemma gives a matrix-form integral expression for MEk(t). The following lemma gives a matrix-form integral expression for MEk(t). Lemma 1. MEk(t) is of the form MEk(t) = Z t 0 e− 2δ−(D0+D1) x IEk µ1 D1v(t −x)dx (16) (16) where v(t) is a matrix with (i, j)-th element being where v(t) is a matrix with (i, j)-th element being vi,j(t) = Ei h e−R t 0 δ(s)dsI(J(t) = j) i , i, j ∈E. vi,j(t) = Ei h e−R t 0 δ(s)dsI(J(t) = j) i , i, j ∈E. Proof. Conditioning on the events that may occur over an inﬁnitesimal interval (0, ∆t), we have Mi,j,Ek(t) = (1 + d0,ii∆t)e−2δi∆tMi,j,Ek(t −∆t) + ∑ l̸=i d0,il∆t e−2δi∆tMl,j,Ek(t −∆t) + m ∑ l=1 d1,il ∆t e−2δi∆t h I(i ∈Ek)µ(1) i El e−R t ∆t δ(s)dsI(J(t) = j) + Ml,j,Ek(t −∆t) i . (17) (17) We can then obtain a matrix-form differential equation for MEk(t) from Equation (17) as follows: We can then obtain a matrix-form differential equation for MEk(t) from Equation (17) as follows: M′ Ek(t) = (D0 + D1 −2δ)MEk(t) + IEkµ1 D1v(t) , (18) (18) with MEk(0) = 0. In fact, it is easy to show that v(t) = e(D0+D1−δ)t, with v(0) = I and v(∞) = 0. Solving Equation (18) gives Equation (16). with MEk(0) = 0. In fact, it is easy to show that v(t) = e(D0+D1−δ)t, with v(0) = I and v(∞) = 0. Solving Equation (18) gives Equation (16). Let qi,j(t) = Pi(J(t) = j). 4. The Covariance of the ADC with Two Different Time Lengths Then Q(t) = qi,j(t) m×m is the transition matrix of the underlying Markov process {J(t)}t≥0 at time t. It follows from Ren (2008) that Q(t) = e(D0+D1)t. Theorem 5. REk(t, t + h) can be expressed as Theorem 5. REk(t, t + h) can be expressed as REk(t, t + h) = V2,Ek(t) + (MEk ◦Q)(t)V1,Ek(h) (19) (19) where (MEk ◦Q)(t) is the Hadamard product of MEk(t) and Q(t), i.e., the (i, j)-th element of (MEk ◦Q)(t) is Mi,j,Ek(t) × qi,j(t), and MEk(t) is given by Equation (16) in Lemma 1. where (MEk ◦Q)(t) is the Hadamard product of MEk(t) and Q(t), i.e., the (i, j)-th element of (MEk ◦Q)(t) is Mi,j,Ek(t) × qi,j(t), and MEk(t) is given by Equation (16) in Lemma 1. Proof. Equation (19) follows immediately from Equations (14) and (15). Proof. Equation (19) follows immediately from Equations (14) and (15). Remark 5. If Ek = E or Ek = {k}, Equation (19) simpliﬁes to the joint moment of S(t) and S(t + h), or the joint moment of Sk(t) and Sk(t + h). Distributions of the ADC 5. The Distributions of the ADC In this section, we investigate the distributions of SEk(t) and its two special cases, S(t) and Sk(t), for Ek = {l1, l2, . . . , lk} ⊆E. To precede, we deﬁne for x ≥0 and i ∈E, Gi(x, t) = Pi(S(t) ≤x) Gi,k(x, t) = Pi(Sk(t) ≤x) Gi,Ek(x, t) = Pi SEk(t) ≤x , 11 of 16 Risks 2018, 6, 59 h the following conditions: with the following conditions: Gi(x, 0) = Gi,k(x, 0) = Gi,Ek(x, 0) = 1, x ≥0 Gi(0, t) = Pi(N(t) = 0) Gi,k(0, t) = Pi(Nk(t) = 0) Gi,Ek(0, t) = Pi NEk(t) = 0 where Nk(t) = ∑ N(t) l=1 I(J(Tl) = k) is the number of claims occurring in state k and NEk(t) = ∑j∈Ek Nj(t) is the number of claims occurring in the subset Ek. Denote where Nk(t) = ∑ N(t) l=1 I(J(Tl) = k) is the number of claims occurring in state k and NEk(t) = ∑j∈Ek Nj(t) is the number of claims occurring in the subset Ek. Denote where Nk(t) = ∑ N(t) l=1 I(J(Tl) = k) is the number of claims occurring in state k and NEk(t) = ∑j∈Ek Nj(t) is the number of claims occurring in the subset Ek. Denote G(x, t) = G1(x, t), G2(x, t), . . . , Gm(x, t) ⊤ Gk(x, t) = G1,k(x, t), G2,k(x, t), . . . , Gm,k(x, t) ⊤ GEk(x, t) = G1,Ek(x, t), G2,Ek(x, t), . . . , Gm,Ek(x, t) ⊤. We present in the theorem below that GEk(x, t) satisﬁes a ﬁrst-order partial integro-differential equation in matrix form. We present in the theorem below that GEk(x, t) satisﬁes a ﬁrst-order partial integro-differential equation in matrix form. We present in the theorem below that GEk(x, t) satisﬁes a ﬁrst-order partial integro-differential equation in matrix form. Theorem 6. GEk(x, t) satisﬁes Theorem 6. 5. The Distributions of the ADC GEk(x, t) satisﬁes ∂GEk(x, t) ∂t −xδ∂GEk(x, t) ∂x = (D0 + D1 −IEk D1)GEk(x, t) + Z x 0 IEk f(y)D1 GEk(x −y, t)dy , (20) ∂GEk(x, t) ∂t −xδ∂GEk(x, t) ∂x = (D0 + D1 −IEk D1)GEk(x, t) + Z x 0 IEk f(y)D1 GEk(x −y, t)dy , (20) (20) with initial conditions GEk(x, 0) = 1, GEk(0, t) = e(D0+D1−IEk D1)t1 (21) (21) where GEk(0, t) is the solution of the differential equation obtained from Equation (20) by setting x = where GEk(0, t) is the solution of the differential equation obtained from Equation (20) by setting x = 0. Proof. Using the same arguments as in Section 2, we have, by conditioning on events that may occur over (0, h], Gi,Ek(x, t) = [1 + d0,iih]Gi,Ek xeδih, t −h + m ∑ j=1,j̸=i d0,ijhGj,Ek xeδih, t −h + m ∑ j=1 d1,ijh Gj,Ek xeδih, t −h , i ̸∈Ek . (22) (22) As Gi,Ek(x, t) is differentiable with respect to x and t, we have x, t) is differentiable with respect to x and t, we have Gi,Ek xeδih, t −h = Gi,Ek(x, t) + δixh∂Gi,Ek(x, t) ∂x −h∂Gi,Ek(x, t) ∂t + o(h) . (23) (23) The justiﬁcation of Equation (23) can be done similarly as that for Equation (5) (see Appendix A). Substituting Equation (23) into Equation (22), rearranging terms, dividing both sides by h, and taking limit as h →0, give ∂Gi,Ek(x, t) ∂t −δix ∂Gi,Ek(x, t) ∂x = m ∑ j=1 d0,ijGj,Ek(x, t) + m ∑ j=1 d1,ijGj,Ek(x, t) , i ̸∈Ek . 12 of 16 Risks 2018, 6, 59 For i ∈Ek = {l1, l2, . . . , lk}, we have For i ∈Ek = {l1, l2, . . . , lk}, we have Gi,Ek(x, t) = [1 + d0,iih]Gi,Ek xeδih, t −h + m ∑ j=1,j̸=i d0,ikhGj,Ek xeδih, t −h + m ∑ j=1 d1,ijh Z xeδih 0 fi(y)Gj,Ek xeδih −y, t −h dy . Gi,Ek(x, t) = [1 + d0,iih]Gi,Ek xeδih, t −h + m ∑ j=1,j̸=i d0,ikhGj,Ek xeδih, t −h + m ∑ j=1 d1,ijh Z xeδih 0 fi(y)Gj,Ek xeδih −y, t −h dy . 5. The Distributions of the ADC Taylor’s expansion gives Taylor’s expansion gives ∂Gi,Ek(x, t) ∂t −δix ∂Gi,Ek(x, t) ∂x = m ∑ j=1 d0,ijGj,Ek(x, t) + m ∑ j=1 d1,ij Z x 0 fi(y)Gj,Ek(x −y, t)dy , i ∈Ek . Equations for i ∈Ek and i ̸∈Ek can then be expressed in matrix form (20). q k ̸ k p ( ) Remark 6. If we set Ek = E and Ek = {k}, respectively, we have the following results: ∂G(x, t) ∂t −xδ∂G(x, t) ∂x = D0G(x, t) + Z x 0 f(y)D1G(x −y, t)dy ∂Gk(x, t) ∂t −xδ∂Gk(x, t) ∂x = (D0 + D1 −Ik D1)Gk(x, t) + Z x 0 Ikf(y)D1Gk(x −y, t)dy , (24) Remark 6. If we set Ek = E and Ek = {k}, respectively, we have the following results: ∂G(x, t) ∂t −xδ∂G(x, t) ∂x = D0G(x, t) + Z x 0 f(y)D1G(x −y, t)dy ∂Gk(x, t) ∂t −xδ∂Gk(x, t) ∂x = (D0 + D1 −Ik D1)Gk(x, t) + Z x 0 Ikf(y)D1Gk(x −y, t)dy , (24) (24) with initial conditions G(x, 0) = 1, G(0, t) = eD0t1 Gk(x, 0) = 1, Gk(0, t) = e(D0+D1−IkD1)t1 . Here, Gk(0, t) is the solution of the differential equation obtained from Equation (24) by setting x = 0. Here, Gk(0, t) is the solution of the differential equation obtained from Equation (24) by setting x = 0. Here, Gk(0, t) is the solution of the differential equation obtained from Equation (24) by setting x = 0. Remark 7. The matrix-form partial integro-differential Equation (20) with the corresponding initial conditions given by Equation (21) may be solved numerically as follows. Remark 7. The matrix-form partial integro-differential Equation (20) with the corresponding initial conditions given by Equation (21) may be solved numerically as follows. (a) For two inﬁnitesimal h1 and h2, we set GEk(lh1, 0) = 1, for l = 1, 2, . . . , and we calculate GEk(0, nh2) using Equation (21) for n = 1, 2, . . . . (a) For two inﬁnitesimal h1 and h2, we set GEk(lh1, 0) = 1, for l = 1, 2, . . . , and we calculate GEk(0, nh2) using Equation (21) for n = 1, 2, . . . . (b) With Equation (20), GEk(lh1, nh2) can be calculated recursively, for n, l = 1, 2, . . . 6. Numerical Illustrations In this section, we consider a two-state Markov-modulated with intensity matrix A =  −1/4 1/4 3/4 −3/4  . We also assume that f1(x) = e−x, f2(x) = 0.5e−0.5x, x > 0, λ1 = 1, λ2 = 2/3, δ1 = 0.03, and δ2 = 0.05. Table 1 gives the ﬁrst moments of S1(t) and S2(t) and their covariance for t = 1, 2, 5, 10, 20, 30, and ∞, given J(0) = 1 and J(0) = 2, respectively, in which the covariances, for i = 1, 2, are calculated by Covi(t) ≜Cov S1(t), S2(t) J(0) = i = Ei S1(t)S2(t) −Ei[S1(t)]Ei[S2(t)]. It shows that, as expected, the expected values of S1(t) and S2(t) (and hence S(t)) are increasing in t given J(0) = i for i = 1, 2. It is not surprised to see that S1(t) and S2(t) are negatively correlated for any t, as claims occurring in the two states compete with each other. Moreover, the larger the time t, the more the negative correlation between S1(t) and S2(t). It shows that, as expected, the expected values of S1(t) and S2(t) (and hence S(t)) are increasing in t given J(0) = i for i = 1, 2. It is not surprised to see that S1(t) and S2(t) are negatively correlated for any t, as claims occurring in the two states compete with each other. Moreover, the larger the time t, the more the negative correlation between S1(t) and S2(t). Figure 1 plots the variances of S(t), S1(t), and S2(t), given J(0) = 1, for 0 ≤t ≤150. The variances all increase with time t. The variance of S(t) is bigger than those of S1(t) and S2(t) for a ﬁxed t. When time t goes to ∞, the three variances converge. Tables 2 and 3 display the covariances of the ADC at time t and t + h, given J(0) = 1, for some selected t values and for h = 1 and h = 5. It is shown that S(t) and S(t + h), S1(t) and S1(t + h), and S2(t) and S2(t + h) are all positively correlated. Moreover, when t increases, the covariances increase; moreover, when h increases, the covariances decrease. When t →∞, the covariances of the pairs S(t) and S(t + h), Si(t), and Si(t + h) converge to the variances of S(∞) and Si(∞), respectively. 5. The Distributions of the ADC , by (b) With Equation (20), GEk(lh1, nh2) can be calculated recursively, for n, l = 1, 2, . . . , by GEk(lh1, nh2) = I −lh2 δ −h2(D0 + D1 −IEkD1) −1 × h GEk(lh1, (n −1)h2) −lh2 δGEk (l −1)h1, nh2 + h2 h1 l−1 ∑ j=0 IEkf(jh1)D1GEk (l −1 −j)h1, nh2 i . Remark 8. If fi(x) = βie−βix, βi > 0, then f(x) = βe−βx, with f′(x) = −βf(x), where β = diag(β1, β2, . . . , βm). Taking partial derivative with respect to x on both sides of Equation (20) and performing some manipulations, we obtain the following matrix-form second-order partial differential equation for GEk(x, t) : Remark 8. If fi(x) = βie−βix, βi > 0, then f(x) = βe−βx, with f′(x) = −βf(x), where β = diag(β1, β2, . . . , βm). Taking partial derivative with respect to x on both sides of Equation (20) and performing some manipulations, we obtain the following matrix-form second-order partial differential equation for GEk(x, t) : ∂2GEk(x, t) ∂t∂x −xδ∂2GEk(x, t) ∂x2 + IEkβ∂GEk(x, t) ∂t + δ −D0 −D1 + IEk(xδβ + D1) ∂GEk(x, t) ∂x −IEk(βD0 + D1)GEk(x, t) = 0 . (25) Risks 2018, 6, 59 13 of 16 13 of 16 This partial differential equation can also be solved numerically by using forward ﬁnite difference methods. This partial differential equation can also be solved numerically by using forward ﬁnite difference methods. Remark 9. Li et al. (2015) show that, when δ(s) = 0, Gi(x, t) can be used to ﬁnd an expression for the density This partial differential equation can also be solved numerically by using forward ﬁnite difference methods. Remark 9. Li et al. (2015) show that, when δ(s) = 0, Gi(x, t) can be used to ﬁnd an expression for the density of the time of ruin in a MAP risk model. 6. Numerical Illustrations 6. Numerical Illustrations Similar patterns should be expected for J(0) = 2. Finally, we display in Figure 2 the numerical values of the distribution function of S(t) with initial state i, Gi(x, t) = Pi(S(t) ≤x), for t = 1 and 4, 0 ≤x ≤25, and i = 1, 2. Note that G(x, t) = (G1(x, t), G2(x, t))⊤satisﬁes the partial differential Equation (25); its solution can be obtained numerically. From the graph, it shows clearly that the probability of S(t) being bigger than a ﬁxed x is smaller for small values of t as expected. For most x values, G1(x, t) is bigger than G2(x, t) due to the fact that the underlying Markov process in our example tends to stay in state 1 more often than staying at state 2. Table 1. Expected values and covariances of S1(t) and S2(t). t J(0) = 1 J(0) = 2 E1[S1(t)] E1[S2(t)] Cov1(t) E2[S1(t)] E2[S2(t)] Cov2(t) 1 0.8948 0.1196 −0.0599 0.2690 0.9444 −0.1412 2 1.6665 0.3607 −0.2832 0.8117 1.4717 −0.5475 5 3.7056 1.1998 −1.3303 2.6996 2.4452 −1.8361 10 6.6248 2.4695 −2.9252 5.5563 3.6966 −3.4208 20 11.1330 4.4336 −5.0170 9.9757 5.6221 −5.4630 30 14.3123 5.8188 −6.1938 13.0922 6.9800 −6.6142 ∞ 21.9178 9.1324 −7.9012 20.5479 10.2283 −8.2962 Table 1. Expected values and covariances of S1(t) and S2(t). 14 of 16 Risks 2018, 6, 59 Figure 1. Variances of S(t), S1(t) and S2(t) with initial state J(0) = 1. Figure 1. Variances of S(t), S1(t) and S2(t) with initial state J(0) = 1. Figure 1. Variances of S(t), S1(t) and S2(t) with initial state J(0) = 1. Table 2. Covariances of discounted claims at t and t + 1. t J(0) = 1 Cov1 S(t), S(t + 1) Cov1(S1(t), S1(t + 1)) Cov1(S2(t), S2(t + 1)) 1 1.9327 1.7143 0.5328 2 3.9024 3.2169 1.7021 5 9.5545 7.3372 5.8448 10 17.0771 12.8965 11.4471 20 26.6637 20.2686 18.0782 30 31.9796 24.5961 21.2571 ∞ 40.3073 32.2449 23.8648 Table 3. Covariances of discounted claims at t and t + 5. t J(0) = 1 Cov1 S(t), S(t + 5) Cov1(S1(t), S1(t + 5)) Cov1(S2(t), S2(t + 5)) 1 0.8651 1.2213 0.4437 2 1.3181 2.0228 1.4775 5 3.4481 4.5219 5.2676 10 7.5945 8.5121 10.5187 20 15.2651 14.9882 16.9435 30 21.6039 19.7868 20.2186 ∞ 40.3073 32.2449 23.8648 (a) (b) Figure 2. Distribution functions of S(1) in (a) and S(4) in (b) for J(0) = 1, 2. Table 2. Author Contributions: The two authors contribute equally to this article Acknowledgments: The authors would like to thank two anonymous reviewers for providing helpful comments and suggestions that improved the presentation of this paper. This research for Dr. Yi Lu was supported by the Natural Science and Engineering Research Council (NSERC) of Canada (grant number 611467). Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Appendix A. Justiﬁcation of Equation (5) It is easy to see that the partial derivation of iL(ξ1, ξ2, . . . , ξm; t) in Equation (2) with respect to ξi exists for i ∈E. For its partial derivation with respect to t, we have ∂iL(ξ1, ξ2, . . . , ξm; t) ∂t = −lim h→0 iL(ξ1, ξ2, . . . , ξm; t) −iL(ξ1, ξ2, . . . , ξm; t + h) h = −lim h→0 1 hEi h e−∑m j=1 ξjSj(t) 1 −e−∑m j=1 ξj(Sj(t+h)−Sj(t))i . (ξ ξ ξ ) ∂t = −lim h→0 (ξ ξ ξ ) (ξ ξ ξ ) h = −lim h→0 1 hEi h e−∑m j=1 ξjSj(t) 1 −e−∑m j=1 ξj(Sj(t+h)−Sj(t))i . Now, under certain regularity conditions, we obtain lim h→0 Ei " e−∑m j=1 ξjSj(t) 1 −e−∑m j=1 ξj(Sj(t+h)−Sj(t)) h # ≤lim h→0 Ei " 1 −e−∑m j=1 ξj(Sj(t+h)−Sj(t)) h # = lim h→0 1 −Ei h e−∑m j=1 ξj(Sj(t+h)−Sj(t))i h . Let ξ = max(ξ1, ξ2, . . . , ξm). Then limh→0 1−Ei e −∑m j=1 ξj(Sj(t+h)−Sj(t)) h ≤ limh→0 1−Ei e −ξ ∑m j=1(Sj(t+h)−Sj(t)) h = limh→0 1−Ei[e−ξ(S(t+h)−S(t))] h = limh→0 1−Ei[e−ξS(h)] h . (A1) Since Ei h e−ξS(h)i = [1 + d0,iih] + m ∑ k=1,k̸=i d0,ikh + m ∑ k=1 d1,ikh ˆfi ξe−δih + o(h) = 1 + m ∑ k=1 d0,ikh + m ∑ k=1 d1,ikh ˆfi ξe−δih + o(h) = 1 + m ∑ k=1 (d0,ik + d1,ik)h + m ∑ k=1 d1,ikh h ˆfi ξe−δih −1 i + o(h) = 1 + m ∑ k=1 d1,ikh h ˆfi ξe−δih −1 i + o(h) , it follows from Equation (A1) that lim h→0 1 −Ei h e−∑m j=1 ξj(Sj(t+h)−Sj(t))i h ≤lim h→0 1 −Ei h e−ξS(h)i h = m ∑ k=1 d1,ik ! h 1 −ˆfi ξ i , Now, under certain regularity conditions, we obtain Now, under certain regularity conditions, we obtain lim h→0 Ei " e−∑m j=1 ξjSj(t) 1 −e−∑m j=1 ξj(Sj(t+h)−Sj(t)) h # ≤lim h→0 Ei " 1 −e−∑m j=1 ξj(Sj(t+h)−Sj(t)) h # = lim h→0 1 −Ei h e−∑m j=1 ξj(Sj(t+h)−Sj(t))i h . lim h→0 Ei " e−∑m j=1 ξjSj(t) 1 −e−∑m j=1 ξj(Sj(t+h)−Sj(t)) h # ≤lim h→0 Ei " 1 −e−∑m j=1 ξj(Sj(t+h)−Sj(t)) h # = lim h→0 1 −Ei h e−∑m j=1 ξj(Sj(t+h)−Sj(t))i h . 6. Numerical Illustrations Covariances of discounted claims at t and t + 1. Table 3. Covariances of discounted claims at t and t + 5. (b) (a) Figure 2. Distribution functions of S(1) in (a) and S(4) in (b) for J(0) = 1, 2. Risks 2018, 6, 59 15 of 16 15 of 16 Author Contributions: The two authors contribute equally to this article. Author Contributions: The two authors contribute equally to this article. Appendix A. Justiﬁcation of Equation (5) lim h→0 Ei " e−∑m j=1 ξjSj(t) 1 −e−∑m j=1 ξj(Sj(t+h)−Sj(t)) h # ≤lim h→0 Ei " 1 −e−∑m j=1 ξj(Sj(t+h)−Sj(t)) h # = lim h→0 1 −Ei h e−∑m j=1 ξj(Sj(t+h)−Sj(t))i h . Let ξ = max(ξ1, ξ2, . . . , ξm). Then limh→0 1−Ei e −∑m j=1 ξj(Sj(t+h)−Sj(t)) h ≤ limh→0 1−Ei e −ξ ∑m j=1(Sj(t+h)−Sj(t)) h = limh→0 1−Ei[e−ξ(S(t+h)−S(t))] h = limh→0 1−Ei[e−ξS(h)] h . (A1) (A1) Since Ei h e−ξS(h)i = [1 + d0,iih] + m ∑ k=1,k̸=i d0,ikh + m ∑ k=1 d1,ikh ˆfi ξe−δih + o(h) = 1 + m ∑ k=1 d0,ikh + m ∑ k=1 d1,ikh ˆfi ξe−δih + o(h) = 1 + m ∑ k=1 (d0,ik + d1,ik)h + m ∑ k=1 d1,ikh h ˆfi ξe−δih −1 i + o(h) = 1 + m ∑ k=1 d1,ikh h ˆfi ξe−δih −1 i + o(h) , it follows from Equation (A1) that it follows from Equation (A1) that it follows from Equation (A1) that it follows from Equation (A1) that lim h→0 1 −Ei h e−∑m j=1 ξj(Sj(t+h)−Sj(t))i h ≤lim h→0 1 −Ei h e−ξS(h)i h = m ∑ k=1 d1,ik ! h 1 −ˆfi ξ i , which justiﬁes the existence of the partial derivation of iL(ξ1, ξ2, . . . , ξm; t) with respect to t. which justiﬁes the existence of the partial derivation of iL(ξ1, ξ2, . . . , ξm; t) with respect to t. Risks 2018, 6, 59 16 of 16 16 of 16 References Asmussen, Søren. 2003. Applied Probability and Queues. New York: Springer. Barges, Mathieu, Hélene Cossette, Stéphane Loisel, and Etienne Marceau. 2011. 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W4248975246.txt	https://www.researchsquare.com/article/rs-233847/latest.pdf	en		Improving performance intelligence for governing an integrated health and social care delivery network: a case study on the Amsterdam Noord district	Research Square (Research Square)	2,021	cc-by	5,942	Improving performance intelligence for governing an integrated health and social care delivery network: a case study on the Amsterdam Noord district Véronique LLC Bos (  v.l.bos@amsterdamumc.nl ) Amsterdam UMC, University of Amsterdam, Department of Public and Occupational Health, Amsterdam Public Health research institute Niek S Klazinga Amsterdam UMC, University of Amsterdam, Department of Public and Occupational Health, Amsterdam Public Health research institute Dionne S Kringos Amsterdam UMC, University of Amsterdam, Department of Public and Occupational Health, Amsterdam Public Health research institute Research Article Keywords: Performance intelligence, performance measurement, integrated care, governance, health services, the Netherlands, Triple Aim, Amsterdam Noord, network, population management Posted Date: February 24th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-233847/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/15 Abstract Background: A guiding principle of a successful integrated health and social care delivery network is to establish a governance approach based on learning, grounded in a data and knowledge infrastructure. The ‘Krijtmolen Alliantie’ is a network of health and social care providers with the ambition to realize such a performance intelligence driven governance model in line with the Triple Aim. This study seeks to identify what performance intelligence is available and how it can be improved. Methods: This case study was conducted in the district of Amsterdam Noord, the Netherlands, and employed 23 semi-structured interviews with stakeholders in health and social care, a feasibility analysis of available administrative data, and a reflection meeting with board members of the ‘Krijtmolen Alliantie’. Information needs for performance intelligence by the stakeholders were mapped and a data landscape of the district covered by the network was drafted. Finally, in the reflection meeting with board members of the ‘Krijtmolen Alliantie’ the information needs and data landscape were aligned with governing needs, resulting in priority domains around which to strengthen the data infrastructure for governance of the integrated health and social care delivery network. Results: The ‘Krijtmolen Alliantie’ encompasses a network of providers with a diverse range of catchment areas. There are indicators on population health and welfare, however they have limited actionability for providers due to a misalignment with their respective catchment areas. There is a barrier in data exchange between health and social care providers. It is difficult to construct one indicator for per capita cost in the Dutch health data infrastructure as health and social care are subdivided in financing siloes. Priority domains for improvement of performance intelligence for the ‘Krijtmolen Alliantie’ are: 1) Per capita and per patient cost data integration that would allow combined accountability through bundling of financing, and 2) combined patient experience and outcome measures to reflect network quality of care and patient experience performance. Conclusion: Available performance intelligence lacks actionability for the governance of integrated care networks. Our recommendation is to align performance intelligence with the regional governance responsibilities of stakeholders for health and social care delivery. Background The delivery of health and social care in integrated care networks has become a necessity to provide people centred care(1). Concepts like Population Management and the Triple Aim, redirect measuring performance of the delivery of health and social care from single performance domains by siloed organizations, towards measuring the performance of integrated care networks(2, 3). Performance intelligence is an essential tool to govern integrated delivery systems, to redefine value of health and social care provision, and monitor outcomes (4–8). Performance intelligence is defined as “the structured approach to acting on health policies, using knowledge and information generated by the application of scientific methods to comparable healthcare data to systematically measure indicators of health systems Page 2/15 performance”(9). The actionability, defined as fitness for purpose and use, of performance intelligence is important in order for it to contribute to governance(10). However, knowledge on how to create actionable performance intelligence to guide governance of integrated care networks is still limited (11, 12). There is no ‘off the shelf’ framework available to measure performance of integrated care delivery (13), and lessons on hindering and facilitating factors to achieve improved outcomes in national and local level integrated care approaches illustrate the complexity worldwide (14–18). An awareness of the particularities of the local setting when implementing integrated care (19), and including perceptions of stakeholders involved in coordinating care delivery across organizations, is known to be important (20). In the Netherlands, healthcare reforms in 2007 and 2015 decentralized social care to the level of the municipalities, and ‘de Juiste Zorg op de Juiste Plek’ (the right care at the right place) launched by the Ministry of Health, Welfare and Sport in 2018 is one of the strategies to organise health and social care needs of the patient closer to home(21). The ‘Krijtmolen Alliantie’ (KMA) is a network of health and social care providers in the Amsterdam Noord district in the Netherlands who have been cooperating for about 10 years with the goal to realize a better experience of care for patients, improved health outcomes for the population, and lower per capita costs in line with the Triple Aim proposed by the Institute for Healthcare Improvement (2). Recently the KMA has decided to strengthen their cooperation through enhancing their shared performance intelligence. This paper provides a case study on how to prioritize domains to strengthen performance intelligence in integrated care networks. The objective of this research is to investigate the perceived need for performance intelligence among the members and stakeholders of an integrated care network, explore possibilities for generating performance intelligence given the current data landscape, and identify priority domains for improvement of performance intelligence to govern the integrated care network. To do so we address the following questions: 1) Who are the members of the KMA and their stakeholders in the health and social care district Amsterdam Noord, and what are their roles and information needs? 2) What data is currently collected by health and social care providers and their stakeholders in the district covered by the network, which exchanges of information already take place and what performance intelligence is available to support the Triple Aim goals? And, 3) What priority domains to improve performance intelligence can be identified in Amsterdam Noord? Methods Research setting The KMA serves the Amsterdam Noord district, one of the 8 districts in Amsterdam. The district is geographically separated from the rest of the city of Amsterdam by the IJ river and has a population of 97.200 inhabitants(22). High social care needs in combination with an already existing alliance of health and social care providers makes Amsterdam Noord an interesting case study for districts and regions that wish to establish performance intelligence to support further integration of health and social care governance. In Table 1a brief description of the research setting is provided. Page 3/15 Table 1 Key characteristics of the Amsterdam Noord district(22) Characteristics Inhabitants (2019) Geography (2019) Social economic status Health provision (2019) Health and social care usage 65 + years of age (Gemeentezorgspiegel, 2018) Amsterdam Noord Benchmarks* Total 97.200 862.987 65 + years of age 15% 13% 19% Under 20 years of age 23% 19% 22% Neighbourhoods 15 99 Area (ha) 6382.85 16473.34 Postal codes 1020– 1039 Average income per household per year (x€1.000; 2017) 34.5 39.0 Unemployment rate (%) in labour force (2018) 6 5 Percentage low education level (15– 74 year olds; 2017) 34 24 Hospitals 1 General practitioner practices 27 Pharmacies 15 Long-term Care Act (WLZ) average cost per inhabitant €3,532 €2.999 Social Support Act (WMO) average cost per inhabitant €963 €815 Healthcare Insurance Act (ZVW) average cost per inhabitant €6,690 €6.133 AMS (including Noord) AMS (excl. Noord) Netherlands €3.532 €5.732 *Benchmarks given: N = Amsterdam Noord district, NL = Netherlands, AMS = city of Amsterdam, AMSN = city of Amsterdam excluding Amsterdam Noord district. Page 4/15 Data collection A mixed method study was conducted using semi-structured interviews (n = 23) with KMA members and stakeholders of the Amsterdam Noord district, a feasibility analysis exploring available administrative data, and a reflection meeting with the governing board of the KMA. The data was collected from November 2019 to May 2020. For the semi-structured interviews (n = 23) we used stratified purposive sampling (23). The interviewees needed to represent a) KMA members (n = 11), and b) key stakeholders in health and social care in the Amsterdam Noord district (n = 12) (citizens/patients, healthcare financiers, and other health and social care providers). The interviewee list was validated with the chair of the KMA (see appendix 1 for the validated interviewee lists). Interviewees were invited via the KMA secretariat and were informed, and asked to consent to contribute to the content of the research and the processing of the interview data. The interviews were held in Dutch, approximately 30 minutes per interview, audio recorded, transcribed, and shared with the interviewees to check for accuracy. The interview guide was structured in line with the Triple Aim vision of the network for the district and contained questions on the provider organizations and their role in Amsterdam Noord, what data is collected, what information is currently used to govern services delivery, if there are any initiatives for exchanging data or integrated governance, and what is the current need for performance intelligence (for the translated interview guide see appendix 2). The database used for the feasibility analysis was the ‘Gemeentezorgspiegel’ database in collaboration with Vektis (the healthcare information centre that functions as the national claims data centre for health care consumption reimbursed via the Dutch Health Insurance Act) and the municipality of Amsterdam. The database consists of claims data from health insurance companies and public health authorities representing yearly about 75.3 billion euro (about 96% the Dutch national health budget in 2018) (24). The data consists of reimbursements within the Long-term Care Act (WLZ); regulating care for citizens with a permanent 24 hour care need, the Social Support Act (WMO); regulating municipal responsibility for supplementing citizens in their care need, and the Healthcare insurance Act (ZVW); regulating the right to medical specialist care and health insurance for all citizens. The reflection meeting with the board members of the KMA (n = 7) was organized digitally, as alternative to an in-person meeting, due to COVID-19 restrictions, in the presence of three researchers, one to present the findings, one to moderate the discussion, and one to take notes of the discussion. Analysis To answer research question 1, pieces of transcript were grouped that relate to the role of the interviewed organization and their catchment areas in a table. Then all pieces of transcript containing content on information needs were extracted and an analytic induction process using key words and statements was used to define needs for performance intelligence. To answer research question 2, pieces of transcript were extracted that relate to the data organizations collect, what exchanges of data are taking place and what information is used for governance. The extracts were categorized using an inductive approach. To get more insight in available cost data this analysis was supplemented with a feasibility analysis exploring available administrative data on Page 5/15 purchased health and social care delivery for the purpose of governing an integrated care network. This feasibility analysis aimed to identify, using an available administrative data source, what performance intelligence on costs is feasible for the Amsterdam Noord district. First, using the interface of the database, the available indicators, metadata, source information, and possible breakdowns were studied to assess what performance intelligence is currently available for the municipality of Amsterdam. Then via a series of meetings with Vektis a feasibility analysis for the Amsterdam Noord district was constructed from the database focusing on health and social service consumption. In the analysis specific attention was given to what level of data (neighbourhood, quarter or district) is informative to govern integrated care and the (im)possibilities of combining data from different sources (e.g. Social Support Act data with Healthcare insurance Act data), breakdowns (e.g. age groups), and benchmarks (e.g. Amsterdam Noord district compared to the municipality of Amsterdam excluding the Noord district). Finally, to answer research question 3, study findings were shared with the governing body of the KMA and a reflection meeting was organized to define priority domains for improving performance intelligence to govern the integrated care network. Notes were inducted to key messages from the discussion by one researcher and further complemented by the two researchers present in the reflection meeting. Results The Krijtmolen Alliantie and their stakeholders: performance intelligence needs for the integrated care network in the Amsterdam Noord district At the time of this study the Krijtmolen Alliantie had 13 member organizations representing health and social care provision organizations active in the Amsterdam Noord district. Selected stakeholders for interviews (n = 12) include the health insurer Zilveren Kruis, and the municipality of Amsterdam (both funders in health and social care in the Amsterdam Noord district), patient and client representation, the Dutch Healthcare Authority (NZa), general practitioners, and the ambulant pharmacy of the local hospital (see appendix 1 for details on interviewees and catchment areas). KMA members reported working with a variety of catchment areas ranging from provision at neighbourhood level within the district, to province level. The KMA members and stakeholders serve different proportions of the population of Amsterdam Noord either due to the nature of their provision (for example: welfare aid to those in financial problems) and/or due to sharing the market in the district (for example with competing elderly care providers Amstelring, Cordaan and Evean). Multiple KMA members report overlapping service provision, stating potential for competition, as well as beneficial alignment, between providers. KMA members and stakeholders showed aligned needs for performance intelligence. The most commonly expressed needs for performance intelligence among interviewees were related to: 1) population data and outcomes adjusted to their catchment area, 2) information on the alignment between providers, 3) outcomes of (multiple-provider) interventions, and 4) an overview of health and social care information of and for the citizen/patient. Population data and outcomes adjusted to catchment areas Page 6/15 Most health and social care providers report a need for information about the population they serve, either to be able to better define and support their target group, or as an outcome indicator for their work. Only few referred to the availability of population data in the Amsterdam Noord region, and the ones that did, found it hard to align the information with their practice as it is presented on an aggregated level and not tailored to their catchment areas. Information on the alignment between providers Most interviewees reported a lack of information on the alignment between providers. KMA members report that they are not always aware if other providers are involved with their patient/client. Most interviewees mention there is a barrier between social care information and health care information either on micro (individual patient), meso (between organizations) or macro (alignment social and health budgets) level. Some interviewees emphasized the importance of the correlation between the two, stating that coordinating health and social care in an integrated way can change decisions for individual treatment and adjust service provision on a meso and macro level. Information on outcomes of (multiple-provider) interventions Interviewees report a need for insights into the efficacy and efficiency of implemented multi-provider interventions. This performance intelligence can generate a learning curve on these interventions (mostly stated by providers of care) or can be used for accountability purposes (mostly stated by financers and patient representatives). Overview of health and social care information for the citizen/patient The interviewees representing patients in the region reported a need for an overview of health and social care data. They describe having limited information on possible health and social care providers in their region as well as lacking an overview of their own health and social care usage. Available information is compartmentalized per provider in, among other things, bilateral digital patient portals and limited in content (e.g. summarized letters or appointment dates). The interviewees representing patients in the region report that their main source for health and social care information comes from direct contact with their doctor, general practitioner (GP) or specialist, and experiences of friends and family. Available data (infrastructure) on health and social care in the Amsterdam Noord district and existing exchanges of data Data collected in health and social care organizations Interviewees describe data collection within their organizations that can be categorized as being used for five different purposes: 1) Identification and contact details of clients (name, address, personal identifier); 2) Treatment support (what are the needs of the client, what treatment is given). Every provider makes their own comprehensive analysis of the client, even after referral from other health and social care providers. Reasons mentioned to do this were incomplete information from referral in order to start health or social care provision, trust in referral information, and to encompass changes over time that might have occurred; 3) General management information for the organization, e.g. wellbeing of employees (e.g. absence due to illness of employees), quality (e.g. near misses, complaints), service (e.g. time until Page 7/15 phone is being answered), production (e.g. amount of treatments done or clients seen), and finances (e.g. costs and benefits); 4) Financial accountability (different per stakeholder varying from minute writing of professionals to complex coded accounting); and 5) Quality of care outcome measurements (e.g. routine outcome measurements, patient reported outcome measures). When done, providers collect their own outcome measures, aimed at particular interventions within the institution and its target population. Many state to obtain most information, even in the case of a referral, from the patient themselves. Data exchange between health and social care organizations All health and social care providers exchange information mostly on an aggregated (organizational) meso-level with their funders and their relevant overseeing authorities (e.g. the Dutch Health Authority and/or Inspectorate of Health and Youth (IGJ)) in line with Dutch legislation. Structural exchange of information between health and social care providers (e.g. including patient identifier) is limited. The general practitioners, functioning as gate keepers, exchange most information with other providers via referrals (e.g. the referral from general practitioners to medical specialists through a referral data exchange programme (Zorgdomein)). However, general practitioners state an incomplete exchange of information with elderly care providers and social care providers, noting this is not standardized and only available circumstantially or on the initiative of certain care professionals. Health insurance companies have the possibility to integrate data on patient-level from different providers, however strictly monitored by privacy laws, and only for the percentage of patients that they finance (their market share). A couple of providers of those interviewed have agreements to exchange anonymized aggregated data with academic networks to contribute to national-level data for performance measurement of the overall health system, for example exchanges with the Academic Collaborative Centre for Elderly Care, the Academic Collaborative Centre for General Practice and the Netherlands Institute for Health Services Research (NIVEL). There is potential to exchange health and social care data on citizen level as most providers use personal identifiers in their registration. The patient is the only point where all data for an individual comes together, however, this information is fragmented in bilateral communication between patient and providers and often summarized in referral letters or discharge letters formats. Patients can choose between one of the, at the time of this study, 29 accredited providers which all offer a ‘personal health environment’ (persoonlijke gezondheidsomgeving; PGO), to gather all of their individual healthcare data. Furthermore, there is a national healthcare data exchange platform (Landelijk Schakelpunt; LSP). This platform gives an overview to the patient what data (for example medication list) has been requested by providers. This platform, however, does not contain content data on care provided. Most health and social care providers report the existence of a patient portal or information exchange platform to communicate and/or share data bilaterally. All health and social care providers report internal data collection supporting their care provision. As providers do their own purchasing for digital support software, there is a great variety in digital support software and suppliers used by health and social care providers. An example being that there are eight different systems used by general practitioners in the district. Available performance intelligence to support Triple Aim goals Page 8/15 On population health The descriptive data in the ‘Gemeentezorgspiegel’ and public data from the municipality of Amsterdam and the Public Health Service of Amsterdam (GGD) include more than 100 indicators on population health and welfare available about the Amsterdam Noord district. Only few of the interviewed KMA members mentioned this information and the ones that did, expressed to experience difficulties to use this information. Only one social care provider mentioned the use of neighbourhood-level data on income from the publicly available data combined with their own data gives them actionable insights. Multiple interviewees refer to the reports made by independent research institutes or the municipality. These reports give insights, but there remains a gap towards actionability, not aligning to the catchment areas or information needs of providers. On costs per capita Financial and usage data on health and social care are siloed and costs per patient or inhabitant over the whole spectrum of care are not integrated on patient identifier level due to privacy concerns, reducing the value for use. Costs for long-term care (Wet Langdurige Zorg; WLZ), medical care (Zorgverzekeringswet; Zvw) and social support (Wet Maatschappelijke ondersteuning; WMO) can be shown separately with breakdowns (e.g. age groups) and benchmarks (e.g. municipality vs national). The absence of integration of costs on personal identifier level makes it difficult to construct one indicator on per capita costs for governing integrated care delivery purposes. Using either cost per inhabitant or cost per patient as an indicator for costs in the district, measure different things and show different outcomes. In the feasibility analysis of the Health Insurance Act claims, which includes the reimbursement of hospital care, GP’s, pharmaceuticals and mental healthcare the benchmark between Amsterdam Noord and Amsterdam excluding the Noord district, showed relative less costs per patient, but more cost per inhabitant. At the time of the study there is still a lag of about one year in reporting based on this database. To support governance of integrated care networks, information will have to be timelier. On care experiences Experience of care is documented in bilateral relations between KMA members and patients through patient satisfaction surveys, requests to (anonymously) review care givers or organizations and compliments and complaints reporting systems. There is a national system for reviewing healthcare providers called ‘Zorgkaart Nederland’(25), however this system also measures patient experience per siloed provider (organization or specific professional). There is no data collection that gives insight in integrated patient experience in the Amsterdam Noord district. Governing an integrated health and social care district – priority domains for improvement of performance intelligence In the reflection meeting with the board of the KMA, 7 members were represented. Reflecting on the findings, the fragmented data collection mirrors the fragmented accountability and financing siloes currently applied in the Netherlands. As one member put it: “die fragmentatie, ook in verantwoording, nekt Page 9/15 ons”, loosely translated: “this fragmentation, also in accountability, is our downfall”. Governing to incentivize the Triple Aim in an integrated health and social care provision network requires an integrated data infrastructure that aligns with the governing structure. Board members emphasise that there is a willingness to integrate care provision, however this must be aligned with external expectations in accountability and financing. Board members reflect that to counteract the individual accountability and financing siloes, boldness is needed to step away from the norms and set up a performance intelligence dashboard that reflects the governing ambition. Multi-year contracts of involved parties are needed to create such movement space to innovate. Making the best use of available data is the first step towards building performance intelligence. The areas that were highlighted in the reflection meeting as potential domains for improving performance intelligence to govern the integrated care network were: costs, effectiveness & safety, patient experiences & outcomes, usage and process of care provision, cooperation & capacity in the network, evaluation of interventions, and population outcomes. There were some important reflections on these domains. The analytical capacity needed to process data into pieces of information and performance intelligence for a person centred approach, outreaches the capacity of one organization. The fragmentation in bilateral patient portals and patient experience measures per organization does not reflect a person centred approach. The current Dutch funding system is making use of budgets per organization, which does not allow for changing capacity needs within an integrated care network to carry over funding from one organization to the other. The timeliness of Dutch administrative reporting systems for care, needs to improve in order to create indicators that are actionable for governance. The reflection meeting defined costs (combined accountability through bundling financial incentives) and patient experiences & outcomes (measured in an integrated approach) as priority domains to improve the performance intelligence of their integrated care network. Discussion Our findings show that the performance intelligence in order to govern regional integrated provision of health and social care in the Netherlands is still showing considerable barriers on both data availability and translating data into actionable indicators for governance. It is known that integrated care can contribute to a more people centred approach(26). Our findings suggest that intentions for integrating health and social care services are there, but the data and information structure needed to govern such a network are not. The identified barriers in data availability and data alignment for effective governance in the integrated care network in our case study can partially be explained by the competitive market structure introduced in healthcare in the Netherlands in 2006 and 2008(27). However, as the Netherlands, and health systems worldwide, are changing to more cooperative integrated networks, data infrastructures should align to support governance in networks with actionable performance intelligence. The importance of an integrated data infrastructure, feeding agreed upon performance intelligence for decision making has become even more clear in the light of the COVID-19 pandemic(28). Integrated national data infrastructures like the Danish national patient registry have been able to inform decision makers on the effect of the pandemic on non-COVID-19 related care needs, and thus were able to make a more informed decision on COVID-19 measures from an integrated perspective (29). Our Page 10/15 recommendation is to align performance intelligence with the regionalized responsibilities for governance of health and social care. Our findings show that care providers may have become reluctant to share essential data for integrating health and social care treatment, hindering the development of an integrated data and knowledge infrastructure, essential to govern effectively across organizational silos. We acknowledge the importance of protecting sensitive health and social care data. The bottleneck for governance does not seem to be with having to identify the specific patient, but the need to integrate data on patient level to gain actionable insights. This leaves room to organize data for effective regional governance within the scope of the General Data Protection Regulation (GDPR) taking into account the privacy of the patient (30). Strengths and limitations KMA members were well represented in the study by interviews and the reflection meeting, and their most important stakeholders were able to contribute. At this moment youth care and independent pharmacies are not part of the KMA alliance, and therefore not taken into account in this study. This study is done in the Dutch context, however can be an inspiration to other high income countries moving from a competitive healthcare market towards regional governance of integrated care. Conclusion As health systems are changing to more cooperative integrated networks, data infrastructures should align in order to support governance on performance intelligence. Available information lacks actionability for the governance of integrated care networks. In this case study an information need was observed in the following areas: 1) population data and outcomes adjusted to catchment areas of providers, 2) data on alignment between providers, 3) outcomes of (multiple-provider) interventions, and 4) an overview of health and social care information of and for the citizen/patient. There are indicators on population health and welfare, however they have limited actionability for providers due to a misalignment with their respective catchment areas. Indicators to measure integrated cost per capita and per patient, and integrated patient experience/outcomes indicators are lacking. Priority domains to improve the performance intelligence of the integrated care network proposed by this case study are: 1) Per capita and per patient cost data integration that would allow combined accountability through bundling of financing, and 2) combined patient experience and outcome measures to reflect network quality of care and patient experience performance. Multiple-year contractual arrangements with funding agencies, integrated regional data performance infrastructure, and formalized regional patient and citizen representation could facilitate the further development of integrated health and social care delivery networks in the Netherlands. List Of Abbreviations Page 11/15 GGD Gemeentelijke Gezondheidsdienst GP General Practitioner IGJ Inspectie voor Gezondheidszorg en Jeugd KMA Krijtmolen Alliantie LSP Landelijk Schakelpunt NZa Nederlandse Zorgautoriteit PGO Persoonlijke Gezondheidsomgeving WLZ Wet Langdurige zorg WMO Wet Maatschappelijke ondersteuning ZVW Zorgverzekeringswet Declarations Ethics approval and consent to participate This research proposal has been reviewed and approved by the Medical Ethics Review Committee of the Academic Medical Center prior to data collection (reference number W19_358 # 19.422 app.). All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects in this study or, if subjects are under 18, from a parent and/or legal guardian. All interviewees were verbally asked for consent and signed consent forms to participate in this study. Consent for publication Not applicable. This manuscript does not contain any non-public data traceable to an individual in any form. Availability of data and materials The data that support the findings of this study are stored at the AMR research B.V. and available for quality inspection. However restrictions apply to the public availability of this data. Interview transcriptions and reflection meeting notes cannot be anonymized due to the thick content of the material. Anonymized data used for the feasibility analysis are available from the authors upon reasonable request and with permission of the KMA, Vektis and the municipality of Amsterdam. Competing interests The authors declare that they have no competing interests. Page 12/15 Funding This research group received a funding of €10.000 from the Voucher ‘Juiste Zorg op de Juiste Plek’ from ‘the Netherlands Organisation for Health Research and Development’ (ZonMw). This Voucher went towards the ‘Gemeentezorgspiegel’ data availability, expertise and analysis for Amsterdam Noord district by Vektis. The funding body, ZonMw, had no role in the design of the study and the collection, analysis and interpretation of data and writing of the manuscript. Authors’ contribution This manuscript was drafted and executed by VB under close collaboration and supervision of DK and NK. VB conducted the interviews. VB, DK and NK collaborated with Vektis to conduct the feasibility analysis. VB, DK and NK conducted the reflection meeting. All Authors reviewed the research proposal and manuscript. Acknowledgements The authors thank all patient representatives, health and social care professionals, data experts and CEOs who were willing to reflect on defining and measuring performance so openly during the interviews and in the reflection meeting. References 1. Viktoria Stein K, Barbazza ES, Tello J, Kluge H. 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https://openalex.org/W3196753126	https://bmcinfectdis.biomedcentral.com/counter/pdf/10.1186/s12879-021-06596-5	English	null	Assessment of COVID-19 progression on day 5 from symptoms onset	BMC infectious diseases	2,021	cc-by	6,671	© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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: A major limitation of current predictive prognostic models in patients with COVID-19 is the hetero- geneity of population in terms of disease stage and duration. This study aims at identifying a panel of clinical and laboratory parameters that at day-5 of symptoms onset could predict disease progression in hospitalized patients with COVID-19. Methods: Prospective cohort study on hospitalized adult patients with COVID-19. Patient-level epidemiological, clinical, and laboratory data were collected at fixed time-points: day 5, 10, and 15 from symptoms onset. COVID-19 progression was defined as in-hospital death and/or transfer to ICU and/or respiratory failure (PaO2/FiO2 ratio < 200) within day-11 of symptoms onset. Multivariate regression was performed to identify predictors of COVID-19 progres- sion. A model assessed at day-5 of symptoms onset including male sex, age > 65 years, dyspnoea, cardiovascular disease, and at least three abnormal laboratory parameters among CRP (> 80 U/L), ALT (> 40 U/L), NLR (> 4.5), LDH (> 250 U/L), and CK (> 80 U/L) was proposed. Discrimination power was assessed by computing area under the receiver operating characteristic (AUC) values. Results: A total of 235 patients with COVID-19 were prospectively included in a 3-month period. The majority of patients were male (148, 63%) and the mean age was 71 (SD 15.9). One hundred and ninety patients (81%) suffered from at least one underlying illness, most frequently cardiovascular disease (47%), neurological/psychiatric disorders (35%), and diabetes (21%). Among them 88 (37%) experienced COVID-19 progression. The proposed model showed an AUC of 0.73 (95% CI 0.66–0.81) for predicting disease progression by day-11. Conclusion: An easy-to-use panel of laboratory/clinical parameters computed at day-5 of symptoms onset predicts, with fair discrimination ability, COVID-19 progression. Assessment of these features at day-5 of symptoms onset could facilitate clinicians’ decision making. The model can also play a role as a tool to increase homogeneity of population in clinical trials on COVID-19 treatment in hospitalized patients. Keywords: COVID-19, Disease progression, Outcome, Public health Assessment of COVID‑19 progression on day 5 from symptoms onset Elisa Gentilotti1, Alessia Savoldi1, Monica Compri1, Anna Górska1, Pasquale De Nardo1, Alessandro Visentin1, Giorgia Be1, Elisa Razzaboni1, Nicola Soriolo1, Dario Meneghin1, Domenico Girelli2, Claudio Micheletto3, Sara Mehrabi4, Elda Righi1 and Evelina Tacconelli1 Introductionh The global Coronavirus disease 2019 (COVID-19) pan- demic is challenging healthcare systems worldwide [1]. As of June 2021, the number of patients confirmed to have COVID-19 has exceeded 190 million in 191 coun- tries, with more than 4 million deaths [2]. The critical disease, described in up to 20% of hospitalised patients Correspondence: pasquale.denardo@univr.it 1 Division of Infectious Diseases, Department of Diagnostics and Public Health, University of Verona, P.le L.A. Scuro 10, 37134 Verona, Italy Full list of author information is available at the end of the article Gentilotti et al. BMC Infect Dis (2021) 21:883 https://doi.org/10.1186/s12879-021-06596-5 Open Access © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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. Gentilotti et al. BMC Infect Dis (2021) 21:883 Gentilotti et al. BMC Infect Dis (2021) 21:883 Page 2 of 8 [3, 4], is associated with high case fatality rate and leads to substantial increase in the demand for hospital beds and shortage of medical equipment. Up to January 2021, the hospital and intensive care unit (ICU) occupancy and the new admission due to COVID-19 increased in sev- eral European countries, reaching 82% of the peak ICU occupancy observed during the pandemic [5]. Descrip- tive studies on the natural course of COVID-19 have revealed that the disease progression occurs typically at an early stage of the illness, usually within 7–14 days after the onset of symptoms [6, 7] with or without acute severe respiratory distress syndrome [8]. Given the lack of standardized treatments for patients with COVID-19, the identification of patients at higher risk of short-term complications is of utmost importance to ensure the best possible clinical care and to optimise resource allocation. Since the beginning of the pandemic, several prognostic predictive models combining various clinical and labo- ratory parameters have been developed to estimate risk of patients experiencing poor outcomes. A systematic review of ten prognostic models observed that the most reported predictors of disease progression and mortality were age, sex, C-reactive protein (CRP), lactic dehydroge- nase (LDH) and lymphocyte count [9]. guardian on enrolment. Statistical analysis Descriptive statistics included means with standard deviations (SD) and frequency analyses (percentages) for categorical variables at day 5 after onset of symp- toms. T-test for independent samples, χ2 test, or Fish- er’s test were applied to compare differences between patients with or without disease progression. Logistic regression was performed to evaluate univariate and multivariate associations with the outcome. Results were presented as odds ratio (OR) and 95% confidence interval (95% CI). A p-value less than 0.05 was deemed as statistically significant. In order to enter the model as categorical variable, the cut-off of each laboratory parameter was set at the best point of sensitivity and specificity identified through the receiver operating characteristic (ROC) curve. The selected parameters with correspondent cut-offs were: CRP > 80 mg/L, neu- trophil lymphocyte ratio (NLR) > 4.5, LDH > 250 U/L, Infection with SARS-CoV-2 was microbiologically defined by a positive real-time reverse- transcriptase polymerase chain reaction (RT-PCR) assay from nasopharyngeal swab. Outcomes COVID-19 progression was defined as PaO2/FiO2 ratio < 200 and/or transfer to ICU and/or in-hospital death occurring by day-11 after symptoms onset. This study aims at identifying a panel of clinical and laboratory parameters that could support the prediction of disease progression based on symptoms onset in hos- pitalized patients with COVID-19. Data collection l l d Patient-level demographic (age, sex, ethnicity), chronic underlying diseases (hypertension and/or coronary artery disease, diabetes, dyslipidaemia, pulmonary dis- ease (chronic obstructive pulmonary disease, active tuberculosis, asthma or obstructive sleep apnoea syn- drome), chronic kidney disease, neoplastic disease, thy- roid disorders, neurological/psychiatric disorders, liver diseases, obesity (body mass index higher than 30), and clinical parameters (body temperature, respiratory rate, blood pressure, heart rate, peripheral oxygen saturation) were gathered at study inclusion and entered into a pre- defined case report form. Laboratory parameters, includ- ing lymphocytes (reference range: 120–400 109/L) and neutrophils (reference range: 180–800 109/L) count, LDH (reference range: 135–214 U/L), CRP (reference range: < 5 mg/L), aspartate aminotransferase (AST, reference range: 5–45 U/L), alanine aminotransferase (ALT, refer- ence range: 5–45 U/L)), creatine kinase (CK, reference range: 30–200 U/L) were extracted from routine blood testing at fixed time points: at hospital admission, day-5 (± 1) and day-10 (± 1) from onset of symptoms and in case of transfer to ICU/sub-intensive unit, discharge, and death. All the published models were computed using the trend of clinical and laboratory information starting from the admission day onward [10–12], with the result that the data was related to a wide range of disease length as opposed to specific time points. To the best of our knowl- edge, no prognostic models have been developed taking into account a homogeneous time point across patients, such as the onset of symptoms, for the assessment of dis- ease progression. Study design and participantsh This is a prospective cohort study. All adult patients (aged ≥ 18 years) with microbiologically confirmed SARS-CoV-2 infection admitted from 1st March to 31st May, 2020 at the University Hospital of Verona were pro- spectively followed during the hospital stay and included in the COVID 19-VR registry (registered on ClinicalTri- als.gov, 18/05/2020, Number: NCT04497194). The study protocol is performed in accordance with the relevant guidelines. The study was approved by the hospital Insti- tutional Review Board (“Comitato etico per la speri- mentazione clinica delle province di Verona e Rovigo”, 2577CESC). A written informed consent for study partic- ipation was obtained from the patients or from the legal Gentilotti et al. BMC Infect Dis (2021) 21:883 Page 3 of 8 had final responsibility for the decision to submit for publication. CK > 80 U/L, and ALT > 40 U/L. Candidate predictors to enter the logistic multivariate regression were varia- bles with p < 0.05 in univariate analysis. Variables inde- pendently associated with the outcome at multivariate analysis together with those deemed as critically rel- evant in accordance with literature evidence and clini- cal experience were selected to fit the prediction model. The performance of the model was assessed by calculat- ing the ROC curves and the corresponding area under the curve (AUC) values. The Hosmer–Lemeshow test was applied to assess the goodness-of-fit. All statisti- cal analyses were carried out using International Busi- ness Machines (IBM) Statistical Package for the Social Sciences (SPSS) 21 (IBM Corporation: Armonk, NY, 10504). Results A total of 235 patients with COVID-19 were prospec- tively included in the cohort. Demographic and clinical characteristics of patients enrolled in the study are dis- played in Table 1. The majority of patients were male (148, 63%) and the mean age was 71 (SD 15.9). One hun- dred and ninety patients (81%) suffered from at least one underlying illness, most frequently cardiovascular dis- ease (47%), neurological/psychiatric disorders (35%), and diabetes (21%). The majority of patients had fever prior to admission (193, 82%). Cough and dyspnoea were reported in 106 (45%) and 87 (37%) cases, respectively. The mean time from onset of symptoms and admission to hospital was 3.6 days (SD 3.2), while mean length of hospital stay was 13.2 days (SD 11.5). Patients received antiviral therapy according to national and regional recommendations (see Table 1). Steroids (methylprednisolone or dexametha- sone), if prescribed, were administered for at least 5 days. a Disease progression is a composite outcome defined as: death and/or transfer to ICU within day-11 after symptom onset and/or PaO2/FiO2 ratio < 200 on day 10 after symptom onset. Results are presented as mean (SD) and mean difference (95% CI) or frequencies (%) and OD (95% CI) as appropriate Role of the funding sourceh The funders of the study did not have any role in data collection and in study design, data analysis, data inter- pretation, or writing of the report. The correspond- ing author had full access to all data in the study and Table 1 Demographic and clinical characteristics of patients by COVID-19 disease progressiona BMI: body mass index a Disease progression is a composite outcome defined as: death and/or transfer to ICU within day-11 after symptom onset and/or PaO2/FiO2 ratio < 200 on day 10 after symptom onset. Role of the funding sourceh best point of sensitivity and specificity identified through the ROC curve, an association between disease progres- sion and the above mentioned variables showing values above the cut-offs was observed (CRP: OR 7.38; 95% CI 3.83–14.23, p < 0.000; NLR: OR 3.43; 95% CI 1.77–6.64, p < 0.000; LDH: OR 6.70; 95% CI 2.14–21.04, p = 0.001; and CK: OR 3.83; 95% CI 1.56–9.39, p = 0.003) (Table 2). Eighty-eight (37%) patients experienced poor outcome, e.g. PaO2/FiO2 ratio < 200 (37 patients, 42%), transfer to ICU (30 patients, 34%), and death (47 patients, 53%) by day-11 after symptoms onset. At univariate analysis (Table 1), patients with poor outcome were more likely to be older than 65 years (OR 2.02; 95% CI 1.12–3.65 years, p = 0.020), to suffer from cardiovascular disease (OR 2.85; 95% CI 1.55–5.23, p = 0.001), and to have experi- enced dyspnoea during the 5 days prior to admission (OR 2.83; 95% CI 1.63–4.92, p < 0.000). Blood test performed by day-5 of symptoms onset showed that mean values of CRP, NLR, LDH and CK were significantly higher com- pared with patients not experiencing progression of COVID-19 (Fig. 1). After applying the cut-offs set at the Multivariate logistic regression showed that at day 5 presence of cardiovascular disease (OR 2.29; CI 95% 1.12–4.68; p = 0.023), dyspnoea (OR 2.40; 95% CI 1.19– 4.84; p = 0.015) and at least three abnormal laboratory findings among CRP > 80 U/L, ALT > 40 U/L, NLR > 4.5, LDH > 250 U/L, and CK > 80 U/L (OR 2.80; 95% CI 1.35–5.81; p = 0.006) were independently associated with COVID-19 progression (Table 3). A model combining the above mentioned variables with age > 65 years old and male sex showed an AUC of 0.73 (95% CI 0.66–0.81) for Fig. 1 Comparison of laboratory parameters at day-5 from symptoms onset between patents with (red) and without (blue) COVID-19 disease progression. Disease progression is a composite outcome defined as: death and/or transfer to ICU by day-11 after symptom onset, and/or PaO2/FiO2 ratio < 200 on day 10 after symptom onset. CRP C-reactive protein, NLR neutrophils to lymphocytes ratio, LDH lactate dehydrogenase, CK creatine kinase, AST aspartate aminotransferase, ALT alanine transaminase. p-values are reported at the top of each box plot Fig. 1 Comparison of laboratory parameters at day-5 from symptoms onset between patents with (red) and without (blue) COVID-19 disease progression. Role of the funding sourceh Results are presented as mean (SD) and mean difference (95% CI) or frequencies (%) and OD (95% CI) as appropriate TOT (n = 235) Disease progressiona Univariate No (n = 147) Yes (n = 88) OR (95% CI) p-value Age (years) > 65 157 (67%) 90 (61%) 67 (76%) 2.02 (1.12 to 3.65) 0.020 Gender (female) 87 (37%) 58 (40%) 29 (33%) 0.75 (0.43 to 1.31) 0.318 Comorbidities (any) 190 (81%) 119 (81%) 71 (81%) 0.98 (0.50 to 1.92) 0.959 Diabetes 40/190 (21%) 27/119 (23%) 13/71 (18%) 0.76 (0.37 to 1.59) 0.475 Cancer 33/190 (17%) 22/119 (19%) 11/71 (16%) 0.81 (0.37 to 1.79) 0.599 Cardiovascular diseases 90/190 (47%) 45/119 (38%) 45/71 (63%) 2.85 (1.55 to 5.23) 0.001 Renal diseases 39/190 (21%) 20/119 (17%) 19/71 (27%) 0.81 (0.89 to 3.69) 0.103 Respiratory diseases 34/190 (18%) 21/119 (18%) 13/71 (18%) 1.05 (0.49 to 2.25) 0.908 Liver diseases 6/190 (3%) 4/119 (3%) 2/71 (3%) 0.83 (0.15 to 4.67) 0.836 BMI > 30 16/190 (8%) 12/119 (10%) 4/71 (6%) 0.53 (0.17 to 1.72) 0.292 Neurologic/Psychiatric diseases 66/190 (35%) 38/119 (32%) 28/71 (39%) 1.39 (0.75 to 2.56) 0.294 Symptoms Fever 193 (82%) 120 (82%) 73 (83%) 1.09 (0.55 to 2.19) 0.798 Cough 106 (45%) 68 (46%) 38 (43%) 0.88 (0.52 to 1.50) 0.646 Dyspnoea 87 (37%) 41 (28%) 46 (52%) 2.83 (1.63 to 4.92) 0.000 Treatment Steroids (at least 5 days) 38/183 (21%) 26/124 (21%) 12/59 (20%) 0.96 (0.45 to 2.07) 0.922 Non-invasive ventilation 26 (11%) 17 (12%) 9 (10%) 0.87 (0.37 to 2.05) 0.752 Invasive mechanical ventilation 30 (13%) 2 (1%) 28 (32%) 33.83 (7.81 to 156.53) 0.000 Outcomes (days), mean (SD) Time from symptoms onset to admission 3.6 (3.2) 3.2 (2.4) 4.0 (3.9) 0.78 (− 0.08 to 1.63) 0.076 Length hospital stay 13.2 (11.5) 12.4 (11.2) 14.3 (12) 1.82 (− 1.24 to 4.87) 0.243 a Disease progression is a composite outcome defined as: death and/or transfer to ICU within day-11 after symptom onset and/or PaO2/FiO2 ratio < 200 on day 10 after symptom onset. Results are presented as mean (SD) and mean difference (95% CI) or frequencies (%) and OD (95% CI) as appropriate Gentilotti et al. BMC Infect Dis (2021) 21:883 Page 4 of 8 In 26 (11%) cases non-invasive ventilation was required, while 30 (13%) patients underwent invasive mechanical ventilation. a Disease progression is a composite outcome defined as: death and/or transfer to ICU within day-11 after symptom onset, and/or PaO2/FiO2 ratio < 200 on day 10 after symptom onset b Within: CRP > 80 mg/L; NLR > 4.5; LDH > 250 U/L; CK > 80 U/L; ALT > 40 U/L. Results are presented as mean (SD) and mean difference (95% CI) or frequencies (%) and OD (95% CI), as appropriate n, NLR neutrophils to lymphocytes ratio, LDH lactate dehydrogenase, CK creatine kinase, AST aspartate aminotransferase, ALT alanine Role of the funding sourceh Table 3 Multivariate analysis of clinical characteristics and laboratory parameters by COVID-19 progression a Within: CRP > 80 mg/L; NLR > 4.5; LDH > 250 U/L; CK > 80 U/L; ALT > 40 U/L OR (95% CI) p-value Age (years) > 65 1.53 (0.63 to 3.67) 0.345 Cardiovascular diseases 2.29 (1.12 to 4.68) 0.023 Dyspnoea 2.40 (1.19 to 4.84) 0.015 At least 3 abnormal laboratory valuesa 2.80 (1.35 to 5.81) 0.006 Antibiotic therapy 1.16 (0.57 to 2.36) 0.691 y p Since the start of the pandemic, a wide range of pre- dictive models and scores has been published with the common goal of informing clinical decision making and optimising resource allocation. An observational cohort of 1157 patients acutely admitted to two London hospi- tals analysed several demographics, clinical, laboratory and imaging factors likely to predict mortality, highlight- ing a correlation of male sex, older age, hypertension, chronic lung diseases and higher levels of lymphocytes, CRP and creatinine, among the others, with critical care admission and/or death [7]. A systematic review of ten prognostic models revealed that the most reported pre- dictors of disease progression and mortality were age, sex, CRP, LDH and lymphocyte count [9]. Recently, the predictive value of NLR measured at hospital admission has been assessed in a prospective cohort, showing a high value in predicting disease deterioration, shock and death (all the areas under the curve > 0.80) [13]. The vast major- ity of these models assessed the predictors by considering the hospital admission as baseline time-point [10–13], and share therefore the common drawback of including patients at varying stages of the disease. Fig. 2 COVID-19 disease progression by day-11 after onset of symptoms: Receiver operator characteristic (ROC) curve for discrimination, Area under the curve (AUC) = 0.733 (95% CI 0.655– 0.810). Hosmer–Lemeshow Chi-squared = 8.061, p = 0.427; DF = 8). Variables included in the model: age > 65 years, male sex, presence of cardiovascular disease, at least three abnormal parameters at day-5 after symptom onset within: CRP > 80 mg/L; NLR > 4.5; LDH > 250 U/L; CK > 80 U/L; ALT > 40 U/L, and history of dyspnoea prior to admission In our cohort, more than one-third (37%) of patients experienced disease progression by day-11 of symptoms onset. Role of the funding sourceh Descriptive studies on clinical course of COVID- 19 revealed that the median time from onset of illness to acute respiratory distress syndrome and to ICU admis- sion was 8–12 days and 9.5–12 days, respectively [14]. These findings would seem to indicate that clinical dete- rioration with the need of higher level of care may occur at a very early stage of the disease and suggest that set- ting “onset of symptoms” as baseline time-point leads to inclusion of patients with homogeneous disease length. Fig. 2 COVID-19 disease progression by day-11 after onset of symptoms: Receiver operator characteristic (ROC) curve for discrimination, Area under the curve (AUC) = 0.733 (95% CI 0.655– 0.810). Hosmer–Lemeshow Chi-squared = 8.061, p = 0.427; DF = 8). Variables included in the model: age > 65 years, male sex, presence of cardiovascular disease, at least three abnormal parameters at day-5 after symptom onset within: CRP > 80 mg/L; NLR > 4.5; LDH > 250 U/L; CK > 80 U/L; ALT > 40 U/L, and history of dyspnoea prior to admission Fig. 2 COVID-19 disease progression by day-11 after onset of symptoms: Receiver operator characteristic (ROC) curve for discrimination, Area under the curve (AUC) = 0.733 (95% CI 0.655– 0.810). Hosmer–Lemeshow Chi-squared = 8.061, p = 0.427; DF = 8). Variables included in the model: age > 65 years, male sex, presence of cardiovascular disease, at least three abnormal parameters at day-5 after symptom onset within: CRP > 80 mg/L; NLR > 4.5; LDH > 250 U/L; CK > 80 U/L; ALT > 40 U/L, and history of dyspnoea prior to admission Older age and the presence of cardiovascular comor- bidities were associated with a higher risk of unfavour- able outcome, as highlighted by other studies [7, 15, 16]. Comorbidities, secondary bacterial infections, and altered cellular and humoral immune functions are more common in the elderly, thus increasing the risk of developing severe illness. A meta-analysis of 51 studies including a total of over 48,000 patients with confirmed COVID-19 infection showed that fatal outcomes with COVID-19 infection were strongly associated with dia- betes, hypertension and cardiovascular diseases across all age groups, thus suggesting that the risk of poor out- come associated with cardiovascular diseases may be not predicting disease progression. The Hosmer–Lemeshow goodness of fit test did not reach statistical significance (p = 0.427), indicating a good match of predicted risk over observed risk. Role of the funding sourceh Disease progression is a composite outcome defined as: death and/or transfer to ICU by day-11 after symptom onset, and/or PaO2/FiO2 ratio < 200 on day 10 after symptom onset. CRP C-reactive protein, NLR neutrophils to lymphocytes ratio, LDH lactate dehydrogenase, CK creatine kinase, AST aspartate aminotransferase, ALT alanine transaminase. p-values are reported at the top of each box plot Table 2 Univariate analysis of laboratory parameters by COVID-19 progressiona CRP C-reactive protein, NLR neutrophils to lymphocytes ratio, LDH lactate dehydrogenase, CK creatine kinase, AST aspartate aminotransferase, ALT alanine transaminase a Disease progression is a composite outcome defined as: death and/or transfer to ICU within day-11 after symptom onset, and/or PaO2/FiO2 ratio < 200 on day 10 after symptom onset b Within: CRP > 80 mg/L; NLR > 4.5; LDH > 250 U/L; CK > 80 U/L; ALT > 40 U/L. Results are presented as mean (SD) and mean difference (95% CI) or frequencies (%) and OD (95% CI), as appropriate TOT (n = 235) Disease progressiona Univariate No (n = 147) Yes (n = 88) OR (95% CI) p-value Laboratory parameters CRP > 80 mg/L 87/196 (44%) 34/124 (27%) 53/72 (74%) 7.38 (3.83 to 14.23) 0.000 NLR > 4.5 104/184 (57%) 54/117 (46%) 50/67 (75%) 3.43 (1.77 to 6.64) 0.000 LDH > 250 U/L 68/104 (65%) 37/69 (54%) 31/35 (89%) 6.70 (2.14 to 21.04) 0.001 CK > 80 U/L 56/102 (55%) 29/66 (44%) 27/36 (75%) 3.83 (1.56 to 9.39) 0.003 ALT > 40 U/L 35/148 (24%) 20/94 (21%) 15/54 (28%) 1.42 (0.66 to 3.09) 0.371 At least 3 abnormal labora- tory valuesb 66/210 (31%) 29 (22%) 37 (49%) 3.56 (1.93 to 6.56) 0.000 Table 2 Univariate analysis of laboratory parameters by COVID-19 progressiona Gentilotti et al. BMC Infect Dis (2021) 21:883 Page 5 of 8 Table 3 Multivariate analysis of clinical characteristics and laboratory parameters by COVID-19 progression dyspnoea and at least three abnormal blood parameters among CRP (> 80 U/L), ALT (> 40 U/L), NLR (> 4.5), LDH (> 250 U/L), and CK (> 80 U/L) shows fair discrimi- nation ability (AUC 0.73). To our knowledge, this is the first study assessing predictors of disease progression at specific time-points starting from the onset of COVID- 19 symptoms. Role of the funding sourceh The ROC curve for COVID-19 pro- gression is displayed in Fig. 2. Discussionh This study proposes an easy-to-use panel of five clini- cal and laboratory parameters to aid in the prediction of disease progression in COVID-19 patients. The model including age > 65 years, male sex, cardiovascular disease, Gentilotti et al. BMC Infect Dis (2021) 21:883 Page 6 of 8 affected by age [17]. In the present cohort, no statistically significant differences between male and female were observed with regards to the outcome, while in most of the studies the severity and mortality of COVID-19 have been reported to be higher in males than in females. Sex-related differences in immune system responses to pathogens has been observed, with female eliciting higher immune responses. Furthermore, RNA clearance has been reported to be delayed in male patients with COVID-19 [18]. and transaminases within the 1st week after symptoms onset to evaluate the risk of developing a life-threatening disease. In a recent work, a large group of Italian experts was invited to complete an online survey through the PAPRIKA (Potentially All Pairwise RanKings of all pos- sible Alternatives) method [25] with the aim of deter- mining the weights of several criteria for prioritizing COVID-19 patients for hospitalization [26]. Among a list of criteria, including age, body mass index, comorbidi- ties, findings at chest X-ray, CRP, and duration of symp- toms among others, the highest weights were attributed to PaO2 and peripheral oxygen saturation, denoting the well-known central role of respiratory findings in the assessment of the risk of rapid deterioration of COVID- 19 patients. These findings, alongside those outlined in our study, suggest that in addition to considering symp- toms onset and duration, the typology of symptoms should also be taken into account in an early risk assess- ment of COVID-19 deterioration. Laboratory assessment on day-5 of symptoms onset showed several parameters with significant mean dif- ferences between patients with or without poor out- come. In particular, poor outcome was associated with higher NLR, CRP, LDH, and CK, according with several studies [12, 19]. Lymphopenia and elevated neutrophil count suggest an alteration of lymphocyte function and are associated with elevated secretion of IL-6 and TNF- alpha, since these inflammatory markers contribute to lymphocyte apoptosis as well as decreased proliferation of lymphocytes [20]. Several mechanisms are responsible of this alteration, including direct infection of the lym- phocytes with SARS-CoV-2 virus, causing lymphocyte death or dysfunction. Discussionh Furthermore, the pro-inflamma- tory state with increased cytokines is inversely correlated with the induction of granulopoiesis (resulting in neutro- philia) and suppressed lymphopoiesis in the bone mar- row of patients with SARS‐CoV infection [21]. This study has several limitations. First, the sample size limits the accuracy of the findings. Second, the study was performed in a single centre, hampering the generaliza- bility and the applicability of the results to other settings. Third, the model did not undergo external validation and might be at risk of overfitting. However, we tried to miti- gate overfitting by decreasing the number of predictors. The results from this study highlight the importance of relying on homogeneous populations with same length of disease in order to build the best possible prediction model for disease progression in COVID-19 patients and allow optimal treatment and resource allocation.hi This inflammatory state, when persistent and uncon- trolled, may lead to acute respiratory distress syndrome and rapid deterioration of the clinical conditions. IL-6 upregulates hepatic CRP production, thus suggesting the plausibility of the clinical use of this inflammatory bio- marker as a prognosis predictor for COVID-19 patients [9]. Similarly, LDH blood level increases in case of cell damage and is a marker of various inflammatory states. A systematic review and meta-analysis of 28 studies reporting LDH levels in severe vs. non-sever COVID- 19 patients confirmed that LDH level can be used as a COVID-19 severity marker and is a predictor of survival [22] CK, a marker of muscle damage, has been associ- ated with a more severe COVID-19. Currently, it remains unclear whether increased levels of CK in COVID- 19 patients is due to a virus‐triggered inflammatory response or direct muscle toxicity [23]. Elevation of liver transaminases during SARS-CoV-2 infection has been frequently reported. Possible pathophysiology mecha- nisms include direct effect of the virus, liver injury medi- ated by uncontrolled immune response drug toxicity and ischemic hepatitis due to multiorgan dysfunction [24]. Based on these evidences and on our findings, patients affected by COVID-19 may benefit from blood testing including inflammation markers, complete blood count This study shows how an easy-to-use panel of five lab- oratory and clinical parameters applied in patients with COVID-19 at day-5 of symptoms onset can predict dis- ease progression with fair discriminatory power. It fur- ther suggests that the onset of symptoms might represent a useful and reliable baseline time-point for develop- ing prognostic models. Discussionh The assessment of few variables at the right time may contribute to early identification of patients at major risk of developing life-threatening COVID-19. The model can also play a role as a tool to increase homogeneity of population in clinical trials on COVID-19 treatment in hospitalized patients. A vali- dation study is needed to evaluate whether this model can reliably inform decision making and identify proper level-of-care requirements for hospitalized patients. Acknowledgements We thank Ruth Joanna Davis for the editorial support. This article was accepted as oral presentation at the 31st European Con- gress of Clinical Microbiology & Infectious Diseases, 9–12 July 2021 Author details 1 Division of Infectious Diseases, Department of Diagnostics and Public Health, University of Verona, P.le L.A. Scuro 10, 37134 Verona, Italy. 2 Department of Medicine, Section of Internal Medicine, University of Verona, EuroBlood- Net Referral Center for Iron Metabolism Disorders, Azienda Ospedaliera Universitaria Integrata Verona, 37138 Verona, Italy. 3 Cardio‑Thoracic Depart- ment, Respiratory Unit, Azienda Ospedaliera Universitaria Integrata Verona, 37124 Verona, Italy. 4 Department of Radiology, University of Verona, Piazzale L.A. Scuro, 10, 37100 Verona, Italy. 15. Dorjee K, Kim H, Bonomo E, Dolma R. Prevalence and predictors of death and severe disease in patients hospitalized due to COVID-19: a compre- hensive systematic review and meta-analysis of 77 studies and 38,000 patients. PLoS ONE. 2020;15(12):e0243191. https://doi.org/10.1371/journ al.pone.0243191. 16. Li L, Sun W, Han M, Ying Y, Wang Q. A study on the predictors of disease severity of COVID-19. Med Sci Monit. 2020;26:e927167. https://doi.org/10. 12659/MSM.927167 (Published 2020 Sep 23). Received: 28 April 2021 Accepted: 20 August 2021 17. Bae S, Kim SR, Kim M-N. Impact of cardiovascular disease and risk factors on fatal outcomes in patients with COVID-19 according to age: a system- atic review and meta-analysis. Heart. 2021;107:373–80. https://doi.org/10. 1136/heartjnl-2020-317901. Acknowledgements This article was accepted as oral presentation at the 31st European Con- gress of Clinical Microbiology & Infectious Diseases, 9–12 July 2021 Gentilotti et al. BMC Infect Dis (2021) 21:883 Page 7 of 8 Availability of data and materials 10. Shang Y, Liu T, Wei Y, Li J, Shao L, Liu M, et al. Scoring systems for predict- ing mortality for severe patients with COVID-19. EClinicalMedicine. 2020;3(24):100426. https://doi.org/10.1016/j.eclinm.2020.100426. 10. Shang Y, Liu T, Wei Y, Li J, Shao L, Liu M, et al. Scoring systems for predict- ing mortality for severe patients with COVID-19. EClinicalMedicine. 2020;3(24):100426. https://doi.org/10.1016/j.eclinm.2020.100426. The datasets analysed during the current study are available from the cor- responding author on reasonable request. 11. Wendel Garcia PD, Fumeaux T, Guerci P, Heuberger DM, Montomoli J, Roche-Campo F, et al. Prognostic factors associated with mortality risk and disease progression in 639 critically ill patients with COVID-19 in Europe: initial report of the international RISC-19-ICU prospective obser- vational cohort. 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Presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with COVID-19 in the New York City Area. JAMA. 2020;323:2052. 4. Richardson S, Hirsch JS, Narasimhan M, et al. Presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with COVID-19 in the New York City Area. JAMA. 2020;323:2052. 22. Szarpak L, Ruetzler K, Safiejko K, et al. Lactate dehydrogenase level as a COVID-19 severity marker. Am J Emerg Med. 2020. https://doi.org/10. 1016/j.ajem.2020.11.025 (published online ahead of print, 2020 Nov 15). y 5. European Centre for Disease Prevention and Control. COVID-19 surveil- lance report. https://covid19-surveillance-report.ecdc.europa.eu/. Accessed 20 Jan 2021. 5. European Centre for Disease Prevention and Control. COVID-19 surveil- lance report. https://covid19-surveillance-report.ecdc.europa.eu/. Accessed 20 Jan 2021. 6. Cohen PA, Hall LE, John JN, Rapoport AB. The early natural history of SARS-CoV-2 infection: clinical observations from an urban, ambulatory COVID-19 clinic. Mayo Clin Proc. 2020;95(6):1124–6. https://doi.org/10. 1016/j.mayocp.2020.04.010. 23. The authors declare no competing interests. 14. Centers for Disease Control and Prevention. Interim clinical guidance for management of patients with confirmed Coronavirus Disease (COVID- 19). https://www.cdc.gov/coronavirus/2019-ncov/hcp/clinical-guidance- management-patients.html#:~:text=Among%20patients%20in%20mul tiple%20early,median%20time%20from%20onset%20of. Accessed 20 Jan 2021. Ethics approval and consent to participate The study was approved by the hospital Institutional Review Board (“Comitato etico per la sperimentazione clinica delle province di Verona e Rovigo”, 2577CESC). A written informed consent for study participation was obtained from the patients or from the legal guardian on enrolment. The study protocol is performed in accordance with the relevant guidelines. Further information are available at ClinicalTrials.gov NCT04497194. 12. Mesas AE, Cavero-Redondo I, Álvarez-Bueno C, Sarriá Cabrera MA, Maf- feideAndrade S, Sequí-Dominguez I, Martínez-Vizcaíno V. Predictors of in-hospital COVID-19 mortality: a comprehensive systematic review and meta-analysis exploring differences by age, sex and health conditions. PLoS ONE. 2020;15(11):e0241742. https://doi.org/10.1371/journal.pone. 0241742. Consent for publication 13. Zeng ZY, Feng SD, Chen GP, Wu JN. Predictive value of the neutrophil to lymphocyte ratio for disease deterioration and serious adverse outcomes in patients with COVID-19: a prospective cohort study. BMC Infect Dis. 2021;21(1):80. https://doi.org/10.1186/s12879-021-05796-3. All the authors gave their consent for the publication. Authors’ contributions 7. Galloway JB, Norton S, Barker RD, et al. A clinical risk score to identify patients with COVID-19 at high risk of critical care admission or death: an observational cohort study. J Infect. 2020. https://doi.org/10.1016/j.jinf. 2020.05.064 (published online ahead of print, 2020 May 29). EG, AV, MC, GB, ER, NS, DM and SM collected the data. EG performed the statis- tical analysis and wrote the first draft of the manuscript. ET, EG, DG, CM and ER contributed in the conceptualisation of the study. PDN reviewed the literature, edited and revised all the draft of the manuscript. AG did the visualization. AS and MC reviewed the manuscript. ET supervised the work, provided advice to the statistical analysis, and reviewed all the drafts of the manuscript. All authors read and approved the final manuscript. 8. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Yet al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020;395(10223):497–506. https://doi.org/10.1016/S0140-6736(20)30183- 5. Erratum in: Lancet. 2020 Jan 30. (Epub 2020 Jan 24). 8. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Yet al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020;395(10223):497–506. https://doi.org/10.1016/S0140-6736(20)30183- 5. Erratum in: Lancet. 2020 Jan 30. (Epub 2020 Jan 24). edited and revised all the draft of the manuscript. AG did the visualization. AS and MC reviewed the manuscript. ET supervised the work, provided advice to the statistical analysis, and reviewed all the drafts of the manuscript. All authors read and approved the final manuscript. 9. Wynants L, Van Calster B, Collins GS, et al. Prediction models for diagnosis and prognosis of covid-19 infection: systematic review and critical appraisal [published correction appears in BMJ. 2020 Jun 3;369:m2204]. BMJ. 2020;369:m1328. https://doi.org/10.1136/bmj.m1328. (Published 2020 Apr 7) Funding This study is part of React-COVID-19 project funded by Fondazione CARIVERONA. This study is part of React-COVID-19 project funded by Fondazione CARIVERONA. 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https://openalex.org/W3030354598	https://europepmc.org/articles/pmc7273463?pdf=render	English	null	Resuscitation for Cardiac Arrest and Postcardiac Arrest Care	Emergency medicine international	2,020	cc-by	1,610	Editorial Resuscitation for Cardiac Arrest and Postcardiac Arrest Care Yan-Ren Lin ,1,2,3 Aristomenis K. Exadaktylos,4 Kee-Chong Ng,5 and John M. Ryan6 1Department of Emergency and Critical Care Medicine, Changhua Christian Hospital, Changhua, Taiwan 2School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 3School of Medicine, Chung Shan Medical University, Taichung, Taiwan 4Department of Emergency Medicine, Bern University Hospital, Freiburgstrasse, Bern, Switzerland 5Department of Emergency Medicine, KK Women’s and Children’s Hospital, Singapore 6Emergency Department, St Vincent’s University Hospital, Elm Park, Dublin, Ireland Yan-Ren Lin ,1,2,3 Aristomenis K. Exadaktylos,4 Kee-Chong Ng,5 and John M. Ry 1Department of Emergency and Critical Care Medicine, Changhua Christian Hospital, Changhua, Taiwan 2School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 3School of Medicine, Chung Shan Medical University, Taichung, Taiwan 4Department of Emergency Medicine, Bern University Hospital, Freiburgstrasse, Bern, Switzerland 5Department of Emergency Medicine, KK Women’s and Children’s Hospital, Singapore 6Emergency Department, St Vincent’s University Hospital, Elm Park, Dublin, Ireland Correspondence should be addressed to Yan-Ren Lin; h6213.lac@gmail.com Correspondence should be addressed to Yan-Ren Lin; h6213.lac@gmail.com Received 9 May 2020; Accepted 11 May 2020; Published 27 May 2020 Received 9 May 2020; Accepted 11 May 2020; Published 27 May 2020 Copyright © 2020 Yan-Ren Lin et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A well-coordinated team, both in prehospital and in-hospital resuscitation, will save lives. For example, high performance cardiopulmonary resuscitation (HPCPR) and extracorporeal cardiopulmonary resuscitation (ECPR), which were both established by closely coordinated teamwork, were recently demonstrated to shorten interruptive time of chest com- pression and reduce the postcardiac arrest syndrome, re- spectively [1–4]. Some leading councils (i.e., American Heart Association (AHA) and European Resuscitation Council (ERC)) pointed out the importance of group monitoring in performing resuscitation and emphasized the quality of postcardiac arrest care. Recently, AHA revised the golden guidelines for cardiac arrest resuscitation and postcardiac arrest care on January 2, 2018 [5]. Several leading journals also recently discussed how to increase the quality and outcome in the postcardiac arrest care (including strategic application of ECPR and hypothermia in treating pediatric or traumatic patients), thus making this topic hot and timely [1, 4, 6, 7]. Te updated knowledge globally guides the treatment strategies of critical and emergency care. All new guidelines and knowledge can be clinically applied, chal- lenged, and improved upon. Hindawi Emergency Medicine International Volume 2020, Article ID 4053960, 2 pages https://doi.org/10.1155/2020/4053960 Hindawi Emergency Medicine International Volume 2020, Article ID 4053960, 2 pages https://doi.org/10.1155/2020/4053960 Hindawi Conflicts of Interest Te editors declare that they have no conﬂicts of interest regarding the publication of this special issue. Acknowledgments Te editors appreciate all authors who made contribution for this special issue. Tey hope this collection of articles will be useful to the scientiﬁc community. Yan-Ren Lin Aristomenis K. Exadaktylos Kee-Chong Ng John M. Ryan Editorial Resuscitation for Cardiac Arrest and Postcardiac Arrest Care Te prognosis of an OHCA patient was more likely to be aﬀected by the building structure and use rather than the ﬂoor height. [2] H. J. Park, W. J. Jeong, H. J. Moon et al., “Factors associated with high-quality cardiopulmonary resuscitation performed by bystander,” Emergency Medicine International, vol. 2020, Ar- ticle ID 8356201, 6 pages, 2020. [3] Y. R. Lin, M. H. Wu, T. Y. Chen et al., “Time to epinephrine treatment is associated with the risk of mortality in children who achieve sustained ROSC after traumatic out-of-hospital cardiac arrest,” Critical Care, vol. 23, no. 1, 2019. [4] C. Adler, T. Heller, F. Schregel et al., “TIMP-2/IGFBP7 predicts acute kidney injury in out-of-hospital cardiac arrest survivors,” Critical Care, vol. 22, no. 1, 2018. [5] M. E. Kleinman, Z. D. Goldberger, T. Rea et al., “American heart association focused update on adult basic life support and cardiopulmonary resuscitation quality: an update to the American heart association guidelines for cardiopulmonary resuscitation and emergency cardiovascular care,” Circulation, vol. 137, no. 1, pp. e7–e13, 2017. [6] J. J. Lin, Y. J. Lin, S. H. Hsia et al., “Early clinical predictors of neurological outcome in children with asphyxial out-of-hos- pital cardiac arrest treated with therapeutic hypothermia,” Frontiers in Pediatrics, vol. 7, p. 534, 2020. [7] E. Corral Torres, A. Hernandez-Tejedor, R. Suarez Bustamante, R. de Elias Hernandez, I. Casado Florez, and A. San Juan Linares, “Prognostic value of venous blood analysis at the start of CPR in non-traumatic out-of-hospital cardiac arrest: as- sociation with ROSC and the neurological outcome,” Critical Care, vol. 24, no. 1, 2020. Tis special issue highlights several articles that report improvements in OHCA-related oxygenation, airway management, resuscitation strategies, and outcomes. We strongly believe that readers will gain useful information from this issue. Editorial Resuscitation for Cardiac Arrest and Postcardiac Arrest Care pointed out that Injury Severity Score, Glasgow Coma Scale, and Revised Trauma Score were all associated with increased mortality, prolonged intensive care unit stay, and longer length of hospital stay. Kim et al. administered the Mini-Mental State Examination (MMSE) to 92 cardiac arrest survivors who were treated with targeted temperature management immediately after regaining consciousness. Cognitive impairments were common im- mediately after patients regained consciousness but recov- ered substantially before intensive care unit discharge. Moreover, they also reported hypoalbuminemia was com- mon after cardiac arrest, and the serum albumin level at admission was associated with poor neurological outcomes at 6 months after cardiac arrest in patients treated with targeted temperature management. Choi et al. analyzed 6,335 OHCA patients. Tey found that both the EMS re- sponse times to OHCA events in high-rise buildings and the probability of a neurologically favorable discharge diﬀered obviously between homes and public places. Te prognosis of an OHCA patient was more likely to be aﬀected by the building structure and use rather than the ﬂoor height. Hsu Chen et al. analyzed the risk of new-onset heart failure of 49,101 nontraumatic OHCA adult patients. Tey found that, in patients aged from 61 to 75 years, a history of myocardial infarction or cardiomyopathy, and ischemic heart disease or infection as comorbidities, occurring during hospitalization were strong risk factors for new-onset heart failure. However, extracorporeal membrane oxygenation could decrease this risk. Most heart failure events occurred within 60 days after OHCA. For young traumatic OHCA patients, Huang et al. pointed out that Injury Severity Score, Glasgow Coma Scale, and Revised Trauma Score were all associated with increased mortality, prolonged intensive care unit stay, and longer length of hospital stay. Kim et al. administered the Mini-Mental State Examination (MMSE) to 92 cardiac arrest survivors who were treated with targeted temperature management immediately after regaining consciousness. Cognitive impairments were common im- mediately after patients regained consciousness but recov- ered substantially before intensive care unit discharge. Moreover, they also reported hypoalbuminemia was com- mon after cardiac arrest, and the serum albumin level at admission was associated with poor neurological outcomes at 6 months after cardiac arrest in patients treated with targeted temperature management. Choi et al. analyzed 6,335 OHCA patients. Tey found that both the EMS re- sponse times to OHCA events in high-rise buildings and the probability of a neurologically favorable discharge diﬀered obviously between homes and public places. Editorial Resuscitation for Cardiac Arrest and Postcardiac Arrest Care In this special issue, we would like to provide an opportunity to introduce various related works discussing resuscitation for cardiac arrest and post- cardiac arrest care. greater level of oxygenation and less lung and brain injuries than intermittent positive pressure ventilation. Huang et al. performed a meta-analysis study and concluded that ad- ministering of high-ﬂow nasal cannula therapy in the emergency department (ED) for respiratory failure patients might decrease the intubation rate compared with con- ventional oxygen therapy. Cho et al. investigated whether those capnometer readings could be easily aﬀected by ﬂuid exposure during treatment of critically ill patients. Tey prospectively compared the diﬀerences of the ETCO2 level between proximal or distal connecting direct connect catheter mount (DCCM) and found that application of DCCM onto the capnometer setting seems to be eﬀective in reducing capnometer malfunctioning under ﬂuid exposing conditions. Lee et al. investigated three-dimensional shapes and deformability of red blood cells (RBCs) during and after asphyxia cardiac arrest in rats. In their study, quantitative phase imaging results revealed that RBC membrane ﬂuc- tuations, sphericity, and surface area did not change sig- niﬁcantly during CPR or after return of spontaneous circulation (ROSC) compared with initial values. Second, the association between deﬁnitive airway es- tablishment and outcomes of patients were also discussed in this special issue. Choi et al. reported that the mean intu- bation time was signiﬁcantly shorter in using I-gel blind intubation than using I-gel bronchoscopic intubation or Macintosh laryngoscope. In the ﬁeld of oxygenation and cardiopulmonary re- suscitation (CPR) or respiratory failure, Lee et al. introduced an animal study and demonstrated that continuous ﬂow insuﬄation of oxygen using a one-way valve resulted in a Te other studies focused on the short/long-term out- comes of out-of-hospital cardiac arrest (OHCA) patients. Emergency Medicine International 2 Hsu Chen et al. analyzed the risk of new-onset heart failure of 49,101 nontraumatic OHCA adult patients. Tey found that, in patients aged from 61 to 75 years, a history of myocardial infarction or cardiomyopathy, and ischemic heart disease or infection as comorbidities, occurring during hospitalization were strong risk factors for new-onset heart failure. However, extracorporeal membrane oxygenation could decrease this risk. Most heart failure events occurred within 60 days after OHCA. For young traumatic OHCA patients, Huang et al. References [1] E. Gregers, J. Kjaergaard, F. Lippert et al., “Refractory out-of- hospital cardiac arrest with ongoing cardiopulmonary resus- citation at hospital arrival—survival and neurological outcome without extracorporeal cardiopulmonary resuscitation,” Crit- ical Care, vol. 22, no. 1, 2018.
https://openalex.org/W4288114870	https://zenodo.org/records/3386279/files/035LB.pdf	English	null	"Spoken Portrait" - Head Description	Zenodo (CERN European Organization for Nuclear Research)	2,019	cc-by	3,860	Lucia Cerasela Balan Faculty of Economic Sciences and Public Administration, Law specialization from Suceava, Romania Master in Criminal Sciences at ”Dimitrie Cantemir” Christian University of Bucharest, Lucya_9212@yahoo.com Lucia Cerasela Balan Lucia Cerasela Balan Faculty of Economic Sciences and Public Administration, Law specialization from Suceava, Romania Master in Criminal Sciences at ”Dimitrie Cantemir” Christian University of Bucharest, Lucya_9212@yahoo.com ABSTRACT: The description of the signs referred to in the Specialty literature and the ”Spoken Portrait” Method, has the characteristics of the body in its entirety, the emphasis being placed on the anatomic features of the face, being covered in the description of the volume, shape, position and color of the observed parts, each element being appreciated in the report with other anatomical elements that make up the described ensemble. Descriptions differ depending on who performs them. In some cases, the description of an individual by the eyewitness may be incomplete or even erroneous because of objective or subjective circumstances that prevent good perception. As a person can be distinguished from another, in a Forensic Sciences and yet identified way, it is necessary to describe his anatomical characteristics according to scientific methods, using appropriate terminology and precise criteria for dimensioning. In this sense, the identification is carried out by means of specialized methods and techniques in the framework of criminal prosecution activities, carried out according to tactical rules specific to the hearing of the witnesses or victims of the offense. KEYWORDS: color, figure, forensic identification, signs, spoken portrait Introduction The foundation of the portrait was established in the XV Century by Leonardo da Vinci, who systematized the organs of the human body, appreciating them to be diverse: straight, concave, convex. It indicates that the technique of describing the portrait is the way to retain and reproduce the profile of a human figure after being seen once (Lucard 1932, 119). Every person has a certain, objective, invariable individuality in its essence and easily identifiable. It must be borne in mind that man is constantly and continuously evolving his particular features. Body features can also allow for changes that are largely conditioned by man's aspiration, such as the port of beard, mustache, the development of corporeality, the change in hair color, etc. Diseases or trauma suffered by the human body, certain surgical interventions may give rise to changes that are distinctive signs of that person. And age gives rise to changes that relate to the dimensions of certain organs or parts of the body, except those that have a bone or cartilage structure. The identification of persons by the portrait method is based on the principles of forensic identification, which is based on the thesis of didactic materialism, according to which all the objects of the material world are identical by themselves, they are individual and their characteristics are not repeated (Engels 1966, 180). In describing the signals of a person, a precise and unitary terminology must be used, in a logical sequence. The description of the signals is done from the front or from the frontal plane and from the profile or from the lateral plane. The features of the head and face hold the main place in the realization of the spoken portrait and will be completed with the description of the whole body and of the clothing (Buzatu 2013, 92). The hair Hair on the head is described by: color, nature, thread direction, insertion and hair loss. The color of the hair is marked according to the variants of shades: black-blue, black, dark brown, light brown, blond-red, blonde-gold, gray blond, platinum, gray and white. Depending on the nature of the hair, it is classified as: thick and slim hair, the right hair - medium as thread thickness, curly hair, slick hair, curly hair. The hair port is determined by the type of combing presented in various shapes: right-hand, left-hand, middle-right, short-cut, full-grained, completely knotless. The hairline direction depends on its length. As direction, the thread can be oriented straight, forward, backward, left or right. Hair density is appreciated as dense, medium, rare, and intermediate. The insertion of hair on a person's forehead can be: straight, convex or concave. Cheeks can be appreciated as: large, appropriate or small, indicating their frontal, parietal or occipital position. Also, the data on how to cut the hair in front of the ears, indicating whether or not they are preferred, what is the size and form of their completion. Forehead Forehead can be viewed both from the front and the profile, insisting on the height, width, contour, inclination and peculiarities. The height of the forehead is appreciated as tripartite: high, medium or small relative to the distance between the hair and the root of the nose. The middle form is considered to be the normal height of the forehead. As regards the width of the forehead, the description is made after a tripartite classification: narrow, medium or wide, after the distance between the two temples. Leaning of the forehead is established by reporting the real inclination to an imaginary vertical plane that runs through the base and root of the nose. Depending on this imaginary plane, the forehead is considered to be vertical, bulging, prominent, oblique, or fugitive. To consider that the forehead position is normal, it should be slightly inclined. Among the peculiarities we can mention in the forehead description: the protrusion of the arches, the frontal sinus, the frontal bosses and the curved profile. Arcades can be large, medium or large. Their size varies with the angle of inclination of the bone protrusion located above the nose, on the center of the forehead, and the frontal bones that also constitute bone protrusions are located in the lateral sides of the forehead. They can be remarked as peculiarities: excessive lifting, ridding the forehead, deep grooves in the depth of it. As the constituent elements of the forehead are the arches and can be appreciated according to the size: large, medium, small and are reported by the degree of inclination of forehead. Head description In Forensic practice and theory, the main elements of the face are described according to their location, starting with those located in the upper area, continuing with the middle and the lower ones. The figure of a person is examined from profile or front and includes face areas, respectively: a. The frontal area, limited to the top of the hair insertion area and continuing to the root of the nose; ; b. The nasal area between the root of the nose and the base of the nose; . The mouth area includes the face portion between the base of the nose and the chin tip. c. The mouth area includes the face portion between the base of the nose and the chin tip. Five areas, namely forehead, eyes, nose, mouth and chin, can be considered, but the description will not be limited to them. Each constituent element of the figure will be described separately with the characteristic or particular elements, respectively: 216 Eyebrows They are hairstyles placed above the eyes and are appreciated by their length, thickness, thickness, arc, direction, or position occupied by eyeball or color. In some cases, the eyebrows may be missing altogether, or they may be made up of a few splinters or may also be cosmetically tattooed. By length, the eyebrows can be long, medium or short, as well as thin, well-suited, very often quite stiff. Depending on the arc, they may be straight, arched upwards or downwards, serpentine, angular, and after the direction they are straight, oblique up or down. The distance between them can be: merged, close or distant. The hair color of the person concerned is taken into account when describing the color of the eyebrows, with the indication that capillary hair discolours faster. As peculiarities, the eyebrows may be lowered, overburdened, or exhibiting specific forms due to scars, depilation, or pins. Nose It is the olfactory organ, with an important role in sketching human physiognomy as a dominant element in the composition of the face and which can be observed and retained after the size of the nasal pyramid after the contour line or base. The first segment of the respiratory apparatus is formed by the nose, in the form of a downward- facing pyramid, somewhat limited by the cross-section of the eyebrows, and at the bottom of the upper lip region. pp p g There are some exceptions that differ in some breeds, namely: the right nose is specific to the Greeks, the Romanian aquilla, the African Americans have a wider nose in the lower part of the nose, and the Europeans have a light nose. The nose is examined both from the front and the profile. The elements that play an important role in making the portrait are: the root of the nose, the contour line, the base of the nose, the height and width, the protrusion of the nasal pyramid and the possible particularities. Observation should be related to the position it has been assessed (Voinea 1999, 10). The root of the nose represents the hollow between the eye and eyebrow arches, and is appreciated in terms of depth; according to this principle they meet: the root, the middle root or the small root. According to the frontal observation, the root of the nose may be wide, suitable or narrow. The nose of the nose begins from the deepest part of the nose root and ends at its base. It is generally seen from the profile and can take various forms such as rectilinear, aquiline, corrugated, concave, etc. The base of the nose is described both in the aspect of the direction and the width of the nose, the latter being the width of the nose. The imaginary line that starts from the point of connection of the nostril with the human figure to the tip of the nose determines the direction of the base of the nose. The direction can be seen from the profile and can be horizontal, raised or lowered. The height and width of the nose is observed and appreciated, looking individually in the profile, being also replicated by the vertical line joining the root of the nose with the base of the nose base with the naso-labial groove. The eyes They represent the organs of sight and are located in the orbital cavity at the level of the base. In the composition of the eyeball, the retina, a photoreceptor formation, elements of it that assure the projection of the light on the retina. The ocular bulb consists of three membranes: sclerotic, retina, choroid, cornea, aqueous humor, lens and glassy humor. From the visible parts we mention the pupil, iris and sclerotica or the white of the eyes. The eyelids have a protective role and therefore their eyes RAIS Conference Proceedings, August 19-20, 2019 217 are protected by them. The eyelids close the base of the orbit, delineate the palpinal crack that opens during the operation of the visual apparatus and closes during sleep. In making the portrait, the eyes are presented in shape, position, color, interocular space, eyelid peculiarities, genes, depth in orbit (Stancu 2010,174). Each individual has an iris characteristic pigmentation that cannot be easily recalled or appreciated, because in some opinions it is influenced by light. According to other opinions, this pigmentation remains stable in adults (Voinea 1999,10). y g g p p g ( ) According to the orifice, the eyes can be described as righteous, with external or inner commands raised or lowered, by size they can be large, medium or small. Orbital placement is appreciated from the profile, so the eyes can be clogged in orbits, prominent or normal. The iris, the colored strip around the pupil, is the one that defines the color of the eyes. The most common eye colors are: black, brown, blue, gray, green, and in some cases may be discolored. Among the peculiarities that are recorded as particular signs we can remember his eyes, the missing eye, the different color of the two eyes, the presence of cataracts, the opacity of the cornea, the total lack of color. Also interested in eyelids, genes, eye orbits or interocular space. The eyelids are presented in length, breadth, description, and whether they are large, medium or small. The genes are described by orientation, length or length and can be: Straight, oriented up or down; long, suitable or short; frequent or rare. Orbits of eyes are valued in terms of their height and depth: high or low, hollow or full. Interaction space is appreciated according to the distance between the two eyes and may be small, very small, appropriate, large or very large. RAIS Conference Proceedings, August 19-20, 2019 The color of the nose generally has the same hue with the rest of the face, but not in all cases. Sometimes the color of the nose may be different, especially at the tip of the nose, which may appear under a red, red-brown, violet shade, indicating some skin diseases, a state of debilitating or even excessive drinking. The color of the nose generally has the same hue with the rest of the face, but not in all cases. Sometimes the color of the nose may be different, especially at the tip of the nose, which may appear under a red, red-brown, violet shade, indicating some skin diseases, a state of debilitating or even excessive drinking. Among the peculiarities are scars, warts, excessive pore expansion, congenital or accidental malformations. Chin The beard is the lowest part of the human figure, it continues the face and gives it the form that characterizes it. It can be examined both from the front and from the profile of the inclination, height, width and its peculiarities. From the profile we can see a slanting or receding, vertical, preeminent chin. As peculiarities, we can remember the bilobate, elongated, double, prominent chub with chub (Ciopraga, Iacobuţă 2001, 196). ( p g ţ ) Beard and mustaches are hair accessories located in the mouth, described in terms of shape, size, position or color. The beard is considered large when it encompasses the whole face, its hair being long, small or barby when the hair is short on the entire face. It may also appear as a beak, a bundle of yarn, located at the bottom of the chin. By shape, the beard may be a sailor, a collar, a beard full, etc. Musts can be large with corners raised or lowered, cut small on the lip, or small after the width of the nose, arched in the shape of the swallowtail, etc. The mouth area It includes the lower part of the face, from the subchannel groove to the top of the chin, including the mouth, lips, teeth and chin and forms the lower third of the human figure (Dragomir, Niţu and Boalbeş 2008, 243). The mouth is examined from the front and from the profile, both as a general aspect and as a peculiarity. As a general aspect, the size and orientation of the corners are described. The size of the mouth is appreciated by three gradations such as: small, medium, large but there may also be intermediate variations between them. The mouths of the mouth and the comics can be lifted up, very high, horizontal or appropriate, lowered or very lowered. They may also experience a unilateral deviation to the right or to the left following a habit, congenital illness or malformation. Lips malformations, considered as peculiarities, are: rabbit lip, scarring, elevation of the median lower lip, lower lip hanging. The teeth are positioned in the alveolar edge of the two maxillaries and are grouped as follows: 8 incisors, 4 canines, 8 premolars and 12 molars. They can be described in terms of size, distance, position, color, degree of wear and can be: long, outward, distant, missing, fake, dressed. In general, the incisors are described, namely their shape, the distance between them, if they are out, the way the root is dressed by the gum, because they are discovered and can be sensed during speech or laughter. A number of abnormalities can be observed, some of them congenital: incisors discovered, distant, welded, inclined outward or inward, incisive; but also acquired abnormalities such as the absence of one or more incisors, horizontal fracture, wear, caries. Nose Under the height, it may be large, medium or small, and broad, medium or narrow width. The nostrils' conformity is noted with certain characteristics, without the use of gradations of size, appreciating them in the particularities of: glued, dilated, turned, tightened, raised or flattened on one side. The tip of the nose is noted as a peculiarity without the use of gradations such as: thin, thick, bilobed, deviated to the right, diverted to the left, etc. 218 RAIS Conference Proceedings, August 19-20, 2019 Ear It is appreciated as one of the most informative organs of the face. It is of particular importance because of its position on the contour of the head, but also its shape, size or other morphological elements (Doroş 1996, 205). It is recorded that the ear has a double character, i.e. it is immutable in form, from birth to death and variable, in other words, two similar ears do not meet (Cârjan, and Chiper 2009, 187). Head position is parallel to the cranial bone, but in some people the ears may be located at the top or bottom. The outer ear pavilion consists of a set of cartilages, namely five ridges and three depressions. The characteristic elements are: helix, antihelix, tragus, antitragus, lobe or ear, conca or auditory aperture, digital depression and internal depression. They present various varieties of shape, position or proportions as will be briefly presented: 219 RAIS Conference Proceedings, August 19-20, 2019 The helix, the ridge that surrounds the ear pavilion from the front to the rear; is composed of four parts: original, anterior, superior, inferior; may be large, medium or small. Antihelix, the cartilage cartilage located inside the ear, parallel to the upper and posterior helix; may be complete, apparently only in the upper area or absent. Tragus, protrusion from cartilage tissue positioned under the original helix that partially covers the auditory aperture; the volume is rated as high, medium or small; can be sharp, bifurcated or prominent. Antitragus, prominence of the cartilage located in the lower part of the ear shell and can be described as: concave, convex, bulging, straight. The lob or ear, the fleshy lower part of the ear pavilion, which has various shapes, sizes, and with respect to the contour of the free part of the lobe can be presented as square, scattered, angular, in the echery, etc. y Conca, the depression formed in the mouth of the auditory aperture, which is presented as a funnel and is appreciated being related to the other elements of the ear pavilion. y Conca, the depression formed in the mouth of the auditory aperture, which is presented as a funnel and is appreciated being related to the other elements of the ear pavilion. The digital depression is located behind the helix before and under the top helix tal depression is located behind the helix before and under the top helix. Ear The internal depression is located between the posterior upper helix and the earlobe's antihelix with an elongated shape (Doros 1996, 205). As peculiarities can be mentioned the bifurcated tragus, the sharp ears in the upper part, the notched edge, the ears very close to the head etc. Wrinkles are leather creases that can be caused by either aging or expression. They are described by depth, shape, number, and the area of the face where they are positioned. Wrinkles can be corrected through surgical procedures, women are most often used in such procedures, and this should be mentioned if it is noticeable (Popa 2011, 25). Skin color or chromaticity peculiarities vary by race. The color varies from white to black and is essential for making the portrait spoken. It should be noted that some people will change the color of their skin by exposure to the sun, its discoloration, the use of make-up or make-up, aspect to be considered. Conclusions When a person's spoken portrait is drawn, the characterization of the signs will be made according to size, shape, position, and some and color. Size is shown by comparison with the size of other parts of the body and will be noted with small, mediocre and great; when the detail is particularly small or very large it will be marked with very little or very large. y y g The shape is reproduced by the linear outer contour of the organ to be presented to fit into one of the geometric figures that is similar: round, oval, diamond, triangle, rectangle. The color will be recorded for details that have this attribute, such as the skin, hair, eyes or other organs or parts of organs showing a particular coloring to the rest of the skin. The position of a human body detail will be judged relative to the horizontal or vertical plane or neighboring areas, the notation being horizontal, vertical or oblique. When identifying a person's signals, certain factors that affect the accuracy of their observation and retention must be taken into account. The main factors responsible for this influence are the following: the distance from which it is viewed, the place where the person is located, the time when the observation is performed and the factors that may influence: day, night, atmospheric conditions, visibility, duration of observation, as well as its clothing, the ability to perceive and the appreciation of the observer. The primordial assimilation of the principles of the portrait spoken by the police and their skillful application allow a visible and coherent description of the anatomical and functional signs of the different people. RAIS Conference Proceedings, August 19-20, 2019 220 ş, gy, q ș yg p p Dragomir, Ilie; Niţu, Adrian and Boalbeş, Marian. 2008. Criminalistic Guidance for Gendarmes. Bucharest: Ministry of the Interior Publishing House. , q y Popa, Gheorghe. 2011. Methods and techniques of forensic identification. Bucharest: AIT Laboratoires Publishing House. Stancu Emilian 2010 Forensic Treaty Bucharest Universul Juridic Publishing House ncu, Emilian. 2010. Forensic Treaty. Bucharest. Universul Juridic Publishing House. D 1999 ”T h i f P i S k d ” I C l M 4 Voinea, Dan. 1999. ”Technique of Portraiture Speaked.” In Criminalistic Magazine, no 4. References Buzatu, Nicoleta-Elena. 2013. Forensics. Bucharest: Pro Universitaria Publishing House. Cârjan, Lazăr and Chiper, Mihai. 2009. Criminalistics, Tradition and Modernism. Bucharest: Curtea V House. Doraş, Simion Gh. 1996. Criminology, Forensic Techniques. vol. 1. Chișinău: Editorial-Polygraphic Enterprise. gy q ș yg p p Dragomir, Ilie; Niţu, Adrian and Boalbeş, Marian. 2008. Criminalistic Guidance for Gendarmes. Bucharest: Ministry of the Interior Publishing House. Engels, Fr. 1966. Dialectics of Nature. Bucharest: Politics Publishing House. Locard, Edmond. 1932. Traite de Criminalistique. Vol. III. Lyon. Popa, Gheorghe. 2011. Methods and techniques of forensic identification. Bucharest: AIT Laboratoire Stancu, Emilian. 2010. Forensic Treaty. Bucharest. Universul Juridic Publishing House. Voinea, Dan. 1999. ”Technique of Portraiture Speaked.” In Criminalistic Magazine, no 4. Voinea, Dan. 1999. ”Technique of Portraiture Speaked.” In Criminalistic Magazine, no 4. Voinea, Dan. 1999. ”Technique of Portraiture Speaked.” In Criminalistic Magazine, no 4.
https://openalex.org/W2981267886	https://peerj.com/articles/ochem-1.pdf	English	null	Direct multicomponent synthesis of benzocoumarins	null	2,019	cc-by	8,127	ABSTRACT A straightforward and versatile protocol for the synthesis of dibenzo[b,d]pyran-6-ones from readily available 3-carbonylcoumarins is reported. Our strategy is based on a reaction cascade of successive [4+1] and [4+2] cycloadditions that occur in one single operation. This work illustrates the unprecedented use of a multicomponent reaction of isocyanides for the preparation of this biologically relevant type of compounds. Notably, in this highly convergent and atom-economic process, one new single and two new double carbon-carbon bonds are formed in a simple synthetic operation. y Keywords Cycloadditions, Multicomponent reactions, Isocyanides, Benzocoumarins, Natural products, Diels-alder reaction, Tandem reactions How to cite this article Bornadiego A, Díaz J, Marcos CF. 2019. Direct multicomponent synthesis of benzocoumarins. PeerJ Organic Chemistry 1:e1 http://doi.org/10.7717/peerj-ochem.1 Direct multicomponent synthesis of benzocoumarins Ana Bornadiego, Jesús Díaz and Carlos Fernández Marcos Laboratory of Bioorganic Chemistry & Membrane Biophysics (L.O.B.O.), School of Veterinary Sciences, University of Extremadura, Cáceres, Spain Laboratory of Bioorganic Chemistry & Membrane Biophysics (L.O.B.O.), School of Veterinary Sciences, University of Extremadura, Cáceres, Spain INTRODUCTION Coumarins or 2H-chromen-2-ones represent an important class of compounds, which are ubiquitous structures as secondary metabolites in plants and many species of fungi and bacteria. The parent member of this class, coumarin itself, was first isolated by Vogel in 1820 from the tonka bean (Dipteryx odorata) (Vogel, 1820). Since then, many different simple and polycyclic coumarins have been discovered and extensively studied due to their potent and singular biological activities (Stefanachi et al., 2018; Medina et al., 2015; Borges et al., 2005; Lavoie et al., 2019) and their photophysic properties (Wagner, 2009; Trenor et al., 2004). Among them, benzo[c]coumarins (1; Fig. 1) have emerged as privileged structures in drug discovery (Garazd & Garazd, 2016; Mao et al., 2014). Relevant examples include mycotoxin alternariol (2), (Solfrizzo, 2017) antioxidant and anticancer ellagic acid (3), (Ceci et al., 2018) synthetic cannabinoid agonists cannabilactones (4), (Khanolkar et al., 2007) antimalarial dioncolactone (5) (Fran¸cois et al., 2016) and the glucoside derivatives with antitumor properties gilvocarcins (6), (Tomita, Takahashi & Tamaoki, 1982) chrysomycins (7), (Matson et al., 1989) and ravidomycins (8; Fig. 1) (Yamashita et al., 1998). Submitted 20 May 2019 Accepted 16 July 2019 Published 15 October 2019 Corresponding authors Jesús Díaz, jdal@unex.es Carlos Fernández Marcos, cfernan@unex.es Academic editor Robert Smith Additional Information and Declarations can be found on page 11 DOI 10.7717/peerj-ochem.1 Copyright 2019 B di l Submitted 20 May 2019 Accepted 16 July 2019 Published 15 October 2019 Corresponding authors Jesús Díaz, jdal@unex.es Carlos Fernández Marcos, cfernan@unex.es Academic editor Robert Smith Additional Information and Declarations can be found on page 11 DOI 10.7717/peerj-ochem.1 Copyright 2019 Bornadiego et al. Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS Thus, in the last few years, several research groups have directed their efforts to develop efficient syntheses of benzo[c]coumarins. Most approaches rely upon the construction of B ring of benzo[c]courmarin (1), either by lactonization of a biaryl (9; Fig. 2, path a) (Morack, Metternich & Gilmour, 2018; Ramirez, Bosque & Gonzalez-Gomez, 2015; Luo et al., 2013) or by the formation of biaryl bond starting from a bicyclic ester (10; Fig. 2, path b) (Ortiz Villamizar et al., 2017). These methods usually involve the use of UV light, transition metal catalysts, and toxic oxidizing agents, limiting their actual applicability. Other efficient Copyright 2019 Bornadiego et al. Distributed under Creative Commons CC-BY 4.0 Figure 1 The structure of benzo[c]coumarin and some natural and synthetic bioactive analogues. INTRODUCTION Full-size DOI: 10.7717/peerjochem.1/fig-1 Figure 1 The structure of benzo[c]coumarin and some natural and synthetic bioactive analogues. Full-size DOI: 10.7717/peerjochem.1/fig-1 alternative approaches reported in literature involve tandem cycloaddition reactions to form C ring of benzo[c]courmarin 1 (Fig. 2, path c) (Pottie et al., 2011; He et al., 2012). In this latter case, the Diels-Alder reaction is especially useful, as it offers the greatest potential for diversity (Pottie et al., 2011). Unfortunately, harsh reaction conditions and the use of starting materials that are difficult to synthesize are generally required (Pottie et al., 2011). Herein we describe a facile synthesis of derivatives of benzo[c]coumarins (1a-n) by the Diels-Alder reaction of aminofuranocoumarins (11) prepared in situ from 3- carbonylcoumarins (12) and isocyanides (13). This approach has the advantages of an easy and concise construction of 2-aminofuranes by the [4+1] cycloaddition reaction of α,β-unsaturated carbonyl compounds with isocyanides, (Chatani et al., 2003; Ito, Kato & Saegusa, 1982; Bornadiego, Díaz & Marcos, 2019; Bornadiego, Díaz & Marcos, 2015; Bornadiego, Díaz & Marcos, 2014; Neo et al., 2013; Bornadiego, Díaz & Marcos, 2019) and the high reactivity of 2-aminofuranes in Diels-Alder reactions (Padwa et al., 1997). MATERIALS & METHODS General synthetic techniques Materials. Methanol was dried by distillation over CaH2, immediately prior to use. Ethanol was freshly distilled from magnesium ethoxide, prepared from magnesium turnings in the presence of iodine. All other reagents were purchased from commercial sources and used as received. Liquid reagents were measured using positive-displacement micropipettes with disposable tips and pistons. Thin layer chromatography was performed on aluminum plates, using 254 nm UV light or a mixture of p-anisaldehyde (2.5%), acetic acid (1%) and H2SO4 (3.4%) in 95% ethanol as developer. 2/14 Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 2/14 Figure 2 Strategies for the synthesis of benzo[c]coumarins. Full-size DOI: 10.7717/peerjochem.1/fig-2 Figure 2 Strategies for the synthesis of benzo[c]coumarins. Full-size DOI: 10.7717/peerjochem.1/fig-2 Instrumentation. Melting points are uncorrected. IR spectra were recorded as KBr pellets. Proton and carbon-13 nuclear magnetic resonance (1H NMR or 13C NMR) spectra were obtained on a 400 MHz or 500 MHz spectrometer. The assignments of signals in 13C NMR were made by DEPT. High Resolution Mass Spectra (HRMS) were recorded using a 6520 Accurate Mass QTOF LC/MS Spectrometer. Experiments under microwave irradiation were performed in closed vials, using a focused single-mode microwave reactor CEM Discover BenchMate, provided with an IR internal thermal probe. Synthesis of 3-carbonylcoumarins (12a-d) y y ( ) Acyl acetate 16 (3.3 mmol) and piperidine (15% mol) were added to a solution of salicylaldehyde (15, three mmol) in dry methanol (three mL). The mixture was stirred at rt and the progress of the reaction was followed by tlc. After 24–72 h the solvent was evaporated and the resulting solid was washed with cold cyclohexane to give chromones (12a-d), which were used in the following reactions without further purification. 3-Benzoyl-2H-chromen-2-one (12a); (Specht, Martic & Farid, 1982) (72 h, 661 mg, 88%). Obtained as a white solid; mp: 137–138 ◦C (lit (Specht, Martic & Farid, 1982) 137–139 ◦C); IR (cm−1): 3408, 1714, 1657, 1609, 1567; 1H-NMR (CDCl3, 500 MHz) δ 8.09 (s, 1H), 7.90 (d, J = 7.23 Hz, 2H), 7.67–7.60 (m, 3H), 7.49 (t, J = 7.1 Hz, 2H), 7.42 (d, J = 8.1 Hz, 1H), 7.36 (t, J = 7.2 Hz, 1H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 191.8 (C), 158.5 (C), 154.9 (C), 145.6 (CH), 136.3 (C), 134.0 (CH), 133.8 (CH), 129.7 (CH), 129.3 (CH), 128.7 (CH), 127.2 (C), 125.1 (CH), 118.3 (C), 117.1 (CH) ppm. Acyl acetate 16 (3.3 mmol) and piperidine (15% mol) were added to a solution of salicylaldehyde (15, three mmol) in dry methanol (three mL). The mixture was stirred at rt and the progress of the reaction was followed by tlc. After 24–72 h the solvent was evaporated and the resulting solid was washed with cold cyclohexane to give chromones (12a-d), which were used in the following reactions without further purification. 3-Benzoyl-2H-chromen-2-one (12a); (Specht, Martic & Farid, 1982) (72 h, 661 mg, 88%). Obtained as a white solid; mp: 137–138 ◦C (lit (Specht, Martic & Farid, 1982) 137–139 ◦C); IR (cm−1): 3408, 1714, 1657, 1609, 1567; 1H-NMR (CDCl3, 500 MHz) δ 8.09 (s, 1H), 7.90 (d, J = 7.23 Hz, 2H), 7.67–7.60 (m, 3H), 7.49 (t, J = 7.1 Hz, 2H), 7.42 (d, J = 8.1 Hz, 1H), 7.36 (t, J = 7.2 Hz, 1H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 191.8 (C), 158.5 (C), 154.9 (C), 145.6 (CH), 136.3 (C), 134.0 (CH), 133.8 (CH), 129.7 (CH), 129.3 (CH), 128.7 (CH), 127.2 (C), 125.1 (CH), 118.3 (C), 117.1 (CH) ppm. pp 3-Benzoyl-6-bromo-2H-chromen-2-one (12b); (Wang et al., 2012) (24 h, 465 mg, 47%). Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 Synthesis of 3-carbonylcoumarins (12a-d) 232–233 ◦C); IR (cm−1): 3435, 3042, 1735, 1675, 1608, 1550; 1H-NMR (CDCl3, 500 MHz) δ 8.40 (s, 1H), 7.78 (s, 1H), 7.73 (d, J = 8.27 Hz, 1H), 7.27 (s, 1H), 2.72 (s, 3H) ppm; 13C{1H}-NMR (CDCl3, 126 MHz) δ 195.1 (C), 158.7 (C), 154.3 (C), 146.1 (CH), 137.2 (CH), 132.3 (CH), 125.7 (C), 119.9 (C), 118.6 (CH), 117.7 (C), 30.6 (CH3) ppm. 3-Acetyl-6-bromo-2H-chromen-2-one (12c); (Parveen, Khan & Ahmed, 2019) (24 h, 360 mg, 45%). Obtained as a light yellow solid; mp: 224–228 ◦C (lit (Parveen, Khan & Ahmed, 2019). 232–233 ◦C); IR (cm−1): 3435, 3042, 1735, 1675, 1608, 1550; 1H-NMR (CDCl3, 500 MHz) δ 8.40 (s, 1H), 7.78 (s, 1H), 7.73 (d, J = 8.27 Hz, 1H), 7.27 (s, 1H), 2.72 (s, 3H) ppm; 13C{1H}-NMR (CDCl3, 126 MHz) δ 195.1 (C), 158.7 (C), 154.3 (C), 146.1 (CH), 137.2 (CH), 132.3 (CH), 125.7 (C), 119.9 (C), 118.6 (CH), 117.7 (C), 30.6 (CH3) ppm. Ethyl 6-bromo-2-oxo-2H-chromene-3-carboxylate (12d); (Volmajer et al., 2005) (24 h, 197 mg, 22%). Obtained as a white solid; mp: 177 ◦C (lit. 180–181 ◦C; (Volmajer et al., 2005) 172 ◦C (Santos-Contreras et al., 2007)); 3449, 3070, 2974, 1753, 1704, 1617, 1599; 1H-NMR (CDCl3, 500 MHz) δ 8.43 (s, 1H), 7.75 (s, 1H), 7.72 (d, J = 8.79 Hz, 1H), 7.25 (d, J = 8.79 Hz, 1H), 4.42 (q, J = 7.12 Hz, 2H), 1.41 (t, J = 7. 11 Hz, 3H) ppm; 13C{1H}-NMR (CDCl3, 126 MHz) δ 162.8 (C), 156.0 (C), 154.1 (C), 147.1 (CH), 137.0 (CH), 131.7 (CH), 119.7 (C), 119.5 (C), 118.7 (CH), 117.5 (C), 62.3 (CH2), 14.3 (CH3) ppm. Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 Synthesis of 3-carbonylcoumarins (12a-d) Obtained as a white solid; mp: 171–176 ◦C (lit (Wang et al., 2012) 171–172 ◦C); IR (cm−1): 3-Benzoyl-6-bromo-2H-chromen-2-one (12b); (Wang et al., 2012) (24 h, 465 mg, 47%). Obtained as a white solid; mp: 171–176 ◦C (lit (Wang et al., 2012) 171–172 ◦C); IR (cm−1): Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 3/14 3/14 3413, 3069, 1717, 1656, 1619, 1598; 1H-NMR (CDCl3, 500 MHz) δ 7.97 (s, 1H), 7.87 (d, J = 7.43 Hz, 2H), 7.72 (d, J = 7.13 Hz, 2H), 7.63 (t, J = 7 Hz, 1H), 7.49 (t, J = 7.4 Hz, 2H), 7.30 (d, J = 9.06 Hz, 1H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 191.2 (C), 157.8 (C), 153.6 (C), 143.9 (CH), 136.4 (CH), 136.0 (C), 134.2 (CH), 131.4 (CH), 129.7 (CH), 128.8 (CH), 128.2 (C), 119.8 (C), 118.8 (CH), 117.7 (C) ppm. 3-Acetyl-6-bromo-2H-chromen-2-one (12c); (Parveen, Khan & Ahmed, 2019) (24 h, 360 mg, 45%). Obtained as a light yellow solid; mp: 224–228 ◦C (lit (Parveen, Khan & Ahmed, 2019). 232–233 ◦C); IR (cm−1): 3435, 3042, 1735, 1675, 1608, 1550; 1H-NMR (CDCl3, 500 MHz) δ 8.40 (s, 1H), 7.78 (s, 1H), 7.73 (d, J = 8.27 Hz, 1H), 7.27 (s, 1H), 2.72 (s, 3H) ppm; 13C{1H}-NMR (CDCl3, 126 MHz) δ 195.1 (C), 158.7 (C), 154.3 (C), 146.1 (CH), 137.2 (CH), 132.3 (CH), 125.7 (C), 119.9 (C), 118.6 (CH), 117.7 (C), 30.6 (CH3) ppm. h l b h b l ( d) ( l j l ) ( h 3413, 3069, 1717, 1656, 1619, 1598; 1H-NMR (CDCl3, 500 MHz) δ 7.97 (s, 1H), 7.87 (d, J = 7.43 Hz, 2H), 7.72 (d, J = 7.13 Hz, 2H), 7.63 (t, J = 7 Hz, 1H), 7.49 (t, J = 7.4 Hz, 2H), 7.30 (d, J = 9.06 Hz, 1H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 191.2 (C), 157.8 (C), 153.6 (C), 143.9 (CH), 136.4 (CH), 136.0 (C), 134.2 (CH), 131.4 (CH), 129.7 (CH), 128.8 (CH), 128.2 (C), 119.8 (C), 118.8 (CH), 117.7 (C) ppm. ( ) ( ) ( ) ( ) ( ) pp 3-Acetyl-6-bromo-2H-chromen-2-one (12c); (Parveen, Khan & Ahmed, 2019) (24 h, 360 mg, 45%). Obtained as a light yellow solid; mp: 224–228 ◦C (lit (Parveen, Khan & Ahmed, 2019). General procedure for the synthesis of 6H-benzo[c]chromen-6-ones (1a-n) ( ) Maleimide derivative (14, 0.36 mmol) and isocyanide (13, 0.36 mmol) were successively added to a solution of coumarin (12, 0.30 mmol) in dry ethanol (two mL). The reaction mixture was stirred under N2 atmosphere, at 100 ◦C until completion, as judged by tlc. Then, 1 N HCl (20 mL) was added and the mixture stirred 1 h to hydrolyse any excess of isocyanide. The resulting mixture was extracted with CH2Cl2 and the organic phase was dried (Na2SO4) and concentrated in the rotary evaporator. In the case of obtaining a solid, this was washed with cold cyclohexane to give the pure products 1i, 1k and 1l. In all the other cases, the crude was subjected to flash column chromatography (SiO2 12 g cartridge, cyclohexane to cyclohexane-ethyl acetate 8:2), to give the pure products 1a-h; 1j and 1m-n. 11-(Cyclohexylamino)-7,9-diphenylchromeno[3,4-f ]isoindole-6,8,10(9H)-trione (1a); (15 h, 124 mg, 80%). Obtained as a fluorescent orange solid; mp: 243–244 ◦C; IR (cm−1): 3311, 2930, 2850, 1746, 1706, 1600, 1501; 1H-NMR (CDCl3, 500 MHz) δ 8.89 (dd, J = 8.2, 1.2 Hz, 1H), 7.51 (t, J = 8.5 Hz, 1H) , 7.47–7.42 (m, 5H), 7.41–7.39 (m, 2H), 7.38–7.34 (m, 2H), 7.33 (m, 1H), 7.30–7.26 (m, 2H), 6.70 (d, J = 11.0 Hz, 1H), 3.43–3.27 (m, 1H), 1.90 (m, 2H), 1.73 (m, 2H), 1.39–1.30 (m, 2H), 1.27–1.13 (m, 4H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 168.7 (C), 164.7 (C), 158.6 (C), 150.4 (C), 145.6 (C), 136.3 (C), 136.2 (C), 133.3 (C), 131.5 (CH), 131.2 (C), 129.1 (CH), 128.3 (CH), 128.2 (CH), 128.0 (CH), 127.9 (CH), 126.6 (CH), 126.4 (C), 126.0 (CH), 124.1 (CH), 120.8 (C), 118.5 (C), 117.3 (CH), 56.1 (CH), 33.9 (CH2), 25.5 (CH2), 24.8 (CH2) ppm; HRMS (ESI+-QTOF) m/z: [M + H]+ Calcd for C33H27N2O4: 515.1966; found: 515.1975. Maleimide derivative (14, 0.36 mmol) and isocyanide (13, 0.36 mmol) were successively added to a solution of coumarin (12, 0.30 mmol) in dry ethanol (two mL). The reaction mixture was stirred under N2 atmosphere, at 100 ◦C until completion, as judged by tlc. Then, 1 N HCl (20 mL) was added and the mixture stirred 1 h to hydrolyse any excess of isocyanide. The resulting mixture was extracted with CH2Cl2 and the organic phase was dried (Na2SO4) and concentrated in the rotary evaporator. In the case of obtaining a solid, this was washed with cold cyclohexane to give the pure products 1i, 1k and 1l. General procedure for the synthesis of 6H-benzo[c]chromen-6-ones (1a-n) Obtained as a fluorescent orange solid; mp: 177–179 ◦C; IR (cm−1): 3346, 2929, 1737, 1702, 1609, 1500; 1H-NMR (CDCl3, 500 MHz) δ8.69 (d, J = 8.23 Hz, 1H), 7.49 (t, J = 8.2 Hz, 1H), 7.46–7.44 (m, 3H), 7.33 (t, J = 8.2 Hz, 2H), 7.25–7.23 (m, 2H), 6.64 (bs, NH), 3.09 (m, 5H), 1.62 (q, J = 7.3 Hz, 2H), 1.32–1.24 (m, 4H), 0.89–0,86 (m, 3H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 169.6 (C), 166.1 (C), 158.7 (C), 150.5 (C), 146.2 (C), 136.5 (C), 135.3 (C), 132.0 (C), 131.1 (CH), 128.5 (C), 128.2 (CH), 128.0 (CH), 127.9 (CH), 126.4 (CH), 126.1 (C), 124.2 (CH), 120.2 (C), 118.3 (C), 117.1 (CH), 49.4 (CH2), 30.6 (CH2), 29.0 (CH2), 24.0 (CH3), 22.5 (CH2), 14.0 (CH3) ppm; HRMS (ESI+-QTOF) m/z: [M + H]+ Calcd for C28H24N2O4: 441.1809; found: 441.1830. J = 8.2 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.45 (m, 3H), 7.32 (d, J = 8.0 Hz, 2H), 7.24 (m, 2H), 6.50 (d, J = 11.0 Hz, 1H), 3.29 (m, 1H), 3.09 (s, 3H), 1.87 (m, 2H), 1.72 (m, 2H), 1.54 (m, 1H), 1.37–1.11 (m, 5H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 169.6 (C), 166.0 (C), 158.7 (C), 150.5 (C), 145.1 (C), 136.5 (C), 135.8 (C), 133.2 (C), 131.4 (CH), 128.2 (CH), 128.0 (CH), 127.9 (CH), 126.1 (CH), 125.9 (C), 124.1 (CH), 121.7 (C), 118.5 (C), 117.2 (CH), 56.2 (CH), 33.9 (CH2), 25.6 (CH2), 24.9 (CH2), 24.0 (CH3) ppm; HRMS (ESI+-QTOF) m/z: [M + H]+ Calcd for C28H25N2O4: 453.1809; found: 453.1816. 9-Methyl-11-(pentylamino)-7-phenylchromeno[3,4-f ]isoindole-6,8,10(9H)-trione 9-Methyl-11-(pentylamino)-7-phenylchromeno[3,4-f ]isoindole-6,8,10(9H)-trione (1d); (59 h, 100 mg, 76%). Obtained as a fluorescent orange solid; mp: 177–179 ◦C; IR (cm−1): 3346, 2929, 1737, 1702, 1609, 1500; 1H-NMR (CDCl3, 500 MHz) δ8.69 (d, J = 8.23 Hz, 1H), 7.49 (t, J = 8.2 Hz, 1H), 7.46–7.44 (m, 3H), 7.33 (t, J = 8.2 Hz, 2H), 7.25–7.23 (m, 2H), 6.64 (bs, NH), 3.09 (m, 5H), 1.62 (q, J = 7.3 Hz, 2H), 1.32–1.24 (m, 4H), 0.89–0,86 (m, 3H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 169.6 (C), 166.1 (C), 158.7 (C), 150.5 (C), 146.2 (C), 136.5 (C), 135.3 (C), 132.0 (C), 131.1 (CH), 128.5 (C), 128.2 (CH), 128.0 (CH), 127.9 (CH), 126.4 (CH), 126.1 (C), 124.2 (CH), 120.2 (C), 118.3 (C), 117.1 (CH), 49.4 (CH2), 30.6 (CH2), 29.0 (CH2), 24.0 (CH3), 22.5 (CH2), 14.0 (CH3) ppm; HRMS (ESI+-QTOF) m/z: [M + H]+ Calcd for C28H24N2O4: 441.1809; found: 441.1830. Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 General procedure for the synthesis of 6H-benzo[c]chromen-6-ones (1a-n) In all the other cases, the crude was subjected to flash column chromatography (SiO2 12 g cartridge, cyclohexane to cyclohexane-ethyl acetate 8:2) to give the pure products 1a-h; 1j and 1m-n Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 4/14 2-Bromo-11-(cyclohexylamino)-7,9-diphenylchromeno[3,4-f ]isoindole-6,8,10(9H)- 2-Bromo-11-(cyclohexylamino)-7,9-diphenylchromeno[3,4-f ]isoindole-6,8,10(9H)- trione (1b); (23 h, 142 mg, 79%). Obtained as a fluorescent orange solid; mp: 142–146 ◦C; IR (cm−1): 3308, 3061, 2927, 2851, 1750, 1707, 1597; 1H-NMR (CDCl3, 500 MHz) δ 9.11 (s, 1H), 7.60 (dd, J = 8.7, 2.3 Hz, 1H), 7.47–7.44 (m, 2H), 7.44 (d, J = 2.5 Hz, 3H), 7.41–7.34 (m, 3H), 7.26 (dd, J = 5.3, 4.2 Hz, 2H), 7.22 (d, J = 8.7 Hz, 1H), 6.69 (d, J = 11.1 Hz, 1H), 3.36–3.25 (m, 1H), 1.97 (m, 2H), 1.76 (m, 2H), 1.42–1.33 (m, 2H), 1.25 (m, 4H) ppm; 13C{1H}-NMR (CDCl3, 126 MHz) δ 168.5 (C), 164.6 (C), 158.0 (C), 149.4 (C), 145.6 (C), 136.4 (C), 136.0 (C), 134.1 (CH), 131.8 (C), 131.2 (C), 129.1 (CH), 128.8 (CH), 128.5 (C), 128.4 (CH), 128.2 (CH), 128.1 (CH), 126.6 (CH), 121.4 (C), 120.0 (C), 118.8 (CH), 117.1 (C), 57.0 (CH), 33.9 (CH2), 25.5 (CH2), 24.9 (CH2) ppm; HRMS (ESI+-QTOF) m/z: [M + H]+ Calcd for C33H26BrN2O4: 593.1071; found: 593.1093. ( ), ( ), ( ), ( ), ( ), ( ), ( ), ( ), 128.4 (CH), 128.2 (CH), 128.1 (CH), 126.6 (CH), 121.4 (C), 120.0 (C), 118.8 (CH), 117.1 (C), 57.0 (CH), 33.9 (CH2), 25.5 (CH2), 24.9 (CH2) ppm; HRMS (ESI+-QTOF) m/z: [M + H]+ Calcd for C33H26BrN2O4: 593.1071; found: 593.1093. 11-(Cyclohexylamino)-9-methyl-7-phenylchromeno[3,4-f ]isoindole-6,8,10(9H)-trione (1c); (59 h, 111 mg, 82%). General procedure for the synthesis of 6H-benzo[c]chromen-6-ones (1a-n) Obtained as a fluorescent yellow solid; mp: 199–205 ◦C; IR (cm−1): 3304, 2924, 2850, 1745, 1698, 1611, 1422; 1H-NMR (CDCl3, 500 MHz) δ 8.90 (d, J = 8.2 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.45 (m, 3H), 7.32 (d, J = 8.0 Hz, 2H), 7.24 (m, 2H), 6.50 (d, J = 11.0 Hz, 1H), 3.29 (m, 1H), 3.09 (s, 3H), 1.87 (m, 2H), 1.72 (m, 2H), 1.54 (m, 1H), 1.37–1.11 (m, 5H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 169.6 (C), 166.0 (C), 158.7 (C), 150.5 (C), 145.1 (C), 136.5 (C), 135.8 (C), 133.2 (C), 131.4 (CH), 128.2 (CH), 128.0 (CH), 127.9 (CH), 126.1 (CH), 125.9 (C), 124.1 (CH), 121.7 (C), 118.5 (C), 117.2 (CH), 56.2 (CH), 33.9 (CH2), 25.6 (CH2), 24.9 (CH2), 24.0 (CH3) ppm; HRMS (ESI+-QTOF) m/z: [M + H]+ Calcd for C28H25N2O4: 453.1809; found: 453.1816. 9-Methyl-11-(pentylamino)-7-phenylchromeno[3 4-f ]isoindole-6 8 10(9H)-trione 11-(Cyclohexylamino)-9-methyl-7-phenylchromeno[3,4-f ]isoindole-6,8,10(9H)-trione (1c); (59 h, 111 mg, 82%). Obtained as a fluorescent yellow solid; mp: 199–205 ◦C; IR (cm−1): 3304, 2924, 2850, 1745, 1698, 1611, 1422; 1H-NMR (CDCl3, 500 MHz) δ 8.90 (d, J = 8.2 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.45 (m, 3H), 7.32 (d, J = 8.0 Hz, 2H), 7.24 (m, 2H), 6.50 (d, J = 11.0 Hz, 1H), 3.29 (m, 1H), 3.09 (s, 3H), 1.87 (m, 2H), 1.72 (m, 2H), 11-(Cyclohexylamino)-9-methyl-7-phenylchromeno[3,4-f ]isoindole-6,8,10(9H)-trione (1c); (59 h, 111 mg, 82%). Obtained as a fluorescent yellow solid; mp: 199–205 ◦C; IR (cm−1): 3304, 2924, 2850, 1745, 1698, 1611, 1422; 1H-NMR (CDCl3, 500 MHz) δ 8.90 (d, J = 8.2 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.45 (m, 3H), 7.32 (d, J = 8.0 Hz, 2H), 7.24 (m, 2H), 6.50 (d, J = 11.0 Hz, 1H), 3.29 (m, 1H), 3.09 (s, 3H), 1.87 (m, 2H), 1.72 (m, 2H), 1.54 (m, 1H), 1.37–1.11 (m, 5H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 169.6 (C), 166.0 (C), 158.7 (C), 150.5 (C), 145.1 (C), 136.5 (C), 135.8 (C), 133.2 (C), 131.4 (CH), 128.2 (CH), 128.0 (CH), 127.9 (CH), 126.1 (CH), 125.9 (C), 124.1 (CH), 121.7 (C), 118.5 (C), 117.2 (CH), 56.2 (CH), 33.9 (CH2), 25.6 (CH2), 24.9 (CH2), 24.0 (CH3) ppm; HRMS (ESI+-QTOF) m/z: [M + H]+ Calcd for C28H25N2O4: 453.1809; found: 453.1816. 9-Methyl-11-(pentylamino)-7-phenylchromeno[3,4-f ]isoindole-6,8,10(9H)-trione (1d); (59 h, 100 mg, 76%). General procedure for the synthesis of 6H-benzo[c]chromen-6-ones (1a-n) Obtained as a fluorescent yellow solid; mp: 107–111 ◦C; IR (cm−1): 3446, 2923, 1769, 1748, 1710, 1597; 1H-NMR (CDCl3, 500 MHz) δ 10.11 (s, 1H), 7.58 (d, J = 8.7 Hz, 1H), 7.46–7.43 (m, 5H), 7.38 (d, J = 7.5 Hz, 3H), 7.29 (m, 2H), 7.17 (d, J = 8.72 Hz, 1H), 5.83 (s, 1H), 1.24 (s, 9H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 167.4 (C), 164.4 (C), 158.0 (C), 149.3 (C), 144.7 (C), 139.2 (C), 138.3 (C), 135.9 (C), 134.5 (CH), 131.1 (C), 130.2 (CH), 129.1 (CH), 128.5 (CH), 128.2 (CH), 128.1 (CH), 128.0 (CH), 127.1 (C), 126.6 (CH), 125.7 (C), 121.6 (C), 118.9 (CH), 116.6 (C), 59.8 (C), 30.1 (CH3) ppm; HRMS (ESI+-QTOF) m/z: [M + H]+ Calcd for C31H24BrN2O4: 567.0914; found: 567.0921. 2-Bromo-9-methyl-11-(pentylamino)-7-phenylchromeno[3,4-f ]isoindole-6,8,10(9H)- trione (1g); (87 h, 98 mg, 63%). Obtained as a fluorescent yellow solid; mp: 164–169 ◦C; IR (cm−1): 3304, 3072, 2930, 2858, 1761, 1744, 1700, 1609, 1425; 1H-NMR (CDCl3, 500 MHz) δ 8.86 (s, 1H), 7.58 (d, J = 8.7 Hz, 1H), 7.47–7.44 (m, 3H), 7.23–7.19 (m, 3H), 6.65 (t, J = 6 Hz, 1H), 3.10 (s, 3H), 3.08 (t, J = 6.88 Hz, 2H), 1.68 (q, J = 7.4 Hz, 2H), 1.37–1.31 (m, 4H), 0.90 (t, J = 7.1 Hz, 3H) ppm;13C{1H}-NMR (CDCl3, 126 MHz) δ 169.4 (C), 165.9 (C), 158.1 (C), 149.4 (C), 146.2 (C), 136.2 (C), 135.6 (C), 133.8 (CH), 130.6 (C), 129.5 (C), 129.0 (CH), 120.2 (CH), 128.0 (CH), 128.0 (CH), 126.2 (C), 120.8 (C), 119.9 (C), 118.8 (CH), 117.2 (C), 49.7 (CH2), 30.5 (CH2), 29.1 (CH2), 24.1 (CH3), 22.5 (CH2), 14.1 (CH3) ppm; HRMS (ESI+-QTOF) [M + H]+ Calcd for C27H24BrN2O4: 519.0914; found: 519.0918. 2-Bromo-11-((2,6-dimethylphenyl)amino)-7-phenylchromeno[3,4-f ]isoindole- 6,8,10(9H)-trione (1h); (67 h, 50 mg, 31%). Obtained as an orange solid; mp: 322–325 ◦C (dec); IR (cm−1): 3319, 1765, 1737, 1710, 1607, 1477; 1H-NMR (CDCl3, 500 MHz) δ 8.96 (s, 1H), 7.77 (s, 1H), 7.46 (m, 3H), 7.28–7.25 (m, 4H), 7.00–6.92 (m, 4H), 2.07 (s, 6H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 169.4 (C), 165.3 (C), 158.3 (C), 148.5 (C), 141.4 (C), 136.7 (C), 135.8 (C), 134.6 (C), 133.2 (CH), 131.1 (C), 130.0 (CH), 129.5 (C), 129.3 (CH), 129.2 (C), 128.4 (CH), 128.2 (CH), 128.1 (CH), 126.4 (C), 125.9 (CH), 117.8 (C), 117.6 (C), 117.4 (C), 115.8 (C), 20.1 (CH3) ppm; HRMS (ESI−-QTOF) [M - H]−Calcd for C29H18BrN2O4: 537.0455; found: 537.0428. 2-Bromo-11-(cyclohexylamino)-7-methyl-9-phenylchromeno[3,4-f ]isoindole- 6,8,10(9H)-trione (1i); (7 h, 158 mg, 99%). Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 General procedure for the synthesis of 6H-benzo[c]chromen-6-ones (1a-n) 11-((2,6-Dimethylphenyl)amino)-7-phenylchromeno[3,4-f ]isoindole-6,8,10(9H)- trione (1e); (69 h, 43 mg, 31%). Obtained as a fluorescent dark orange solid; mp: 310– 312 ◦C (dec); IR (cm−1): 3299, 1765, 1713, 1608, 1505; 1H-NMR (CDCl3, 500 MHz) δ 9.01 (s, 1H), 7.86 (s, 1H), 7.45 (m, 3H), 7.29 (s, 2H), 7.18 (t, J = 7.5 Hz, 1H), 7.16–7.09 (m, 2H), 6.89 (m, 3H), 6.64 (t, J = 7.5 Hz, 1H), 2.04 (s, 6H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 169.6 (C), 165.4 (C), 158.9 (C), 149.6 (C), 141.2 (C), 137.1 (C), 136.1 (C), 135.2 (CH), 134.6 (C), 131.3 (C), 130.8 (C), 130.8 (C), 130.4 (CH), 129.3 (CH), 128.3 (C), 128.7 (CH), 128.4 (CH), 128.0 (CH), 126.4 (CH), 126.3 (CH), 125.5 (CH), 122.1 (CH), 117.5 (C), 116.1 (CH), 115.9 (C), 19.9 (CH3) ppm; HRMS (ESI−-QTOF) m/z: [M - H]− Calcd for C29H19N2O4: 459.1350; found: 459.1365. 5/14 Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 2-Bromo-11-(tert-butylamino)-7,9-diphenylchromeno[3,4-f ]isoindole-6,8,10(9H)- trione (1f); (>100 h, 19 mg, 11%). Obtained as a fluorescent yellow solid; mp: 107–111 ◦C; IR (cm−1): 3446, 2923, 1769, 1748, 1710, 1597; 1H-NMR (CDCl3, 500 MHz) δ 10.11 (s, 1H), 7.58 (d, J = 8.7 Hz, 1H), 7.46–7.43 (m, 5H), 7.38 (d, J = 7.5 Hz, 3H), 7.29 (m, 2H), 7.17 (d, J = 8.72 Hz, 1H), 5.83 (s, 1H), 1.24 (s, 9H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 167.4 (C), 164.4 (C), 158.0 (C), 149.3 (C), 144.7 (C), 139.2 (C), 138.3 (C), 135.9 (C), 134.5 (CH), 131.1 (C), 130.2 (CH), 129.1 (CH), 128.5 (CH), 128.2 (CH), 128.1 (CH), 128.0 (CH), 127.1 (C), 126.6 (CH), 125.7 (C), 121.6 (C), 118.9 (CH), 116.6 (C), 59.8 (C), 30.1 (CH3) ppm; HRMS (ESI+-QTOF) m/z: [M + H]+ Calcd for C31H24BrN2O4: 567.0914; found: 567.0921. 2-Bromo-11-(tert-butylamino)-7,9-diphenylchromeno[3,4-f ]isoindole-6,8,10(9H)- trione (1f); (>100 h, 19 mg, 11%). General procedure for the synthesis of 6H-benzo[c]chromen-6-ones (1a-n) Obtained as a fluorescent orange solid; mp: 337–342 (dec); IR (cm−1): 3441, 2919, 2850, 1739, 1713, 1634; 1H-NMR (CDCl3, 500 MHz) δ 8.77 (s, 1H), 7.72 (s, 1H), 7.30 (s, 1H), 7.01 (d, J = 8.69 Hz, 1H), 6.95–6.88 (m, 4H), 3.12 (s, 3H), 2.01 (s, 6H) ppm; due to the insolubility of compound 1m, it was not possible to record its 13C-NMR spectrum. HRMS (ESI−-QTOF) [M - H]−Calcd for C24H16BrN2O4: 475.0299; found: 475.0326. 2-Bromo-11-((2,6-dimethylphenyl)amino)-7-methylchromeno[3,4-f ]isoindole- 6,8,10(9H)-trione (1m); (18 h, 60 mg, 42%). Obtained as a fluorescent orange solid; mp: 337–342 (dec); IR (cm−1): 3441, 2919, 2850, 1739, 1713, 1634; 1H-NMR (CDCl3, 500 MHz) δ 8.77 (s, 1H), 7.72 (s, 1H), 7.30 (s, 1H), 7.01 (d, J = 8.69 Hz, 1H), 6.95–6.88 (m, 4H), 3.12 (s, 3H), 2.01 (s, 6H) ppm; due to the insolubility of compound 1m, it was not possible to record its 13C-NMR spectrum. HRMS (ESI−-QTOF) [M - H]−Calcd for C24H16BrN2O4: 475.0299; found: 475.0326. 2-Bromo-11-((4-methoxyphenyl)amino)-7-methylchromeno[3,4-f ]isoindole- 6,8,10(9H)-trione (1n); (32 h, 78 mg, 54%). Obtained as a light red oil; IR (cm−1): 3441, 2919, 2850, 1759, 1732, 1702, 1509; 1H-NMR (CDCl3, 500 MHz) δ 8.54 (s, 1H), 8.42 (d, J = 2.26 Hz, 1H), 7.73 (s, 1H), 7.37 (d, J = 8.7 Hz, 1H), 7.09 (d, J = 8.69 Hz, 1H), 6.75–6.68 (m, 4H), 3.71 (s, 3H), 3.16 (s, 3H) ppm; due to the insolubility of compound 1n, it was not possible to record its 13C-NMR spectrum. HRMS (ESI+-QTOF) [M + H]+ Calcd for C23H16BrN2O5: 479.0237; found: 479.0234. General procedure for the synthesis of 6H-benzo[c]chromen-6-ones (1a-n) Obtained as an orange solid; mp: 241–244 ◦C; IR (cm−1): 3439, 2930, 2853, 1758, 1698, 1598, 1500; 1H-NMR (CDCl3, 500 MHz) δ 9.16 (s, 1H), 7.59 (d, J = 8.65 Hz, 1H), 7.54 (t, J = 7.6 Hz, 2H), 7.46–7.42 (m, 3H), 7.23 (d, J = 8.7 Hz, 1H), 6.50 (d, J = 11.14 Hz, 1H), 3.14 (m, 4H), 1.89 (m, 2H), 1.72–1.18 (m, 8H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 168.4 (C), 166.5 (C), 159.2 (C), 149.2 (C), 144.3 (C), 136.4 (C), 134.0 (CH), 132.4 (C), 131.3 (C), 129.3 (CH), 128.9 (CH), 128.6 (CH), 128.3 (C), 127.3 (C), 126.7 (CH), 121.9 (C), 120.1 (C), 118.6 (CH), 117.1 (C), 56.8 (CH), 33.8 (CH2), 25.5 (CH2), 24.9 (CH2), 16.2 (CH3) ppm; HRMS (ESI+-QTOF) [M + H]+ Calcd for C28H24BrN2O4: 531.0914; found: 531.0885. 2-Bromo-11-(tert-butylamino)-7,9-dimethylchromeno[3,4-f ]isoindole-6,8,10(9H)- t i (1j) (27 h 8 6%) Obt i d fl t ll lid 210 213 ◦C IR Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 6/14 7.55 (d, J = 8.72 Hz, 1H), 7.16 (d, J = 8.71 Hz, 1H), 5.52 (s, 1H), 3.19 (s, 3H), 3.14 (s, 3H), 1.13 (s, 9H) ppm; 13C{1H}-NMR (CDCl3, 126 MHz) δ 168.9 (C), 167.5 (C), 159.3 (C), 149.2 (C), 142.6 (C), 138.8 (C), 138.4 (C), 134.2 (CH), 130.6 (CH), 128.6 (C), 128.4 (C), 126.2 (C), 121.7 (C), 118.6 (CH), 116.5 (C), 59.1 (C), 29.9 (CH3), 24.2 (CH3), 16.4 (CH3) ppm; HRMS (ESI+-QTOF) [M + H]+ Calcd for C21H20BrN2O4: 443.0601; found: 443.0585. 7.55 (d, J = 8.72 Hz, 1H), 7.16 (d, J = 8.71 Hz, 1H), 5.52 (s, 1H), 3.19 (s, 3H), 3.14 (s, 3H), 1.13 (s, 9H) ppm; 13C{1H}-NMR (CDCl3, 126 MHz) δ 168.9 (C), 167.5 (C), 159.3 (C), 149.2 (C), 142.6 (C), 138.8 (C), 138.4 (C), 134.2 (CH), 130.6 (CH), 128.6 (C), 128.4 (C), 126.2 (C), 121.7 (C), 118.6 (CH), 116.5 (C), 59.1 (C), 29.9 (CH3), 24.2 (CH3), 16.4 (CH3) ppm; HRMS (ESI+-QTOF) [M + H]+ Calcd for C21H20BrN2O4: 443.0601; found: 443.0585. 2-Bromo-7,9-dimethyl-11-(pentylamino)chromeno[3,4-f ]isoindole-6,8,10(9H)-trione (1k); (13 h, 72 mg, 52%). General procedure for the synthesis of 6H-benzo[c]chromen-6-ones (1a-n) Obtained as a fluorescent orange solid; mp: 139–142 ◦C; IR (cm−1): 3324, 2931, 1740, 1698, 1606, 1436; 1H-NMR (CDCl3, 500 MHz) δ 8.91 (s, 1H), 7.56 (d, J = 8.58 Hz, 1H), 7.21 (d, J = 8.67 Hz, 1H), 6.45 (t, J = 5.3 Hz, 1H), 3.19 (s, 3H), 3.08 (s, 3H), 3.01–2.96 (m, 2H), 1.61 (m, 2H), 1.31–1.30 (m, 4H), 0.88 (m, 3H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 169.3 (C), 167.7 (C), 159.3 (C), 149.2 (C), 145.0 (C), 135.4 (C), 133.7 (CH), 131.2 (C), 129.0 (CH), 128.9 (C), 126.9 (C), 121.4 (C), 119.9 (C), 118.5 (CH), 117.2 (C), 49.5 (CH2), 30.3 (CH2), 29.0 (CH2), 24.1 (CH3), 22.5 (CH2), 16.2 (CH3), 14.0 (CH3) ppm; HRMS (ESI+-QTOF) [M + H]+ Calcd for C22H22BrN2O4: 457.0758; found: 457.0755. 2-Bromo-7,9-dimethyl-11-(pentylamino)chromeno[3,4-f ]isoindole-6,8,10(9H)-trione (1k); (13 h, 72 mg, 52%). Obtained as a fluorescent orange solid; mp: 139–142 ◦C; IR (cm−1): 3324, 2931, 1740, 1698, 1606, 1436; 1H-NMR (CDCl3, 500 MHz) δ 8.91 (s, 1H), 7.56 (d, J = 8.58 Hz, 1H), 7.21 (d, J = 8.67 Hz, 1H), 6.45 (t, J = 5.3 Hz, 1H), 3.19 (s, 3H), 3.08 (s, 3H), 3.01–2.96 (m, 2H), 1.61 (m, 2H), 1.31–1.30 (m, 4H), 0.88 (m, 3H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 169.3 (C), 167.7 (C), 159.3 (C), 149.2 (C), 145.0 (C), 135.4 (C), 133.7 (CH), 131.2 (C), 129.0 (CH), 128.9 (C), 126.9 (C), 121.4 (C), 119.9 (C), 118.5 (CH), 117.2 (C), 49.5 (CH2), 30.3 (CH2), 29.0 (CH2), 24.1 (CH3), 22.5 (CH2), 16.2 (CH3), 14.0 (CH3) ppm; HRMS (ESI+-QTOF) [M + H]+ Calcd for C22H22BrN2O4: 457.0758; found: 457.0755. 11-(Benzylamino)-2-bromo-7-methylchromeno[3,4-f ]isoindole-6,8,10(9H)-trione (1l); (18 h, 107 mg, 77%). Obtained as a fluorescent orange solid; mp: 234–238 ◦C; IR (cm−1): 3431, 1757, 1737, 1709, 1606, 1424; 1H-NMR (CDCl3, 500 MHz) δ 9.02 (s, 1H), 7.78 (s, 1H), 7.54 (d, J = 8.66 Hz, 1H), 7.24–7.17 (m, 4H), 7.12 (d, J = 6.96 Hz, 2H), 6.73 (t, J = 6.3 Hz, 1H), 4.14 (d, J = 6.49 Hz, 2H), 3.02 (s, 3H) ppm; 13C{1H}-NMR (CDCl3, 101 MHz) δ 168.5 (C), 167.0 (C), 159.1 (C), 149.4 (C), 144.6 (C), 137.5 (C), 136.8 (C), 134.2 (CH), 131.9 (C), 129.2 (C), 129.1 (CH), 129.1 (CH), 128.1 (CH), 127.9 (CH), 127.4 (C), 122.4 (C), 119.8 (C), 118.7 (CH), 117.5 (C), 52.9 (CH2), 16.2 (CH3) ppm; HRMS (ESI+-QTOF) Calcd for C23H15BrN2O4H+: 463.0288; found: 463.0289. 2-Bromo-11-((2,6-dimethylphenyl)amino)-7-methylchromeno[3,4-f ]isoindole- 6,8,10(9H)-trione (1m); (18 h, 60 mg, 42%). RESULTS AND DISCUSSION Aminofurans are very reactive dienes that are known to undergo Diels-Alder cycloadditions in relatively mild reaction conditions. We have recently shown that 2-aminofurans, Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 7/14 7/14 Figure 3 Schematic representation of sequential [4+1] and [4+2] cycloaddition reactions leading to the synthesis of 3-carbonylcoumarins. Full-size DOI: 10.7717/peerjochem.1/fig-3 Figure 3 Schematic representation of sequential [4+1] and [4+2] cycloaddition reactions leading to the synthesis of 3-carbonylcoumarins. Full-size DOI: 10.7717/peerjochem.1/fig-3 readily synthesized by a [4+1] cycloaddition of isocyanides and α,β-unsaturated carbonyl compounds, can react with dienophiles in a single operation to give anilines (Bornadiego, Díaz & Marcos, 2019; Bornadiego, Díaz & Marcos, 2015; Bornadiego, Díaz & Marcos, 2014; Neo et al., 2013; Bornadiego, Díaz & Marcos, 2019). Henceforth, we reason that 1-amino- 4H-furo[3,4-c]chromen-4-ones (11) would be suitable intermediate dienes for the Diels- Alder construction benzo[c]coumarin C ring. Accordingly, we propose a multicomponent synthesis of benzo[c]coumarins (1) by the sequential [4+1] / [4+2] cycloaddition of isocyanides (13), 3-carbonylcoumarins (12) and dienophiles (14, Fig. 3). readily synthesized by a [4+1] cycloaddition of isocyanides and α,β-unsaturated carbonyl compounds, can react with dienophiles in a single operation to give anilines (Bornadiego, Díaz & Marcos, 2019; Bornadiego, Díaz & Marcos, 2015; Bornadiego, Díaz & Marcos, 2014; Neo et al., 2013; Bornadiego, Díaz & Marcos, 2019). Henceforth, we reason that 1-amino- 4H-furo[3,4-c]chromen-4-ones (11) would be suitable intermediate dienes for the Diels- Alder construction benzo[c]coumarin C ring. Accordingly, we propose a multicomponent synthesis of benzo[c]coumarins (1) by the sequential [4+1] / [4+2] cycloaddition of isocyanides (13), 3-carbonylcoumarins (12) and dienophiles (14, Fig. 3). The starting carbonylcoumarins (12) were synthesized by the Knoevenagel condensation of salicylaldehydes (15) with different β-ketoesters (16), followed by cyclization, according to slightly modified Farid’s method (Specht, Martic & Farid, 1982). In our case, the reaction was optimally carried out at room temperature in methanol, using piperidine as basic catalyst (Fig. 4). In order to prove our strategy for the synthesis of benzo[c]coumarins (1), we reacted 3-benzoylcoumarin 12a with 1.2 equivalents of cyclohexyl isocyanide 13a and N-phenylmaleimide 14a in THF. After 12 h at 25 ◦C, the reaction medium became yellow- orange and a new highly fluorescent yellow product was evident on tlc. However, the reaction was extraordinarily sluggish at room temperature and it did not reach completion even after 30 days (Table 1, entry 1). Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 RESULTS AND DISCUSSION The reaction was still slow when the temperature was raised to 80 ◦C, but it did conclude after 9 days, allowing the isolation of a new product, 1a, the identity of which was confirmed by 1H-RMN, 13C-RMN, IR and MS (Table 1, entry 2). With the purpose of finding better reaction conditions, we explored the use of different solvents, temperatures, and the addition of catalysts (Table 1). Using a solvent with a higher boiling point, such as dibutyl ether, resulted in a complex mixture, difficult to purify. Fortunately, when we carried out the reaction in ethanol at 100 ◦C, benzocoumarin 1a was obtained in just 15 h with an 80% yield (Table 1, entry 5). Similarly, the reaction Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 8/14 Figure 4 Synthesis of 3-carbonylcoumarins. Full-size DOI: 10.7717/peerjochem.1/fig-4 Table 1 Optimization of the 3-component reaction. Entry Solvent T, ◦C Catalyst, % mol Time Yield, % (Product) 1 THF rt – 30 d IRa 2 THF 80 – 9 d 44 (1a) 3 THF 80 Y(OTf)3, 5 9 d CMb 4 Bu2O 150 – 43 h CMb 5 EtOH 100 – 15 h 80 (1a) 6 EtOH rt 30 d 32 (1b) 7 EtOH 100 23 h 79 (1b) 8 EtOH 100 H2SO4, 20 65 h 74 (1b) 9 EtOH 100 TUc, 20 27 h 75 (1b) Notes. aIR, incomplete reaction. bCM, complex mixture. cTU, 1,3-bis(3,5-bis(trifluoromethyl)phenyl)thiourea. with bromocoumarin 12b afforded the corresponding benzocoumarin 1b with a 79% yield (entry 7). In consequence, the reaction, which is very slow in THF, seems to be favored in ethanol, suggesting the occurrence of charged intermediates that may be stabilized in protic polar solvents. This is in accordance with our theoretical studies (Wu, Xu & Xie, 2005) that show that related syntheses of 2-aminofuranes proceed through a [4+1] stepwise cycloaddition involving a zwitterionic intermediate (Bornadiego, Díaz & Marcos, 2019). On Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 9/14 Table 2 Synthesis of 6H-benzo[c]coumarins. Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 RESULTS AND DISCUSSION Entry Coumarin R1 R2 Dienophile X Isocyanide R3 Time, h Product (yield, %) 1 12a H Ph 14a N-Ph 13a cC6H11 15 1a (80) 2 12a H Ph 14b N-CH3 13a cC6H11 59 1c (82) 3 12a H Ph 14b N-CH3 13b C5H11 59 1d (76) 4 12a H Ph 14c N-H 13c 2,6-diMe-Ph 69 1e (31) 5 12b Br Ph 14a N-Ph 13a cC6H11 23 1b (71) 6 12b Br Ph 14a N-Ph 13d tBu >100 1f (11) 7 12b Br Ph 14b N-CH3 13d tBu >100 NIa 8 12b Br Ph 14b N-CH3 13b C5H11 87 1g (63) 9 12b Br Ph 14c N-H 13c 2,6-diMe-Ph 67 1h (31) 10 12c Br CH3 14a N-Ph 13a cC6H11 7 1i (99) 11 12c Br CH3 14b N-CH3 13d tBu 27 1j (6) 12 12c Br CH3 14b N-CH3 13b C5H11 13 1k (52) 13 12c Br CH3 14d O 13a cC6H11 11 CMb 14 12c Br CH3 14c N-H 13e CH2C6H5 18 1l (77) 15 12c Br CH3 14c N-H 13c 2,6-diMe-Ph 18 1m (42) 16 12c Br CH3 14c N-H 13f 4-MeO-Ph 32 1n (54) 17 12d Br OEt 14a N-Ph 13a cC6H11 >100 NRc Notes. aNI non isolated Entry Coumarin R1 R2 Dienophile X Isocyanide R3 Time, h Product (yield, %) 1 12a H Ph 14a N-Ph 13a cC6H11 15 1a (80) 2 12a H Ph 14b N-CH3 13a cC6H11 59 1c (82) 3 12a H Ph 14b N-CH3 13b C5H11 59 1d (76) 4 12a H Ph 14c N-H 13c 2,6-diMe-Ph 69 1e (31) 5 12b Br Ph 14a N-Ph 13a cC6H11 23 1b (71) 6 12b Br Ph 14a N-Ph 13d tBu >100 1f (11) 7 12b Br Ph 14b N-CH3 13d tBu >100 NIa 8 12b Br Ph 14b N-CH3 13b C5H11 87 1g (63) 9 12b Br Ph 14c N-H 13c 2,6-diMe-Ph 67 1h (31) 10 12c Br CH3 14a N-Ph 13a cC6H11 7 1i (99) 11 12c Br CH3 14b N-CH3 13d tBu 27 1j (6) 12 12c Br CH3 14b N-CH3 13b C5H11 13 1k (52) 13 12c Br CH3 14d O 13a cC6H11 11 CMb 14 12c Br CH3 14c N-H 13e CH2C6H5 18 1l (77) 15 12c Br CH3 14c N-H 13c 2,6-diMe-Ph 18 1m (42) 16 12c Br CH3 14c N-H 13f 4-MeO-Ph 32 1n (54) 17 12d Br OEt 14a N-Ph 13a cC6H11 >100 NRc Notes. Competing Interests Carlos Fernández Marcos is an Academic Editor for PeerJ. Carlos Fernández Marcos is an Academic Editor for PeerJ. RESULTS AND DISCUSSION aNI, non isolated. bCM, complex mixture. cNR, no reaction. the other hand, the use of proton donors (entries 8 and 9) or Lewis acid catalysts (entry 3) did not significantly improve the reaction. Thus, the optimal reaction conditions were applied to different combinations of isocyanides (13a-f), dienophiles (14a-d) and 3-carbonylcoumarins (12a-d; Table 2). In most of the cases the products are obtained with moderate to excellent yields. The reaction proceeds equally well with different aromatic and aliphatic substituted 3-carbonylcoumarins. Conversely, as expected, coumarin ester 12d (Table 2, entry 17) does not react. N-substituted and non-substituted maleimides can be used as dienophiles; however, the reaction with maleic anhydride produces a complex mixture of products (entry 13). Aliphatic isocyanides (entries 1–3, 5, 8, 10, 12, 14) require shorter reaction times and result in better yields than less reactive aromatic isocyanides (entries 4, 9, 15, 16). An exception is tert-butyl isocyanide (entries 6, 7, 11), possibly due to steric hindrance. Entry the other hand, the use of proton donors (entries 8 and 9) or Lewis acid catalysts (entry 3) did not significantly improve the reaction. Thus, the optimal reaction conditions were applied to different combinations of isocyanides (13a-f), dienophiles (14a-d) and 3-carbonylcoumarins (12a-d; Table 2). In most of the cases the products are obtained with moderate to excellent yields. The reaction proceeds equally well with different aromatic and aliphatic substituted 3-carbonylcoumarins. Conversely, as expected, coumarin ester 12d (Table 2, entry 17) does not react. N-substituted and non-substituted maleimides can be used as dienophiles; however, the reaction with maleic anhydride produces a complex mixture of products (entry 13). Aliphatic isocyanides (entries 1–3, 5, 8, 10, 12, 14) require shorter reaction times and result in better yields than less reactive aromatic isocyanides (entries 4, 9, 15, 16). An exception is tert-butyl isocyanide (entries 6, 7, 11), possibly due to steric hindrance. Bornadiego et al. (2019), PeerJ Organic Chemistry, DOI 10.7717/peerj-ochem.1 10/14 CONCLUSIONS In conclusion, we have successfully designed a highly convergent and atom-economic synthesis of benzo[c]coumarins. Our strategy is based on the trapping with dienophiles of reactive 2-aminofurans generated in a [4+1] cycloaddition of isocyanides and readily available 3-carbonyl coumarins. In this way, the target compounds are readily obtained in one-pot, in mild conditions with no need of catalysis. Furthermore, different substitution patterns can be easily accessed, as the reaction tolerates a wide choice of the three starting materials. Therefore, this multicomponent approach provides a flexible method to fine tune the properties of the products. Funding This work was supported by the Junta de Extremadura and European Regional Development Fund (IB16095). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability The following information was supplied regarding data availability: The following information was supplied regarding data availability: Raw data are available in the Supplemental Files. Raw data are available in the Supplemental Files. Grant Disclosures The following grant information was disclosed by the authors: Junta de Extremadura and European Regional Development Fund: IB16095. The following grant information was disclosed by the authors: The following grant information was disclosed by the authors: Junta de Extremadura and European Regional Development Fund: IB16095. Junta de Extremadura and European Regional Development Fund: IB16095. Author Contributions • Ana Bornadiego performed the experiments, analyzed the data, prepared figures and/or tables, approved the final draft. • Ana Bornadiego performed the experiments, analyzed the data, prepared figures and/or tables, approved the final draft. • Jesús Díaz conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. • Carlos Fernández Marcos conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. Bornadiego et al. 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https://openalex.org/W4321850976	http://www.ecoeet.com/pdf-157535-87997?filename=Trends in the Use of.pdf	English	null	Trends in the Use of Symbiotics in Morocco during the Years 2020 and 2021	Ecological Engineering & Environmental Technology	2,023	cc-by	2,422	ABSTRACT Several studies have highlighted the importance of these 3 concepts, which are probiotics, prebiotics and symbiotic in daily clinical practice. They play an important role in the treatment/prevention of several diseases and contrib ute to the proper functioning of the digestive and immune systems. The objective of our study is to describe and compare the evolution of the use of symbiotic by the Moroccan population based on the sales rate between the year 2020 and 2021. Data collection was carried out by a means of a questionnaire concerning 15 symbiotic prod ucts most used by the Moroccan population. Our study suggests that some products have seen an increase in sales between 2020 and 2021, such as Lactobaciluscasei which reached 95,500 products in 2021 instead of 69,500 prod ucts in 2020, while the sale of the products decreased such as Bifidobacterium longum which reached 0 products in 2021 instead of 300 products in 2020, and according to the student test there is a significant difference between the average sales during the two successive years of 2020 and 2021 (p = 0,037). Ourresults show that the consumption and the use of symbiotic products by Moroccan populationhave shown significant differences. Keywords: symbiotic, prebiotics, probiotics, sales, Moroccan population. ECOLOGICAL ENGINEERING & ENVIRONMENTAL TECHNOLOGY Received: 2022.12.12 Accepted: 2023.01.27 Published: 2023.02.25 Ecological Engineering & Environmental Technology 2023, 24(3), 109–113 https://doi.org/10.12912/27197050/157535 ISSN 2719-7050, License CC-BY 4.0 Ecological Engineering & Environmental Technology 2023, 24(3), 109–113 https://doi.org/10.12912/27197050/157535 ISSN 2719-7050, License CC-BY 4.0 Mouri Misk1, Alibrahimi Btihaj1, El Ghouddany Safouane1, Ettair Said2, Mehdioui Sakina3, Bour Abdellatif1 1 Laboratory of Biology and Health (LBS), Team of Sciences of Food Nutrition and Health (ESNAS), Faculty of Sciences, Ibn Tofaïl University of Kenitra, Kentira, Morocco 1 Laboratory of Biology and Health (LBS), Team of Sciences of Food Nutrition and Health (ESNAS), Faculty of Sciences, Ibn Tofaïl University of Kenitra, Kentira, Morocco 2 Nutrition and Food Science Research Team, Faculty of Medicine and Pharmacy Mohammed V University of Rabat, Rabat, Marocco 2 Nutrition and Food Science Research Team, Faculty of Medicine and Pharmacy Mohammed V University of Rabat, Rabat, Marocco 3 Geosciences Laboratory, Department of Geology, Faculty of Sciences of Kenitra, Ibn Tofail University, Kenitra, Morocco 3 Geosciences Laboratory, Department of Geology, Faculty of Sciences of Kenitra, Ibn Tofail University, Kenitra, Morocco Corresponding author’s e-mail: mehdiouisakina@gmail.com Trends in the Use of Symbiotics in Morocco During the Years 2020 and 2021 Mouri Misk1, Alibrahimi Btihaj1, El Ghouddany Safouane1, Ettair Said2, Mehdioui Sakina3, Bour Abdellatif1 INTRODUCTION must resist gastric, biliary and pancreatic secre tions to remain alive in the small intestine and colon (Doron and Snydman., 2015). Several stud ies suggest that probiotics play important roles in human health, such as regulating the immune response, rebalancing the intestinal flora and pre venting certain diseases and cancer (Markowiak and Śliżewska., 2017). Prebiotics are defined as non-digestible substances capable of exerting multiple positive virtues in humans, they stimu late the growth and activity of certain microbial species (Gibson and Roberfroid., 1995). The best- known are: inulin, oligofructose, galacto-oligosac charides, lactulose (Fan et al., 2016; Vandenplas et al., 2015; Closa-Monasterolo et al., 2013). Symbiotic are synthetic food products that contain both probiotics and prebiotics (Gibson and Roberfroid., 1995). The main role of this combina tion is to maintain a healthy intestinal microbiota (Rioux et al., 2005). Probiotics are living microor ganisms that, when ingested in adequate quantity, have beneficial effects on health (Hill et al., 2014). Symbiotic are synthetic food products that contain both probiotics and prebiotics (Gibson and Roberfroid., 1995). The main role of this combina tion is to maintain a healthy intestinal microbiota (Rioux et al., 2005). Probiotics are living microor ganisms that, when ingested in adequate quantity, have beneficial effects on health (Hill et al., 2014). Among the microorganisms most commonly used as probiotics are: Lactobacillus and Bifidobac terium, some species of E.coli and Bacillus and the yeast Saccharomyces cerevisiae (Kim et al., 2019; Zawistowska-Rojek and Tyski., 2018). Pro biotics must be neither toxic nor pathogenic, and 109 Ecological Engineering & Environmental Technology 2023, 24(3), 109–113 The relationship between the sales of symbiotic in the years 2020 and 2021 In addition, probiotic-prebiotic couples are currently being tested. For example, the case for the combination of Bifidobacteria/fructo- oligosaccharides, Lactobacilli/Lactilol or Bifidobacteria/galacto-oligosachharides. According to the student test t, the results show that there is a significant difference between averages of sales during the 2 successive years of 2020 and 2021 with p = 0.037 (Table 2). Our work consists to describe the evolution of the use of symbiotic by the Moroccan population during two successive years 2020 and 2021 based on the growth rate of sales of these products. The rate of change in sales between 2021 and 2022 (thousands of Dirhams) The percentage variation in sales between 2020 and 2021is shown in figure 2. We observed that P5 was predominant with 88% of the overall products, followed by P5 with 85% and P12 with 65%. Inversely, P11, P13, P14, P15 presented 0% of the overall market. DISCUSSION The oral consumption of microorganisms as beneficial agents for human health has fascinat ed humanity for many centuries. The concept of probiotics was defined in the first time by Ferdi nand Vergin in 1954 (Markowiak and Śliżewska., 2017). Products containing probiotics have re cently known a great commercial success in Eu rope, in Asia and in other parts of the world. This progress will stimulate the consumption and de velopment of the product. Data collection This cross-sectional study was based on sever al types of symbiotic products. The data concerned the increase in sales throughout the Moroccan ter ritory during the two consecutive years 2020 and 2021. Data collection was based on a question naire distributed by IMS (information manage ment system) and completed by the marketing au thorization holder in Morocco territory (Table 1). Statistical analysis The mean comparison between the sales of the symbiotic was evaluated by the Student test t. Any association with p <0.05 was con sidered statistical significant. The data analysis was entered and performed using the Statistical Package for the Social Science (SPSS) (software version 22.0). The results of the study showed that there is a difference in sales between the symbiotic prod ucts. Some products have seen an increase in sales between 2020 and 2021, as Lactobaciluscasei which reched 95,500 products in 2021 instead of 69,500 products in 2020, while the sale of the products decreased such as Bifidobacterium longum which reached 0 products in 2021 instead of 300 products in 2020, and the major rate in the market is occupied by Bifidobacterium lactis with a sale rate reaching 88% of total sales in the Mo roccan product. The evolution of sales of symbiotic products during 2020 and 2021 According to the results obtained in Fig ure 1, we observed an increase in the sales of products P1 (Corn starch, capsule shell...), P2 (fructoolligosacharide…), P3 (Lactobacilus casei...), P4 (Fiber…), P5 (Bifidobacterium lactis…), P6 (Rhubarb…), P8 (Ocidilactici..), P9 (Lactobacillus rhamnosus…), P12 (Cellu lose..), on the other hand, a decrease in the sales of products P7 (Bifidobacterium longum..), P10 (Lactobacillus helveticus..), and lack in the sales of products P11 (Vitamin D..), P13 (Bulking agent..), P14 (Citral…), P15 (1g of active substance con tains: Enterococcus faecium – 300 mg...). The European population consumes probiot ics in food and in food supplements. The con sumer market for probiotic products is worth > 1,4 billion euros in Western Europe (Saxelin., 2008). The consumption of yoghurts and desserts is the most important, which sales of 1 billion euros. Thus, the food supplements have an esti mated activity of 10% of total probiotic market. A study in the Japan revealed that the sale of probiotic products has grown exponentially, US$5.3 billion in 2005 instead $1.12 billion in 110 Ecological Engineering & Environmental Technology 2023, 24(3), 109–113 Table 1. Symbolization of symbiotic products in this study with their dates of initial sales on the Moroccan market Trade name Symbol of symbiotic in the study Date of initial sales in the Moroccan market Corn starch; capsule shell: hydroxypropylmethylcellulose; bacterial strain Bifidobacterium infantis (strain 35624®); anti-caking agents: magnesium salts of fatty acids, magnesium stearate of fatty acids; saccharose, trehalose; stabilizing agent: sodium citrate P1 01/2018 fructoolligosacharides 958.2 mg/sachet + Lactobacillus casei (PXN® 37TM) ; Lactobacillus rhamnosus PXN® 54TM); Strepococcus thermophylus PXN® 66TM ; Lactobacillus acidophilus PXN® 35TM); Bifidobacterium breve PXN® 25TM ; Bifidobacterium infantis PXN® 27TM; Lactobacillus bulgaricus PXN® 39TM P2 08/2012 Saccharomyces boulardii 282.5 mg per sachet P3 06/2012 Fiber (fructooligosaccharides 25.4 mg), inulin (25.4 mg), Lactobacillus helveticus Rosell-52, Lactobacillus helveticus Lafti® L10, Lactobacillus rhamnosus Rosell-11, Bifidobacterium longum Rosell-175, vitamin D3 (0.75 µg). P4 08/2010 Bifidobacterium lactis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium breve, Lactobacillus acidophilic, Lactobacillus brevis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus lactis Strepococcus thermophylus, Saccharomyces boulardii, Bacillus coagulans, fructo-oligosaccharides (Actilight), inulin, corn maltodextrin (bulking agent), hydroxymethylcellulose (coating agent), magnesium stearate, silicon oxide (anti-caking agent), gelan gum (thickener). The evolution of sales of symbiotic products during 2020 and 2021 P5 10/2017 Rhubarb, guimauve, Pale Pink, Artichoke (support: maltodextrine, antioxydant : acide citrique), Basil, Coriander, yeast, anti-caking agent: tricalcium phosphate; potato dextrin, lactic ferments (support : maltodextrin): Bifidobacterium bifidum, Lactobacillus acidophilus P6 08/2017 Bifidobacterium longum LA101 Lactobacillus helveticus LA102 Lactococcus lactis LA103 Strepococcus thermophylus LA104 P7 03/2010 Lactobacillus plantarum CECT 7484, Lactobacillus plantarum CECT 7485, Pediococcus acidilactici CECT 7483, Excipients: maltodextrin, silicon dioxide, magnesium stearate, hydroxypropyl methylcellulose, titanium dioxide. P8 09/2018 Lactobacillus rhamnosus GG Bifidobacterium lactis + Tormentil root Excipients: maltodextrin, hypromellose (capsule); microcristalline cellullose; titanium dioxide (colorant) ; magnesium stearate(anticlotting agent). P9 04/2019 Bifidobacterium longum LA101, Lactobacillus helveticus LA102, Lactococcus lactis LA103, Strepococcus thermophylus LA104, Lactobacillus Rhamnosus LA801, Vitamine D P10 03/2010 Lactobacillus rhamnosus R0011 Lactobacillus helveticus R0052, Vitamine D P11 06/2016 Bulking agent: cellulose; Lactobacillus acidophilus, Lactobacillus plantarum, Bifidobacterium lactis, Bifidobacterium breve; anti-caking agent: magnesium salts of fatty acids. P12 01/2010 sweetener (xylitol), Lactobacillus reuteri DSM 17938; Lactobacillus reuteri Protectis, vitamin D3 20 µg + flavor enhancer: citric acid. P13 06/2014 Lactobacillus fermentum; Lactobacillus delbrueckii. Fermented culture Lactose monohydrate, casein peptone, yeast extract, sodium acetate trihydrate, dipotassium phosphate anhydrous. Excipients: hydrated colloidal silica, talc, magnesium stearate, anhydrous lactose. Lyophilization adjuvants: lactose monohydrate, calcium carbonate. colorant (capsule): titanium dioxide P14 unknown Lactobacillus acidophilus (DSM 13241); Bifidobacterium animalis subsp. lactis (DSM 15954), + Oligofructose, * bulking agent: cellulose, potato starch, * humectant: fatty acids. P15 06/2013 111 111 Ecological Engineering & Environmental Technology 2023, 24(3), 109–113 Figure 1. The evolution of sales of symbiotic products during 2020 and 2021 Figure 1. The evolution of sales of symbiotic products during 2020 and 2021 Figure 2. The rate of change in sales between 2021 and 2022 (thousands of Dirhams) Table 2. The relationship between the sales of symbiotic in the years 2020 and 2021 Year N Moy / Et P Sales in thousands of units in 2020 Sales in thousands of units in 2021 15 15 13926.66 ± 21258.19 19346.66 ± 30132.15 0.037 Table 2. The relationship between the sales of symbiotic in the years 2020 and 2021 Figure 2. The rate of change in sales between 2021 and 2022 (thousands of Dirhams) CONCLUSION 1997 (Ohama and Ikeda., 2006), additionally, some Japanese hospitals used probiotics for treat ing gastrointestinal disorders, and symptoms re lated to antibiotic use (Amagase., 2008). The consumption and the use of symbiotic by the Moroccan population have shown significant differences. It is necessary to concentrate the ef forts between all the institutions for the sensitiza tion and the awareness by the population on the benefits of probiotics, the consideration of the socio-economic criteria of the person and the fa cilitation of the conditions of importation and the marketing of the products. According to the report revealed by WHO and FAO in November 2018, the global market for probiotic ingredients was valued $1,5 billion in 2016 and is expected to reach $2,15 billion by 2021 (Bornes., 2020). In the same sense, a market research report on probiotics in France for the period of October 2018 to September 2019 revealed that sales of probiotic products increased from about 54 million euros in the period of October 2017 to September 2018 to about 60 million euros in the period of October 2018 to September 2019. That is an increase of about 11% in just 1 year (Bornes., 2020). REFERENCES 1. Amagase H. 2008. Current marketplace for probiot ics: a Japanese perspective. Clin Infect Dis., 46(2), 73–75. 112 Ecological Engineering & Environmental Technology 2023, 24(3), 109–113 2. Bornes F. 2020. Évaluation des probiotiques sur le marché officinal et élaboration d’un guide pratique d’aide au conseil sur les probiotiques pour le phar macien d’officine, 241. 8. Kim S.K., Guevarra R.B., Kim Y.T., Kwon J., Kim H., Cho J.H., Kim H.B., Lee J.H. 2019. Role of Pro biotics in Human Gut Microbiome-Associated Dis eases. J MicrobiolBiotechnol., 29(9), 1335–1340. 9. Markowiak P., Śliżewska K. 2017. Effects of Probi otics, Prebiotics, and Synbiotics on Human Health. Nutrients, 9(9), 1021. 3. Closa M.R., Gispert L.M., Luque V., Ferre N., Rubio T.C., Zaragoza J.M., Escribano J. 2013. Safety and efficacy of inulin and oligofructose supplementation in infant formula: results from a randomized clinical trial. ClinNutr., 32(6), 918–927. 10. Ohama H., Ikeda H., Moriyama H. 2006. Health foods and foods with health claims in Japan. Toxi cology, 221(1), 95–111. 4. Doron S., Snydman D.R. 2015. Risk and safety of probiotics. Clin Infect Dis., 60(2), 129–134. 11. Rioux K.P., Madsen K.L., Fedorak R.N. 2005. The role of enteric microflora in inflammatory bowel disease: human and animal studies with probiotics and prebiotics. GastroenterolClin North Am., 34(3), 4654–4682. 5. Fan C.H., Cao J.H., Zhang F.C. 2016. The prebiotic inulin as a functional food – a review. Eur Rev Med Pharmacol Sci., 20(15), 3262–3265. 6. Gibson G.R., Roberfroid M.B. 1995. Dietary modulation of the human colonic microbiota: in troducing the concept of prebiotics. J Nutr., 125(6), 1401–1412. 12. Saxelin M. 2008. Probiotic formulations and appli cations, the current probiotics market, and changes in the marketplace: a European perspective. Clin Infect Dis., 46(2), 76–79. 7. Hill C., Guarner F., Reid G., Gibson G.R., Meren stein D.J., Pot B., Morelli L., Canani R.B., Flint H.J., Salminen S., Calder P.C., Sanders M.E. 2014. Expert consensus document. The International Sci entific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol., 11(8), 506–514. 13. Vandenplas Y., Zakharova I., Dmitrieva Y. 2015. Oligosaccharides in infant formula: more evidence to validate the role of prebiotics. Br J Nutr., 113(9), 1339–1344. 14. Zawistowska R.A., Tyski S. 2018. Are Probiotic Really Safe for Humans? Pol JMicrobiol., 67(3), 251–258. 113
https://openalex.org/W2323422473	http://www.mathnet.ru/php/getFT.phtml?jrnid=vsgtu&paperid=162&what=fullt&option_lang=rus	Russian	null	О проблеме устойчивости внецентренно сжатых бетонных и железобетонных элементов средней и большой гибкости (до $\lambda=l_0 /i\le 120$)	Vestnik Samarskogo gosudarstvennogo tehničeskogo universiteta. Seriâ: Fiziko-matematičeskie nauki/Vestnik Samarskogo gosudarstvennogo tehničeskogo universiteta. Seriâ Fiziko-matematičeskie nauki	2,003	cc-by	2,113	Math-Net.Ru Общероссийский математический портал В. В. Теряник, О проблеме устойчивости внецентренно сжатых бетонных и же- лезобетонных элементов средней и большой гибкости (до λ = l0/i ≤120), Вестн. Сам. гос. техн. ун-та. Сер. Физ.-мат. науки, 2003, выпуск 19, 167– 169 DOI: 10.14498/vsgtu162 DOI: 10.14498/vsgtu162 Использование Общероссийского математического портала Math-Net.Ru подразумевает, что вы прочитали и согласны с пользовательским соглашением http://www.mathnet.ru/rus/agreement Параметры загрузки: IP: 195.242.1.93 24 октября 2024 г., 08:21:33 Параметры загрузки: IP: 195.242.1.93 24 октября 2024 г., 08:21:33 БИБЛИОГРАФИЧЕСКИЙ СПИСОК 1. Глущенков В. С., Сараев Л. А., Хохрякова Ю. В. Малые упругопластические деформации композиционного ма- териала, хаотически армированного эллипсоидальными включениями // Вестн. СамГУ. 2001. № 2(20). С. 121–125. Поступила 15.02.2003 г. Поступила 15.02.2003 г. 1. Глущенков В. С., Сараев Л. А., Хохрякова Ю. В. Малые упругопластические деформации композиционного ма- териала, хаотически армированного эллипсоидальными включениями // Вестн. СамГУ. 2001. № 2(20). С. 121–125. F Ф К γ ≤ ⋅ , где F - фактическая нагрузка на конструкцию в момент обследования, когда выявлены дефекты и повреждения; Ф - несущая способность конструкции без учета повреждений и дефектов, оп- ределяемая расчетом из условия прочности и устойчивости по фактическим значениям площа- дей сечений Аб, Аs и прочности бетона и стали Rв, Rs; К - коэффициент снижения несущей спо- собности конструкции при наличии дефектов и повреждений, значение которого может быть установлено на основе результатов обследований; γ - коэффициент надежности по материалу. При практических расчетах условие (1) удобно представить в виде (1/ )ФК ( При практических расчетах условие (1) удобно представить в виде (1/ ) F ФК γ ≤ . (2) (2) Чтобы правильно решить вопрос, какие элементы конструкции надо усиливать и по какой при- чине, условие (2) необходимо проверять для каждого элемента конструкции отдельно. Для вне- центренно сжатых элементов: а) из условия прочности – , ) в s пр M γ γ или ( , ) факт в s пр N N γ γ ≤ ; (3) ( , ) факт в s кр N N γ γ ≤ , (4) ( , ) факт в s пр M M γ γ ≤ или ( , ) факт в s пр N N γ γ ≤ ; (3) (3) б) из условия устойчивости – б) из условия устойчивости – ( , ) факт в s кр N N γ γ ≤ , (4) (4) ( , ) факт в s кр N N γ γ ≤ , (4) ( , ) факт в s кр N N γ γ ≤ , ( , ) факт в s кр N N γ γ ≤ , (4) О ПРОБЛЕМЕ УСТОЙЧИВОСТИ ВНЕЦЕНТРЕННО СЖАТЫХ БЕТОННЫХ И ЖЕЛЕЗОБЕТОННЫХ ЭЛЕМЕНТОВ СРЕДНЕЙ И БОЛЬШОЙ ГИБКОСТИ (ДО 0 λ = l /i 120 ≤ ) Проанализированы и обобщены результаты экспериментальных исследований по определению не- сущей способности внецентренно сжатых колонн средней и большой гибкости. Выявлены недос- татки существующей методики расчета. Усовершенствована методика проверки несущей спо- собности внецентренно сжатых колонн из условия прочности и устойчивости с учетом реальных свойств материала. Известно, что для нормальной эксплуатации конструкций должно соблюдаться общее тре- бование строительных норм [1] F Ф К γ ≤ ⋅ , (1) F Ф К γ ≤ ⋅ , (1) где , в s γ γ - коэффициенты надежности по бетону и арматуре. , в s γ - коэффициенты надежности по бетону и арматуре. где , в s γ γ - коэффициенты надежности по бетону и арматуре. Как показано в работе [2], для внецентренно сжатых элементов средней и большой гибко- сти несущая способность при потере устойчивости всегда будет меньше, чем из условия проч- ности пр N . Однако методика определения кр N из условия устойчивости для внецентренно сжа- тых элементов (колонн) при малых и средних эксцентриситетах е0 с учетом гибкости λ разра- ботана пока недостаточно. По имеющейся методике расчета бетонных и железобетонных ко- лонн в СНиПе [1] проверка устойчивости выполняется в косвенном виде и весьма приближен- но. При этом проектировщики не имеют возможности строгой оценки несущей способности таких колонн из условия устойчивости. А для решения вопроса о необходимости усиления сжа- тых колонн следует более точно проверять несущую способность их из условия возможной по- тери устойчивости, особенно это относится к колоннам средней и большой гибкости (3). р у р ( ) Для проверки несущей способности гибких сжатых элементов при 14 λ ≥ рекомендуется использовать условие [1] (5) [ ] расч расч пр в вс в sc s s N е M R А Z R A Z ′ ⋅ ≤ = ⋅ + ⋅ , (5) 167 е для прямоугольного сечения с симметричной арматурой sA и sA′ 0 0 ( ); 2 расч a h M e e a e e N η = ⋅ + − = + . где , в s γ γ - коэффициенты надежности по бетону и арматуре. (6) (6) Здесь η - коэффициент, учитывающий влияние поперечного прогиба колонны на увеличение расчетного момента от N, определяемый по приближенной формуле 1/(1 / ) расч kp N N η = − , (7) (7) 1/(1 / ) расч kp N N η = − , пр N - условная критическая сила для сжатой колонны, определяемая по обобщенной формуле, учитывающей неупругие свойства сжатого бетона и характер действия нагрузки, учитываемого коэффициентом е ϕ : пр N - условная критическая сила для сжатой колонны, определяемая по обобщенной формуле, учитывающей неупругие свойства сжатого бетона и характер действия нагрузки, учитываемого коэффициентом е ϕ : 2 0 6,4 0,11 0,1 , 0,1 / б б кр s l e p E J N I l α ϕ δ ϕ     = + +       +       (8) (8)   0 0 / ; / (0,5 0,01 / 0,01 ) s б e в E E e h l h R α δ = = ≥ − − . (9) где (9) Недостатком методики [1] является приближенная формула (7), которая при больших зна- чениях Nрасч дает неоправданно большое значение η . Теоретически при расч kp N N η → →∞. Чтобы не было большого перерасхода арматуры в правой части условия (4), требования [1] ограничивают максимальное значение этого коэффициента величиной max 2,5 η = , чему соот- ветствует отношение / 0,6 расч кр N N = . В последние годы при строительстве жилых, общественных и промышленных зданий на- чали широко применяться бетоны повышенной и высокой прочности с допустимыми напряже- ниями до 800 МПа. Внедрение высокопрочных бетонов в изготовлении несущих каркасов по- влекло за собой применение конструкций сжатых колонн меньшего сечения по сравнению с сечениями из обычного бетона. При этом гибкости их возрастают, поэтому возникает опас- ность потери устойчивости первоначальной деформации изгиба колонн в плоскости эксцентри- ситета или из плоскости. Таким образом, актуальность разработок по совершенствованию ме- тодики проверки устойчивости таких колонн возрастает. При этом важными факторами в ре- шении задачи устойчивости является более точный учет неупругих свойств материала (бетона), геометрических характеристик сечения и расчетных эксцентриситетов расч e , а также расчетных длин колонн l0, входящих в состав каркаса. За последние пять лет вопрос совершенствования нормативных рекомендаций поднимался многократно проектировщиками и исследователями. Среди выполненных исследований особо надо отметить натурные эксперименты, выполненные Д.О. Астафьевым [2] и В.С.Бабичем [3]. где , в s γ γ - коэффициенты надежности по бетону и арматуре. За последние пять лет вопрос совершенствования нормативных рекомендаций поднимался многократно проектировщиками и исследователями. Среди выполненных исследований особо надо отметить натурные эксперименты, выполненные Д.О. Астафьевым [2] и В.С.Бабичем [3]. Исследования Д.О. Астафьева производились на рамном каркасе с гибкостью колонн 80 λ = . Бабич В.С. для экспериментов использовал одиночные колонны гибкостью 95, варьи- руя эксцентриситеты приложения продольной силы { } 2 1 / 0; 0,5;1 е е = при 1 10 е = см. Общим выводом экспериментальных исследований является доказательство об исчерпании несущей способности колонн вследствие потери устойчивости первоначальной изогнутой оси колонн при достижении критического значения прогиба. При этом найденные значения кр N близко совпадали с уточненными теоретическими значениями Nkp, полученными авторами [4]. В рабо- те В.С. Бабича [3] уточняется формула (7) для определения η , но по структуре она остается такой же. В работе Д.О. Астафьева [2] уточняется формула для определения Nкp с использова- нием коэффициента продольного изгиба e ϕ , зависящего от гибкости λ, относительного эксцен- триситета 0 / m e A W = и класса бетона. Для прямоугольного сечения 2 /6 W bh = , тогда 2 0 0 ( ) 6/ 6 / m e bh bh e h = ⋅ = (10) 2 0 0 ( ) 6/ 6 / m e bh bh e h = ⋅ = . (10) (10) 2 0 0 ( ) 6/ 6 / m e bh bh e h = ⋅ = . Экспериментальные значения Nкp, полученные В.С. Бабичем, близко совпадают с теорети- ческими Nкp , полученными автором из общего условия потери устойчивости (2 рода) внецен- тренно сжатых колонн в виде (11) / 0 dN df = (11) при кубической и квадратной зависимостях: 3 при кубической и квадратной зависимостях: 3 (12) σ = α1ε -α3ε 3 и σ = α1ε - α 2ε 2 . (12) Здесь α1=Еб, α3=Еб/3ε 2 пп , α2 = Еб/ 2ε пп ε пп - относительная деформация бетона при напряже- нии, равном пределу прочности σпп (Rb). Здесь α1=Еб, α3=Еб/3ε 2 пп , α2 = Еб/ 2ε пп ε пп - относительная деформация бетона при напряже- нии, равном пределу прочности σпп (Rb). где , в s γ γ - коэффициенты надежности по бетону и арматуре. 168 Использованы допущения: учитывается работа только восходящих ветвей диаграммы σ - ε; деформация стойки в предельном состоянии по устойчивости происходит по полуволне си- нусоиды до появления трещин; при выводе основных уравнений используется закон плоских сечений; работа сжатых элементов происходит при малых относительных деформациях ε, соот- ветствующих вр σ σ = ; при выявлении физико-механических характеристик используются диа- граммы одноосного напряженного состояния; рассматривается симметричное сечение колонны (в виде прямоугольника или двутавра с одинаковыми полками). ( р у у р ) С учетом принятых допущений для внецентренно сжатой стойки получены уравнения рав- новесия ; z А N dA σ = ∫ 3 3 3 . A A a E M zdA z dA ρ ρ = − ∫ ∫ В результате решения приведенных уравнений получена простая формула для определения N кр, в которой впервые поперечные прогибы внецентренно сжатой стойки учтены более точно по сравнению с [1]: (13) y кр кр н N N k = ⋅ , (13) y кр кр н N N k = ⋅ , где Ν у кр = π2 Еb Jb / ℓ 2 - в упругой стадии работы бетона. Здесь н k - коэффициент, учиты- вающий деформацию внецентренно сжатой стойки с учетом нелинейности материала, значе- ние которого всегда меньше 1 (коэффициент нелинейности); f – поперечный прогиб, опреде- ляемый из решения кубического или квадратного уравнения, полученного из условия (12) с учетом выражения Ν по (13). Доказано, что с увеличением эксцентриситета продольной силы значение коэффициента нелинейности уменьшается. Формула для Nкр может быть использована для построения кривых равновесных состояний N – f. Показана возможность применения формулы (13) для определе- ния Nкр для железобетонных колонн. Произведена оценка несущей способности сжатых эле- ментов различной гибкости по условиям прочности и устойчивости, в том числе для железобе- тонных колонн из серии, испытанных Бабичем С.В. и Астафьевым Д.О. при гибкостях λ = 93 и 78. Результаты сравнения экспериментальных данных с результатами аналитического расчета, полученными по предлагаемой методике, показали незначительные расхождения, что допуска- ет ее применение при решении поверочных и проектировочных задач. Таким образом, совершенствование методики проверки несущей способности внецентрен- но сжатых колонн средней и большой гибкости ( до 120 λ ≤ ) имеет не только теоретическое, но и практическое значение, чтобы правильно решить вопрос об усилении колонн, если ока- жется, что расч kр N N > . где , в s γ γ - коэффициенты надежности по бетону и арматуре. Такое условие может возникнуть при реконструкции существующих зданий со сжатыми ко- лоннами при увеличении расчетных нагрузок от более тяжелого технологического оборудова- ния или при ошибках проектировщика, допущенных в определении Nкp. БИБЛИОГРАФИЧЕСКИЙ СПИСОК 1. СНиП 2.03.01-84* Бетонные и железобетонные конструкции. М. Госстрой России,1999. 75 с. 1. СНиП 2.03.01-84* Бетонные и железобетонные конструкции. М. Госстрой России,1999. 75 с. 2. Астафьев Д.О. Расчет реконструируемых железобетонных конструкций. СПб.: СПб гос. архит.– строит. уни-т, 1995. 158 с. 2. Астафьев Д.О. Расчет реконструируемых железобетонных конструкций. СПб.: СПб гос. архит.– строит. уни-т, 1995. 158 с. 3. Бабич В.С. Исследование и расчет сжатых элементов с переменными эксцентриситетами по длине // Бетон и железобетон. 1992. №10. С.12. 3. Бабич В.С. Исследование и расчет сжатых элементов с переменными эксцентриситетами по длине // Бетон и железобетон. 1992. №10. С.12. 4. Раевский А.Н., Теряник В.В., Мальков А.А. Совершенствование методики определения несущей способности внецентренно сжатых колонн из условия устойчивости и прочности ВНТИЦ. М., 2001. 15с. Деп. в ВНТИЦ. №72200100023. 4. Раевский А.Н., Теряник В.В., Мальков А.А. Совершенствование методики определения несущей способности внецентренно сжатых колонн из условия устойчивости и прочности ВНТИЦ. М., 2001. 15с. Деп. в ВНТИЦ. №72200100023. Поступила 7.05.2003 г. 169
https://openalex.org/W3121110678	https://europepmc.org/articles/pmc7825061?pdf=render	English	null	Therapeutic Nanobodies Targeting Cell Plasma Membrane Transport Proteins: A High-Risk/High-Gain Endeavor	Biomolecules	2,021	cc-by	15,060	Citation: Van Campenhout, R.; Muyldermans, S.; Vinken, M.; Devoogdt, N.; De Groof, T.W. Therapeutic Nanobodies Targeting Cell Plasma Membrane Transport Proteins: A High-Risk/High-Gain Endeavor. Biomolecules 2021, 11, 63. https://doi.org/10.3390/biom11010063 Keywords: nanobodies; cell plasma membrane transport proteins; drug target; therapy Received: 26 November 2020 Accepted: 1 January 2021 Published: 6 January 2021 Received: 26 November 2020 Accepted: 1 January 2021 Published: 6 January 2021 Review Therapeutic Nanobodies Targeting Cell Plasma Membrane Transport Proteins: A High-Risk/High-Gain Endeavor Raf Van Campenhout 1 , Serge Muyldermans 2 , Mathieu Vinken 1,†, Nick Devoogdt 3,† and Timo W.M. De Groof 3,,† 1 Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 B l B l i f h @ b b (R VC ) hi i k @ b b (M V) 1 Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium; raf.van.campenhout@vub.be (R.V.C.); mathieu.vinken@vub.be (M.V.) 2 Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, g p 2 Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium; serge.muyldermans@vub.be g g y 3 In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit Brussel, Laarbeeklaan 103 1090 Brussels, Belgium; ndevoogd@vub.be g g Correspondence: timo.de.groof@vub.be; Tel.: +32-2-6291980 † These authors share equal seniorship. † These authors share equal seniorship. Abstract: Cell plasma membrane proteins are considered as gatekeepers of the cell and play a major role in regulating various processes. Transport proteins constitute a subclass of cell plasma membrane proteins enabling the exchange of molecules and ions between the extracellular environment and the cytosol. A plethora of human pathologies are associated with the altered expression or dysfunction of cell plasma membrane transport proteins, making them interesting therapeutic drug targets. However, the search for therapeutics is challenging, since many drug candidates targeting cell plasma membrane proteins fail in (pre)clinical testing due to inadequate selectivity, speciﬁcity, potency or stability. These latter characteristics are met by nanobodies, which potentially renders them eligible therapeutics targeting cell plasma membrane proteins. Therefore, a therapeutic nanobody-based strategy seems a valid approach to target and modulate the activity of cell plasma membrane transport proteins. This review paper focuses on methodologies to generate cell plasma membrane transport protein-targeting nanobodies, and the advantages and pitfalls while generating these small antibody-derivatives, and discusses several therapeutic nanobodies directed towards transmembrane proteins, including channels and pores, adenosine triphosphate-powered pumps and porters. biomolecules 1. Introduction The cell plasma membrane is an essential structure, as it shapes cyto-architecture and protects cellular integrity. Although the cell plasma membrane features an impermeable double phospholipid layer structure, the passage of ions and biomolecules, such as glucose, glutamate, adenosine triphosphate (ATP) and water-soluble vitamins, through this mem- brane is essential for maintaining cellular homeostasis. Such cross-membrane trafﬁcking of biomolecules and ions between the cellular cytosol and the extracellular environment is facilitated and regulated by membrane transport proteins that are associated with the cell plasma membrane [1,2]. Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- ms in published maps and institutio- nal afﬁliations. Cell plasma membrane proteins form a family of integral membrane proteins that can be grouped in several subclasses, denoted as channels and pores, ATP-powered pumps and porters [3]. By enabling the transport of substances across the cell plasma membrane, these transport proteins are key molecules for the establishment of physiological processes in all vital organs [4]. Moreover, cell plasma membrane transport proteins are also fre- quently involved in disease [2,4]. As such, misfolding, mutations, downregulation or the overexpression of cell plasma membrane transport proteins are implicated in pathological conditions [2,5–7]. Consequently, cell plasma membrane transport proteins have gained Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 63. https://doi.org/10.3390/biom11010063 Biomolecules 2021, 11, 63 2 of 19 considerable interest as potential drug targets [2,5,8,9]. However, the clinical translation of small-molecule drugs targeting these transport proteins is mostly impeded by the lack of se- lectivity, speciﬁcity, potency or stability of these molecules [2,10,11]. Moreover, membrane transport proteins are known to be highly dynamic, resulting in different protein confor- mations, which can interfere with identiﬁcation/screening of drugs targeting cell plasma membrane transport proteins. The use of conventional antibodies and antigen-binding fragments could overcome some of these issues [12–16]. However, the identiﬁcation of cell membrane transport protein-targeting antibodies may be limited due to the large size of antibodies and their generally more concave paratope, making small and hidden cryptic epitopes of these proteins potentially unreachable [17–20]. Nanobodies are a plausible therapeutic alternative for targeting cell plasma membrane transport proteins. 1. Introduction In this review paper, the biology and classiﬁcation of membrane proteins is described, with a special focus on cell plasma membrane transport proteins. Moreover, the charac- teristics of antibodies and a state-of-the-art overview of methods to generate nanobodies against these transport proteins is presented. Finally, the use of transport protein-targeting nanobodies as therapeutic agents for various diseases is discussed. 2. Classiﬁcation of Cell Plasma Membrane Proteins: Focus on Multi-Pass Transmembrane Proteins (D) Multi-pass transmembrane proteins can be divided into different classes based on their function, namely transport proteins and receptors. (E) Transport proteins include channels and pores, ATP-powered pumps and 3 types of porters, namely uniporters, symporters and antiporters. 2. Classiﬁcation of Cell Plasma Membrane Proteins: Focus on Multi-Pass Transmembrane Proteins Cell plasma membrane proteins form a superfamily of proteins that are connected to the outer phospholipid bilayer. These proteins can be classiﬁed based on their structure, topology or their function. Based on the former, cell plasma membrane proteins can be generally categorized into three general classes, namely integral membrane proteins, peripheral membrane proteins and lipid-anchored proteins (Figure 1A) [21]. The class of integral membrane proteins can be further subdivided into two groups based on their interaction with the cell membrane, namely integral monotopic proteins and transmembrane proteins (Figure 1B) [22,23]. Integral monotopic proteins interact with the membrane from one side (i.e., inside or outside) and do not cross the cell plasma membrane. The monotopic proteins are underrepresented in the family of integral membrane proteins, and are performing catalyzing reactions on plasma membrane-resident substrates [24]. In contrast, transmembrane proteins span across the cell plasma membrane at least once. These proteins are made up of one or more hydrophobic transmembrane domain(s), soluble regions at the extracellular and/or intracellular N- and C-termini, and some signal and/or anchor sequences to direct the translocation and correct insertion of these proteins into the cell plasma membrane [22,23]. Based on their topology, the group of transmembrane proteins is classiﬁed into several subgroups (Figure 1C) [22,25]. Type I-IV transmembrane proteins are described as single-pass membrane proteins. Their classiﬁcation is based on the location of the N- and C-terminal ends of the proteins across the cell plasma membrane and the presence of signal peptides and anchor sequences. The structural understanding of these single-pass transmembrane proteins has recently been described in more detail [26]. Other types of transmembrane proteins have a more complex structure, including β-barrel and multi-pass transmembrane proteins [22,27]. The latter feature a complex assembly of both hydrophobic and hydrophilic protein domains allowing multi-pass transmembrane proteins to span the cell plasma membrane more than once [22,23,27]. The structural subgrouping of these proteins is complicated, as structural determination via crystallization or cryogenic electron microscopy remains challenging due to their dynamic and fragile character so that they unfold rapidly upon extraction from their phospholipid bilayer environment [28–30]. However, these multi-pass transmembrane proteins can be divided into different classes based on their function, such as transport proteins and receptors (Figure 1D) [23,27,31,32]. 3 of 19 3 of 19 Biomolecules 2021, 11, 63 Biomolecules 2021, 11, x FO Figure 1. Classification of cell plasma membrane proteins. 2. Classiﬁcation of Cell Plasma Membrane Proteins: Focus on Multi-Pass Transmembrane Proteins A B Peripheral membrane protein Integral membrane protein Lipid-anchored membrane protein Transmembrane protein OUT IN OUT IN C D E Type I Type II Type III Type IV Multi-pass C Pore ATP-powered pump Uniporter Symporter OUT OUT OUT IN IN IN + N N Transport protein N b-barrel N Receptor Channel Antiporter Integral monotopic protein C C C C Figure 1. Classiﬁcation of cell plasma membrane prot be divided in 3 general classes, namely integral memb and lipid anchored proteins. (B) Integral membrane p namely integral monotopic proteins and transmembr family of transmembrane proteins can be classiﬁed in multi-pass transmembrane proteins. (D) Multi-pass different classes based on their function, namely tra proteins include channels and pores, ATP-powered pu A Peripheral membrane protein Integral membrane protein Lipid-anchored membrane protein OUT IN B B Transmembrane protein OUT IN Integral monotopic protein C Type I Type II Type III Type IV Multi-pass C OUT IN N N N b-barrel N C C C C C Type I Type II Type III Type IV N D OUT IN Transport protein Receptor D E niporter Symporter Antiporter E Pore ATP-powered pump Uniporter Symporter OUT IN + Channel Antiporter ATP-powered ATP-powered Antiporter Uniporter Symporter Antiport Figure 1. Classification of cell plasma membrane proteins. (A) Cell plasma membrane proteins can be divided in 3 general classes, namely integral membrane proteins, peripheral membrane proteins and lipid anchored proteins. (B) Integral membrane proteins can be further divided into 2 groups, namely integral monotopic proteins and transmembrane proteins. (C) Based on their topology, the family of transmembrane proteins can be classified in type I, type II, type III, type IV, -barrel and multi-pass transmembrane proteins. (D) Multi-pass transmembrane proteins can be divided into different classes based on their function, namely transport proteins and receptors. (E) Transport proteins include channels and pores, ATP-powered pumps and 3 types of porters, namely uni- porters, symporters and antiporters. Figure 1. Classiﬁcation of cell plasma membrane proteins. (A) Cell plasma membrane proteins can be divided in 3 general classes, namely integral membrane proteins, peripheral membrane proteins and lipid anchored proteins. (B) Integral membrane proteins can be further divided into 2 groups, namely integral monotopic proteins and transmembrane proteins. (C) Based on their topology, the family of transmembrane proteins can be classiﬁed in type I, type II, type III, type IV, β-barrel and multi-pass transmembrane proteins. 2. Classiﬁcation of Cell Plasma Membrane Proteins: Focus on Multi-Pass Transmembrane Proteins (A) Cell plasma membrane proteins can be divided in 3 general classes, namely integral membrane proteins, peripheral membrane proteins and lipid anchored proteins. (B) Integral membrane proteins can be further divided into 2 groups, namely integral monotopic proteins and transmembrane proteins. (C) Based on their topology, the family of transmembrane proteins can be classified in type I, type II, type III, type IV, -barrel and multi-pass transmembrane proteins. (D) Multi-pass transmembrane proteins can be divided into different classes based on their function, namely transport proteins and receptors. (E) Transport proteins include channels and pores, ATP-powered pumps and 3 types of porters, namely uni- porters, symporters and antiporters. A B Peripheral membrane protein Integral membrane protein Lipid-anchored membrane protein Transmembrane protein OUT IN OUT IN C D E Type I Type II Type III Type IV Multi-pass C Pore ATP-powered pump Uniporter Symporter OUT OUT OUT IN IN IN + N N Transport protein N b-barrel N Receptor Channel Antiporter Integral monotopic protein C C C C Figure 1. Classiﬁcation of cell plasma membrane proteins. (A) Cell plasma membrane proteins can be divided in 3 general classes, namely integral membrane proteins, peripheral membrane proteins and lipid anchored proteins. (B) Integral membrane proteins can be further divided into 2 groups, namely integral monotopic proteins and transmembrane proteins. (C) Based on their topology, the family of transmembrane proteins can be classiﬁed in type I, type II, type III, type IV, β-barrel and multi-pass transmembrane proteins. (D) Multi-pass transmembrane proteins can be divided into different classes based on their function, namely transport proteins and receptors. (E) Transport proteins include channels and pores, ATP-powered pumps and 3 types of porters, namely uniporters, symporters and antiporters. Figure 1. Classification of cell plasma membrane pro be divided in 3 general classes, namely integral memb and lipid anchored proteins. (B) Integral membrane namely integral monotopic proteins and transmembr family of transmembrane proteins can be classified in multi-pass transmembrane proteins. (D) Multi-pass different classes based on their function, namely tra proteins include channels and pores, ATP-powered porters, symporters and antiporters. 3.3. Porters Porters mediate the passage of small molecules and ions like glucose and sodium and chloride ions [50]. The binding of substrates induces a conformational change in the porter protein, allowing the movement of these substrates across the cell plasma mem- brane [50]. Porters can be further classiﬁed based upon the number of types of molecules they convey [50,51]. Whereas uniporters transport one type of molecule, symporters and antiporters are classiﬁed as cotransporters that organize the exchange of two different substrates (Figure 1E) [50,51]. The role of porters as drug targets can be demonstrated by the group of solute carriers (SLC) [52,53]. 3.1. Channels and Pores 3.1. Channels and Pores Channels and pores allow the passage of small molecules and ions through a water- ﬁlled tunnel, which is simultaneously exposed to the intracellular and the extracellular environment [33–35]. While channels and pores share a similar structure, their conforma- tion is different. While pores are always open, channels can adopt either an open or a closed conformation (Figure 1E) [34]. Among the many channels are those built up by connexin (Cx) and pannexin (Panx) proteins [7,8,36]. Members of the connexin and pannexin family consist of 4 transmembrane regions, 2 extracellular loops, 1 cytoplasmic loop and an intra- cellular C-terminus and N-terminus [37,38]. At present, more than 20 different connexin isoforms have been identiﬁed in humans [39]. Connexin proteins are widely expressed and are named according to their molecular weight [36]. The best-known family member is Cx43, which is the most abundantly expressed and has been studied extensively because of its role in a variety of cellular processes and diseases [7,8,36,40]. Connexins control cellular communication via the formation of connexin hemichannels, which consist of six connexin molecules, and gap junctions arising from the interaction of two hemichannels of adjacent cells [7,8,36]. Unlike connexins, only three pannexins have yet been identiﬁed, which are named Panx1, Panx2 and Panx3, following their order of discovery [8,36,41]. Pannexins do not form gap junctions but build up hexameric pannexin channels reminiscent of connexin hemichannels [42]. Connexin hemichannels and pannexin channels facilitate paracrine communication and play a central role in the induction and propagation of cell death and inﬂammation [37,43,44]. The pharmacological closing of channels provides therapeutic opportunities in a variety of diseases [8,37,43,44]. However, the current lack of speciﬁc connexin hemichannel and pannexin channel inhibitors hinders clinical exploration in this direction [8,43–45]. 3. Structure and Function of Cell Plasma Membrane Transport Proteins Cell la a e b a e t a o t otei a ti i ate i the t a fe 3. Structure and Function of Cell Plasma Membrane Transport Proteins Cell plasma membrane transport proteins participate in the transfer of substances through the cell plasma membrane, including glutamate, glucose, ATP and cyclic adeno- sine monophosphate as well as ions, such as calcium, potassium and sodium [1,2]. This activity is crucial to maintain cellular homeostasis and plays a key role in the regulation of physiological processes [1,2,4]. In view of the critical role of cell plasma membrane transport proteins, modulation of their basal performance is associated with multiple pa- thologies [2,4,6,7]. Targeting these transport proteins, which include channels and pores, ATP-powered pumps and porters (Figure 1E), would be of therapeutic interest [2,5,8,9]. Cell plasma membrane transport proteins participate in the transfer of substances through the cell plasma membrane, including glutamate, glucose, ATP and cyclic adeno- sine monophosphate as well as ions, such as calcium, potassium and sodium [1,2]. This activity is crucial to maintain cellular homeostasis and plays a key role in the regulation of physiological processes [1,2,4]. In view of the critical role of cell plasma membrane transport proteins, modulation of their basal performance is associated with multiple pathologies [2,4,6,7]. Targeting these transport proteins, which include channels and pores, ATP-powered pumps and porters (Figure 1E), would be of therapeutic interest [2,5,8,9]. Biomolecules 2021, 11, 63 4 of 19 3.2. ATP-Powered Pumps ATP-powered pumps translocate ions and small molecules against their concentra- tion gradient across the cell plasma membrane by utilizing energy from ATP hydrolysis (Figure 1E) [2,46,47]. A well-studied ATP-powered pump is the hydrogen potassium ATPase. The hydrogen potassium ATPase is a heterodimeric protein that is composed of a multi-pass transmembrane subunit and a type II transmembrane subunit [48,49]. Via the hydrolysis of ATP, it facilitates the exchange of hydrogen and potassium ions across the cell plasma membrane [46–49]. By doing so, the hydrogen potassium ATPase is responsible for acid production in the stomach in order to activate digestion. Inactivation of this cell plasma membrane transport protein results in decreased stomach acidity. At present, several pro- ton pump inhibitors, like omeprazole and pantoprazole, are available as drugs for treating gastroesophageal reflux and peptic ulcer disease [46–49]. 4. Antibodies and Nanobodies to Target Cell Plasma Membrane Transport Proteins 4.1. Conventional Antibodies 4. Antibodies and Nanobodies to Target Cell Plasma Membrane Transport Proteins 4.1. Conventional Antibodies The human adaptive immune system generates antibodies to protect against the con- tinuous threat of infection and toxic compounds [54]. Hence, antibodies are naturally occurring therapeutics that speciﬁcally recognize and eliminate antigens. These charac- teristics allow the usage of antibodies as research tools as well as for clinical diagnosis or Biomolecules 2021, 11, 63 5 of 19 therapeutic purposes [54]. Immunoglobulin-G (IgG) is the most abundant antibody isotype circulating in blood of mammals and its structure is highly conserved throughout different species [54]. This Y-shaped protein molecule consists of two identical heavy (H) and two identical light (L) chains (Figure 2A) [55,56]. EVIEW 6 of 19 therapeutic purposes [54]. Immunoglobulin-G (IgG) is the most abundant antibody isotype circulating in blood of mammals and its structure is highly conserved throughout different species [54]. This Y-shaped protein molecule consists of two identical heavy (H) and two identical light (L) chains (Figure 2A) [55,56]. REVIEW 6 of 19 therapeutic purposes [54]. Immunoglobulin-G (IgG) is the most abundant antibody isotype circulating in blood of mammals and its structure is highly conserved throughout different species [54]. This Y-shaped protein molecule consists of two identical heavy (H) and two identical light (L) chains (Figure 2A) [55,56]. REVIEW 6 of 19 Figure 2. Conventional antibodies and nanobodies. (A) Immunoglobulin-G (IgG) with 2 heavy (H) and 2 light (L) chains. The L chain comprises 1 variable (VL) and 1 conserved (CL) domain, whereas the H chain contains 1 variable (VH) and 3 constant (CH1, CH2 and CH3) domains. The paired VH and VL domains form the variable fragment (Fv) and bind to the antigen. The L chain and the first half of the H chain (VH and CH1) are known as the antigen binding fragment (Fab). The CH2 and CH3 domains of the 2 H chains form the crystallizable fragment (Fc). (B) Heavy chain-only antibodies (HCAbs) are smaller than conventional antibodies. They are devoid of L chains and the H chain lacks a CH1 domain. HCAbs rec- ognize antigens via the variable domain of the H chain of HCAbs (VHH), also known as a nanobody. Figure 2. Conventional antibodies and nanobodies. (A) Immunoglobulin-G (IgG) with 2 heavy (H) and 2 light (L) chains. 4. Antibodies and Nanobodies to Target Cell Plasma Membrane Transport Proteins 4.1. Conventional Antibodies The L chain comprises 1 variable (VL) and 1 conserved (CL) domain, whereas the H chain contains 1 variable (VH) and 3 constant (CH1, CH2 and CH3) domains. The paired VH and VL domains form the variable fragment (Fv) and bind to the antigen. The L chain and the ﬁrst half of the H chain (VH and CH1) are known as the antigen binding fragment (Fab). The CH2 and CH3 domains of the 2 H chains form the crystallizable fragment (Fc). (B) Heavy chain-only antibodies (HCAbs) are smaller than conventional antibodies. They are devoid of L chains and the H chain lacks a CH1 domain. HCAbs recognize antigens via the variable domain of the H chain of HCAbs (VHH), also known as a nanobody. Figure 2. Conventional antibodies and nanobodies. (A) Immunoglobulin-G (IgG) with 2 heavy (H) and 2 light (L) chains. The L chain comprises 1 variable (VL) and 1 conserved (CL) domain, whereas the H chain contains 1 variable (VH) and 3 constant (CH1, CH2 and CH3) domains. The paired VH and VL domains form the variable fragment (Fv) and bind to the antigen. The L chain and the first half of the H chain (VH and CH1) are known as the antigen binding fragment (Fab). The CH2 and CH3 domains of the 2 H chains form the crystallizable fragment (Fc). (B) Heavy chain-only antibodies (HCAbs) are smaller than conventional antibodies. They are devoid of L chains and the H chain lacks a CH1 domain. HCAbs rec- ognize antigens via the variable domain of the H chain of HCAbs (VHH), also known as a nanobody. Figure 2. Conventional antibodies and nanobodies. (A) Immunoglobulin-G (IgG) with 2 heavy (H) and 2 light (L) chains. The L chain comprises 1 variable (VL) and 1 conserved (CL) domain, whereas the H chain contains 1 variable (VH) and 3 constant (CH1, CH2 and CH3) domains. The paired VH and VL domains form the variable fragment (Fv) and bind to the antigen. The L chain and the ﬁrst half of the H chain (VH and CH1) are known as the antigen binding fragment (Fab). The CH2 and CH3 domains of the 2 H chains form the crystallizable fragment (Fc). (B) Heavy chain-only antibodies (HCAbs) are smaller than conventional antibodies. They are devoid of L chains and the H chain lacks a CH1 domain. 4. Antibodies and Nanobodies to Target Cell Plasma Membrane Transport Proteins 4.1. Conventional Antibodies HCAbs recognize antigens via the variable domain of the H chain of HCAbs (VHH), also known as a nanobody. 4.2. Nanobodies Camelidae, including Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe, Vicugna pacos and Vicugna vicugna possess, as well as conventional heterotetram- eric antibodies, unique heavy-chain-only antibodies (HCAbs) [63,64]. These HCAbs are smaller than conventional antibodies, as they are devoid of L chains and the CH1 domain is absent from their H chain (Figure 2B). The HCAbs from camelids recognize antigens by only one single variable domain, known as the variable domain of a H chain of HCAbs (VHH). The VHH fragment, also referred to as nanobody, can be produced recombinantly by a variety of host cells, including, bacteria, yeasts, plants and mammalian cells [18–20]. Although nanobodies are the smallest, functional, intact antigen-binding fragments, they are still able to selectively target epitopes selectively and with high affinity. Whereas conventional antibodies and their Fv fragments have a paratope consisting of six CDRs (i.e., three in a VH and three in a VL domain), nanobodies only have three CDRs [18–20]. Nanobodies are believed to have larger CDRs, more mutation hotspots and recombination The L chain comprises one variable (VL) and one constant (CL) domain, whereas the H chain is built up by one variable (VH) and three conserved or constant (CH1, CH2 and CH3) domains. The CH2 and CH3 domains of the two H chains form the crystallizable fragment (Fc), which binds various cell receptors, such as Fc receptors and immune molecules, to generate an adequate immune response [19,55,56]. Furthermore, the two arms of the Y- shaped IgG molecule are known as the antigen-binding fragments (Fabs), of which the extremity comprises the paired VL and VH domains, referred to as the variable fragment (Fv) that associates to antigens [19,55,56]. This process of antigen recognition is mainly mediated by the hypervariable antigen-binding loops or complementarity determining regions (CDRs). Three CDR loops in the VH and three CDR loops in the VL are clustering at one end of the Fv forming the paratope with a surface that is complementary to the epitope, the surface that is recognized on the antigen [19,55,56]. The three parts (i.e., two Fabs and one Fc) of the IgG molecule are linked via a ﬂexible hinge region, located between the CH1 and CH2 domains, permitting independent movement among the three regions [19,55,56]. fﬁ ﬁ 4.2. 4. Antibodies and Nanobodies to Target Cell Plasma Membrane Transport Proteins 4.1. Conventional Antibodies Nanobodies Camelidae, including Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe, Vicugna pacos and Vicugna vicugna possess, as well as conventional heterotetram- eric antibodies, unique heavy-chain-only antibodies (HCAbs) [63,64]. These HCAbs are smaller than conventional antibodies, as they are devoid of L chains and the CH1 domain is absent from their H chain (Figure 2B). The HCAbs from camelids recognize antigens by only one single variable domain, known as the variable domain of a H chain of HCAbs (VHH). The VHH fragment, also referred to as nanobody, can be produced recombinantly by a variety of host cells, including, bacteria, yeasts, plants and mammalian cells [18–20]. Although nanobodies are the smallest, functional, intact antigen-binding fragments, they are still able to selectively target epitopes selectively and with high affinity. Whereas conventional antibodies and their Fv fragments have a paratope consisting of six CDRs (i.e., three in a VH and three in a VL domain), nanobodies only have three CDRs [18–20]. Nanobodies are believed to have larger CDRs, more mutation hotspots and recombination signal sequence mimics to compensate for missing VH-VL combinatorial diversity [65– 67]. Moreover, the smaller size of the footprint and the generally more convex paratope allow nanobodies to target cryptic epitopes, such as the substrate binding site of mem- brane transport proteins, which are less accessible for conventional antibodies and their derivatives such as the Fab [12,19,61]. Furthermore, the single-exon origin (i.e., approxi- mately 360 nucleotides), the intrinsic low immunogenicity, facile blood vessel extravasa- tion, good tissue penetration, robustness upon exposure to extreme conditions and toler- t d i i f b di ff d t f i i it d i i The L chain comprises one variable (VL) and one constant (CL) domain, whereas the H chain is built up by one variable (VH) and three conserved or constant (CH1, CH2 and CH3) domains. The CH2 and CH3 domains of the two H chains form the crystallizable fragment (Fc), which binds various cell receptors, such as Fc receptors and immune molecules, to generate an adequate immune response [19,55,56]. Furthermore, the two arms of the Y- shaped IgG molecule are known as the antigen-binding fragments (Fabs), of which the extremity comprises the paired VL and VH domains, referred to as the variable fragment (Fv) that associates to antigens [19,55,56]. This process of antigen recognition is mainly mediated by the hypervariable antigen-binding loops or complementarity determining regions (CDRs). 4. Antibodies and Nanobodies to Target Cell Plasma Membrane Transport Proteins 4.1. Conventional Antibodies Three CDR loops in the VH and three CDR loops in the VL are clustering at one end of the Fv forming the paratope with a surface that is complementary to the epitope, the surface that is recognized on the antigen [19,55,56]. The three parts (i.e., two Fabs and one Fc) of the IgG molecule are linked via a ﬂexible hinge region, located between the CH1 and CH2 domains, permitting independent movement among the three regions [19,55,56]. Antibodies associate with high afﬁnity to their cognate target and are very speciﬁc to their antigen [20,55]. As antibodies can be raised against virtually any possible molecule, antibodies are already being exploited in the discovery of therapeutics targeting cell plasma membrane transport proteins [12]. In this regard, lifastuzumab–vedotin is an antibody– toxin conjugate, where the antibody part is directed towards SLC34A2, a sodium-dependent phosphate symporter (i.e., multi-pass transmembrane protein). This antibody-toxin is cur- rently tested in clinical trials for the treatment of various cancer types [57,58]. However, the signal sequence mimics to compensate for missing VH-VL combinatorial diversity [65– 67]. Moreover, the smaller size of the footprint and the generally more convex paratope allow nanobodies to target cryptic epitopes, such as the substrate binding site of mem- brane transport proteins, which are less accessible for conventional antibodies and their derivatives such as the Fab [12,19,61]. Furthermore, the single-exon origin (i.e., approxi- mately 360 nucleotides), the intrinsic low immunogenicity, facile blood vessel extravasa- tion, good tissue penetration, robustness upon exposure to extreme conditions and toler- Antibodies associate with high afﬁnity to their cognate target and are very speciﬁc to their antigen [20,55]. As antibodies can be raised against virtually any possible molecule, antibodies are already being exploited in the discovery of therapeutics targeting cell plasma membrane transport proteins [12]. In this regard, lifastuzumab–vedotin is an antibody– toxin conjugate, where the antibody part is directed towards SLC34A2, a sodium-dependent phosphate symporter (i.e., multi-pass transmembrane protein). This antibody-toxin is cur- rently tested in clinical trials for the treatment of various cancer types [57,58]. However, the Biomolecules 2021, 11, 63 6 of 19 use of therapeutic antibodies has also some disadvantages, as the large-scale production of antibody-based therapeutics is an extremely expensive process [59]. In addition, membrane transport proteins feature small and often cryptic epitopes, which could be difﬁcult to target by conventional antibodies due to their large size (i.e., approximately 150 kDa) [12]. 4.2. Nanobodies Camelidae, including Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guani- coe, Vicugna pacos and Vicugna vicugna possess, as well as conventional heterotetrameric antibodies, unique heavy-chain-only antibodies (HCAbs) [63,64]. These HCAbs are smaller than conventional antibodies, as they are devoid of L chains and the CH1 domain is absent from their H chain (Figure 2B). The HCAbs from camelids recognize antigens by only one single variable domain, known as the variable domain of a H chain of HCAbs (VHH). The VHH fragment, also referred to as nanobody, can be produced recombinantly by a variety of host cells, including, bacteria, yeasts, plants and mammalian cells [18–20]. y g y p Although nanobodies are the smallest, functional, intact antigen-binding fragments, they are still able to selectively target epitopes selectively and with high afﬁnity. Whereas conventional antibodies and their Fv fragments have a paratope consisting of six CDRs (i.e., three in a VH and three in a VL domain), nanobodies only have three CDRs [18–20]. Nanobodies are believed to have larger CDRs, more mutation hotspots and recombination signal sequence mimics to compensate for missing VH-VL combinatorial diversity [65–67]. Moreover, the smaller size of the footprint and the generally more convex paratope al- low nanobodies to target cryptic epitopes, such as the substrate binding site of mem- brane transport proteins, which are less accessible for conventional antibodies and their derivatives such as the Fab [12,19,61]. Furthermore, the single-exon origin (i.e., approx- imately 360 nucleotides), the intrinsic low immunogenicity, facile blood vessel extrava- sation, good tissue penetration, robustness upon exposure to extreme conditions and tolerance towards engineering of nanobodies offer advantages for various in vitro and in vivo applications [18–20]. Therapeutic nanobodies targeting cell plasma membrane transport proteins are being developed to interfere with the function of these channels and pores, ATP-powered pumps and porters [2,5,8,9]. Such therapeutic nanobodies may exert these functional effects via different mechanisms. They could block channels and pores or inﬂuence ligand binding (i.e., acting as orthosteric or allosteric modulators) resulting in decreased or enhanced ligand binding [68–70]. Furthermore, nanobodies could exert their therapeutic effect by stabilizing a particular conformational state (i.e., active or inactive) of cell plasma membrane proteins [18]. However, ﬁnding these membrane transport protein-targeting nanobodies is difﬁcult. While protocols to generate nanobodies against soluble proteins are well-established, the identiﬁcation of nanobodies directed towards membrane proteins, such as membrane transport proteins, is more challenging [71]. 4. Antibodies and Nanobodies to Target Cell Plasma Membrane Transport Proteins 4.1. Conventional Antibodies The introduction of antibody-fragments can partially resolve these problems. Smaller antibody entities, in particular, Fabs (i.e., approximately 60 kDa) and single-chain Fvs (i.e., approximately 28 kDa), are interesting alternatives to intact antibodies [55,59,60]. Nev- ertheless, the identiﬁcation of therapeutic antibody(-fragments) recognizing cell plasma membrane transport proteins remains challenging due to the large ﬂat or concave paratope of conventional antibodies, which has limited access to cryptic and conserved sites on these targets [19,61,62]. 4.3. Identiﬁcation of Antigen-Speciﬁc Nanobodies For the identiﬁcation of antigen-speciﬁc nanobodies, it is important to start with high-quality libraries of nanobodies [20]. Gene banks that represent a large number of nanobodies with maximal diversity are envisaged for the retrieval of target-speciﬁc nanobodies. To achieve the latter, different types of libraries (i.e., immune, synthetic and naïve) can be used [20]. Both immune and naïve nanobody libraries are based on naturally occurring HCAbs isolated from the peripheral blood lymphocytes of camelids. Whereas Biomolecules 2021, 11, 63 7 of 19 immunized camelids are used for the generation of immune libraries, the blood of non- immunized camelids is taken to construct naïve libraries. Synthetic libraries, based on a single or few nanobody frameworks that are subjected to diversiﬁcation of the amino acids located in the paratope, have emerged as an alternative to naïve and immune libraries in the last few years [20,72–75]. The employment of immune libraries is a well-established approach to identify a diver- sity of antigen-specific nanobodies with a high success rate [20,76]. Immunizing a camelid with soluble and properly folded proteins mixed with adjuvant is the first step to elicit an affinity matured immune response in the HCAb classes and to generate an immune library [20,76,77]. Following multiple subcutaneous injections of an immunogen, the mRNA extracted from blood lymphocytes of the immunized camelid serves as a template for the reverse transcription to produce cDNA. The nanobody cDNA is amplified by polymerase chain reaction and ligated in a phagemid vector. Finally, bacteria are transformed with the ligated material. In order to ensure high quality, libraries should have a size of around 107–108 individual transformants, of which more than 70% should carry a phagemid with a nanobody-inserted sequence [20,76,77]. To secure a very high (i.e., close to 100%) number of clones with a nanobody insert of the proper length, the use of the Golden Gate cloning strategy might be considered. The nanobody should hereby substitute a lethal ccdB gene in the phagemid to allow bacteria to grow [76]. The antigen-specific nanobodies are retrieved from such large libraries after their expression at the tip of phages and selection by biopan- ning. During biopanning, multiple strategies can be followed to select the nanobodies with the highest affinity and specificity against the target of interest [20,76,77]. Moreover, different enrichment approaches can be developed to select for nanobody characteristics, such as affinity, specificity, blocking of ligands or protein–protein interactions [77,78]. 4.4. Identiﬁcation of Cell Plasma Membrane Protein-Targeting Nanobodies The identiﬁcation of cell plasma membrane protein-binding nanobodies is not an easy task, as the availability of a pure and properly folded target protein is a major requirement for the immunization of camelids and subsequent panning [71,77]. The use of recombinant proteins from the extracellular domain of cell plasma membrane protein for both immu- nization and panning purposes forms an elegant solution to bypass the difﬁculty to obtain an intact cell plasma membrane protein [12,79]. However, this strategy is only practical for single-pass membrane proteins, as multi-pass transmembrane proteins mostly lack a large identiﬁable extracellular domain that can be produced in its native conformation to act as surrogate targets [12,79–84]. Nevertheless, alternative immunization and panning strate- gies have successfully been developed to generate nanobodies targeting both intracellular and extracellular epitopes of multi-pass transmembrane membrane proteins (Table 1). ew of immunization and biopanning strategies for the identiﬁcation of cell plasma membrane-protein Table 1. Overview of immunization and biopanning strategies for the identiﬁcation of cell plasma membrane-protein targeting nanobodies. Table 1. Overview of immunization and biopanning strategies for the identiﬁcation of cell plasma membrane protein targeting nanobodies. Immunization and Biopanning Strategies Advantages Disadvantages References Transfected cells - Presents membrane protein in its native form - Applicable for both immunization and panning procedures - Generation of nanobodies directed towards undesired membrane proteins by the host animal - Requires the construction of transfected cells expressing high levels of the protein of interest [17,71,84–88] Biomolecules 2021, 11, 63 8 of 19 Table 1. Cont. 4.4.1. Transfected Cell Lines One strategy to retrieve nanobodies against multi-pass transmembrane proteins in- volves the utilization of cells that are stably or transiently transfected to express the protein in their plasma membrane. The use of mammalian cell lines ensures the proper folding and native conformation of the cell plasma membrane target of interest [71]. The employment of transfected cells to immunize a camelid will also elicit an immune response against other (immunodominant) components expressed on the host cell surface, which might complicate subsequent selection of target-speciﬁc nanobodies [17,71]. To tackle this shortcoming, it is recommended to use dromedary-derived cells as a host cell for the transfection, since these cells will be less immunogenic in camels or llamas [17,85]. Obviously, stably transfected or transduced cells with a high surface expression level of the transgene will increase the suc- cess rate in ﬁnding target-speciﬁc nanobodies [17,71]. Besides immunizations, transfected or transduced cells can also be used in the subsequent panning procedures [84,86]. The use of cell-based pannings allows the identiﬁcation of nanobodies targeting the extracellular side the cell plasma membrane protein under scrutiny [84]. To avoid the enrichment of nanobodies binding to antigenic components of the host cell, it is recommended to use different cellular backgrounds for the immunizations and panning [17,71]. Moreover, switching to different host cells in consecutive rounds of panning might reduce the retrieval of binders of host cell antigens [86]. In addition, performing a negative selection whereby the assembled library is incubated with a cell line lacking the expression of the protein of interest, prior to incubation with transfected or transduced cells, helps to remove unspeciﬁc binders [87,88]. Table 1. Cont. Table 1. Cont. Table 1. Cont. Immunization and Biopanning Strategies Advantages Disadvantages References Endogenous proteins at the cell surface - Presents membrane protein in its native form - Applicable for both immunization and panning procedures - No need to construct speciﬁc membrane models - Difﬁcult retrieval of desired nanobodies due to a weak immune response of the host animal - Generation of nanobodies directed towards undesired membrane proteins by the host animal - Low expression levels of the antigen of interest [71,100] Endogenous proteins at the cell surface 4.4.2. Membrane Extracts Apart from intact cells, one could consider using the membrane extracts of (transfected) cells for immunization. Besides the solubilization of cell plasma membrane proteins with detergents, cells can also be disrupted to generate membrane vesicles or fragments exposing both extracellular and intracellular epitopes of the transgene [71]. It is clear that high expression levels of the antigen will lead to a better immunization [71]. Membrane extracts are a valid alternative to whole cells when one wants to obtain nanobodies targeting both extracellular and intracellular epitopes. Similar to whole cells, these membrane extracts can be used during panning rounds [71,89,90]. 4.4. Identiﬁcation of Cell Plasma Membrane Protein-Targeting Nanobodies Immunization and Biopanning Strategies Advantages Disadvantages References Membrane extracts - Presents membrane protein in its native form - Enables to identify intracellular and extracellular binders - Applicable for both immunization and panning procedures - Generation of nanobodies directed towards undesired membrane proteins by the host animal - Requires the construction of transfected cells expressing high levels of the protein of interest [71,89,90] Nanodiscs - Presents membrane protein in its native form - Enables to identify intracellular and extracellular binders - Does not imply the risk of retrieving unspeciﬁc nanobodies that target other components present on cells - Might require the construction of transfected cells expressing high levels of the protein of interest - Complex membrane model (antigen- dependent) [91–96] Virus-like particles - Presents membrane protein in its native form - Applicable for both immunization and panning procedures - Does not imply the risk of retrieving unspeciﬁc nanobodies that target other components present on cells - Complex membrane model (antigen- dependent) [71,97–99] cDNA immunization - Presents membrane protein in its native form (in vivo) - No need to construct speciﬁc membrane models - No generation of nanobodies directed towards undesired membrane proteins by the host animal - Difﬁcult retrieval of desired nanobodies due to a weak immune response of the host animal [68,84,100,101] Detergent solubilized proteins - Applicable for both immunization and panning procedures - No need to construct speciﬁc membrane models - Detergents may cause an altered structure and function of the cell plasma membrane proteins [92] Table 1. Cont. Table 1. Cont. 9 of 19 Biomolecules 2021, 11, 63 4.4.4. Virus-Like Particles Virus-like particles (VLPs) mimic viral structural proteins and can be designed to in- corporate cell plasma membrane structures [97]. In addition, VLPs are not infectious as they lack essential genomic material [71,97]. The expression of membrane proteins on VLPs can be recombinantly fabricated in various production platforms [98]. In this light, mammalian cells, such as human embryonic kidney (HEK) cells, can produce structural viral polypro- teins along with the protein of interest [71,98]. The viral proteins’ self-assemble and buds from the host cell to form non-infectious VLPs embedded with the target protein [97,98]. Despite the high costs associated with the production, puriﬁcation and characterization of VLPs, these VLPs offer beneﬁts for the development of nanobodies that target membrane proteins [71,99]. Since this technology can display the extracellular domains of membrane proteins in high concentrations and in a properly folded and stable way without expressing unrelated membrane proteins, VLPs are used to identify target-speciﬁc nanobodies. Thus, the applicability of VLPs is dual as the multiple subcutaneous injection of a camelid with VLPs raises an immune response towards the membrane protein of interest, and/or the panning on VLPs can be performed [71,99]. 4.4.3. Nanodiscs Traditional cell plasma membrane models, like micelles, bicelles and liposomes, are a common source for expressing cell plasma membrane proteins [91]. However, these cell Biomolecules 2021, 11, 63 10 of 19 plasma membrane models face limitations imposed by the employed detergents, causing the structure and function of cell plasma membrane proteins to alter [92]. Hence, the generation of nanodiscs is gaining more attention to maintain membrane proteins in their native form. Preparation of this stable and monodisperse cell plasma membrane model includes osmotic lysis of cells expressing the protein of interest in the presence of phos- pholipids and membrane scaffold proteins [93]. In this way, nanodiscs are produced that display the target protein [93]. The use of nanodiscs as immunogen during immuniza- tions and subsequent panning can identify both intracellular- and extracellular-binding nanobodies [94]. Moreover, employing nanodiscs as immunogen is advantageous over immunizations with intact cells and membrane extracts, since it does not bear the risk of retrieving unspeciﬁc nanobodies that target other membrane proteins that are present on cells [79,95,96]. Nevertheless, the application of nanodiscs warrants some optimization for each antigen as the amount of the target and scaffold protein should be balanced for the successful expression of the target of interest [93,95]. 4.5. Therapeutic Nanobodies Targeting Cell Plasma Membrane Transport Proteins 4.5.1. Channels and Pores 4.4.5. cDNA Immunization Both nanobodies were reformatted into bivalent formats and nanobody Fc-fusion proteins, which resulted in enhanced afﬁnity and potency in the low nanomolar/sub-picomolar range [68]. Further in vitro characterization showed that bivalent formats of both nanobodies were able to modulate P2X7-induced ATP activa- tion on primary mouse macrophages and T cells [68]. Next, the bivalent 13A7 nanobody was reformatted to a half-life extended 13A7 (13A7-HLE) nanobody via the addition of an anti-albumin nanobody to assess the therapeutic potential in vivo. The treatment of mice suffering from allergic contact dermatitis with 13A7-HLE resulted in reduced ear swelling and reduced levels of inﬂammatory cytokines [68]. Moreover, 13A7-HLE treat- ment in an antibody-induced glomerulonephritis model leads to a decrease in inﬂammatory cell inﬁltration and proteinuria in mice [68]. The clinical potential of these P2X7-binding nanobodies was further substantiated by Dano1, a human P2X7-speciﬁc nanobody. Refor- matting of the Dano1 nanobody into a nanobody-Fc fusion resulted in enhanced potency and efﬁcacy compared to the monovalent format and was able to lower the release of the pro-inﬂammatory cytokine IL-1β from endotoxin-exposed human monocytes [68,102]. p y y β p y [ , ] Drugs that selectively target P2X4 channels equally offer therapeutic potential [105,106]. P2X4 channels mainly mediate the trafficking of calcium ions in response to ATP and are involved in different pathologies [105,107–109]. Given the lack of specific and potent P2X4- antagonists, studies have focused on the generation of nanobodies directed towards P2X4 [84]. Llamas were immunized with plasmids encoding either mouse or human P2X4 and P2X4- transfected HEK cells. Following biopanning on HEK cells expressing mouse or human P2X4, several nanobodies targeting extracellular regions of the P2X4 proteins could be retrieved. The specificity of these nanobodies was demonstrated by immunocytochemistry analysis of Chinese hamster ovary cells that were transfected with expression vectors encoding mouse or human P2X4 [84]. Furthermore, seven of the retrieved P2X7-targeting nanobodies were subcloned into bivalent formats to form a bivalent nanobody-rabbit IgG heavy chain antibody. The cross-reactivity of generated constructs was explored by flow cytometry analysis with HEK cells transfected to express mouse, rat or human P2X4. Two of the bivalent nanobody- rabbit Ig heavy-chain antibodies (Nb271-rbhcAb and Nb284-rbhcAb) recognized mouse, rat and human P2X4, whereas the other reformatted nanobodies showed affinity for only one or two of the P2X4 species [84]. Flow cytometry experiments confirmed the binding of endogenous murine P2X4 by Nb271 and Nb325. 4.4.5. cDNA Immunization The immunization of a camelid with cDNA of the target cell plasma membrane protein in an expression vector forms an attractive alternative to the classical protein immunization [100]. The objective is to have the expression vector taken up by host cells, where transcription and translation will expose the membrane protein to the immune system to elicit an immune response against the protein of interest in its native form [84,100]. During the injection of the expression vector in the shoulders and hind limbs of camelids, an electroporation will introduce the DNA to host cells [100]. The main advantage of this strategy is that there is no need to purify the protein or to use cell plasma membrane models to present the antigen to the host animal immune system [100,101]. In this way, the risk of generating undesired binders is circumvented since the camelids’ immune system is only triggered by proteins encoded by the cloned target gene [100]. A disadvantage of this technique might be that the immune response of the host animal fails to elicit a strong immune response to the target [100]. However, this can be overcome by the inclusion of additional boosts with transfected cells expressing the target protein [68]. 4.5. Therapeutic Nanobodies Targeting Cell Plasma Membrane Transport Proteins 4.5.1. Channels and Pores Nanobodies modulating channel activity are of considerable therapeutic relevance (Table 2). A case study in this respect includes nanobodies that block or potentiate gating of P2X7 channels [68,102]. Upon activation, trimeric P2X7 channels mediate the trans- port of calcium, sodium and potassium ions, thereby playing a central role in inﬂam- Biomolecules 2021, 11, 63 11 of 19 11 of 19 matory diseases through inﬂammasome activation and the release of pro-inﬂammatory cytokines [103,104]. Llamas were immunized with HEK cells that stably express mouse or human P2X7 or cDNA encoding mouse and human P2X7 followed by a boost with mouse and human P2X7-transfected HEK cells. Subsequent biopanning on these transfected HEK cells gave rise to the identiﬁcation of 18 different families of P2X7-targeting nanobodies [68]. Of these 18 families, six were able to block or enhance the activation of mouse P2X7 and two blocked ATP-mediated gating of human P2X7. Two of the mouse P2X7-targeting nanobodies were selected for further characterization, namely the antagonistic nanobody 13A7 and the agonistic nanobody 14D5. 4.5.2. ATP-Powered Pumps The zinc-transporting ATPase ZntA from the bacterium Shigella sonnei (SsZntA) is a transport protein from the P-type ATPase family [113]. Targeting and modulating pathogen-derived P-type ATPases is a promising strategy for the development of new antibiotics, antifungals, vaccines and herbicides, as these ATPases play important roles in the survival of pathogens (Table 2) [113]. The immunization of a llama with puriﬁed SsZntA and panning on biotinylated SsZntA resulted in the identiﬁcation of multiple SsZnt- targeting nanobodies that could be grouped into three families [114]. One of the retrieved SsZntA-targeting nanobodies could selectively target the ZntA ATPase and signiﬁcantly reduced its pump function by up to 50% [114]. However, the mechanism-of-action of this inhibition is not yet fully understood [114]. Another example of nanobodies targeting ATP-powered pumps includes the develop- ment of BtuCD-F-targeting nanobodies. The BtuCD-F transporter complex, built up by two multi-transmembrane proteins, participates in vitamin B12 import in bacteria [115,116]. Therefore, the identiﬁcation of drugs that selectively block the uptake of essential nutri- ents forms the basis to design innovative antibiotics [115,116]. Nanobodies binding the periplasmic-binding protein BtuF were identiﬁed by injecting an alpaca with vitamin B12- bound BtuF and subsequent panning against vitamin B12-bound BtuF and apo-BtuF [116]. In total, six different BtuF-binding nanobodies were retrieved that could inhibit vitamin B12 binding to BtuF with inhibition constants ranging from sub-nanomolar to high nanomolar values [116]. Moreover, these BtuF-targeting nanobodies were able to partially inhibit the BtuCD-F-mediated substrate transport in Escherichia coli-derived spheroplasts [116]. Crystallization of the lead nanobody in complex with BtuF revealed that this nanobody exerted its effect by sterically hindering the vitamin-B12-binding pocket of BtuCD-F [116]. 4.4.5. cDNA Immunization P2X4 expressed by mouse peritoneal mast cells and bone-marrow-derived macrophages were specifically targeted by the Nb271- rbhcAb [84]. However, the functional effects of identified P2X4-binding nanobodies have not been thoroughly investigated [84]. Another type of ion-channel-targeting nanobodies are Kv1.3-interacting nanobod- ies [110]. The Kv1.3 channel is a tetrameric structure, comprising multi-pass transmem- brane proteins that mediate the voltage-dependent potassium ion permeability to control the activity of T effector memory cells [111,112]. Blocking these channels is a promising strategy for the treatment of chronic immune diseases like multiple sclerosis and type-1 diabetes mellitus [111,112]. Nanobodies targeting Kv1.3 channels inhibit the activity of human Kv1.3 channels in electrophysiological assays in a dose-dependent manner [110]. The formatting of these blocking nanobodies into bivalent formats improves the afﬁnity of Kv1.3-targeting nanobodies from the low-nanomolar to the sub-nanomolar ratio and the construction of bi- and trivalent structures is beneﬁcial for its functional activity as well [110]. This blocking effect of Kv1.3 binders results from recognizing a previously Biomolecules 2021, 11, 63 12 of 19 unidentiﬁed epitope in the ﬁrst extracellular loop of these multi-pass transmembrane proteins [110]. Moreover, treatment of human T cells with a monovalent, a bivalent and a trivalent Kv1.3-binding nanobody resulted in T cell activation [110]. Finally, the therapeu- tic potential of the Kv1.3-targeting nanobodies was demonstrated by showing a reduction in the ear swelling response upon treatment with both half-life and non-half-life extended bivalent nanobodies in a hypersensitivity rat model [110]. 4.5.3. Porters Currently, nanobodies targeting porters are mainly used as crystallization chaperones [15]. A relevant case study in this respect relates to a SLC-26Dg-targeting nanobody [117]. SLC- 26Dg acts as a symporter by combining the uptake of fumarate with the transport of protons in Deinococcus geothermalis bacteria [117]. Due to its high degree of homology with other SLC-26 proteins, the crystal structure of SLC-26Dg, obtained via the SLC-26Dg-targeting nanobody, has revealed valuable information on the structure and functional behavior of similar multi-transmembrane porters [117]. p The identiﬁcation of nanobodies targeting porters is also promising for the devel- opment of therapeutic agents (Table 2). Vesicular glutamate 1 (VGLUT1) is a multi- transmembrane porter belonging to the SLC17 family that loads glutamate into synap- tic vesicles [118–120]. By doing so, VGLUT1 plays an important role in neurotransmis- sion [119,120]. Moreover, changes in the activity or expression of this symporter is described in diseases, such as schizophrenia and epilepsy [119]. VGLUT1-targeting nanobodies were identiﬁed by immunizing a llama with a truncated and non-glycosylated mutant of the rat VGLUT1 protein. Subsequent biopanning on rat VGLUT1 mutant protein resulted in the identiﬁcation of four nanobodies [121]. Despite being generated with a mutant protein, the anti-VGLUT1 nanobodies could still bind endogenous VGLUT1 expressed on mouse primary cortical neurons [121]. Further characterization of these nanobodies showed that the nanobodies recognized an intracellular, cytoplasmic epitope and were able to inhibit glutamate uptake in proteoliposomes and synaptic vesicles [121]. 13 of 19 Biomolecules 2021, 11, 63 Table 2. Overview of therapeutic nanobodies targeting cell plasma membrane transport proteins. 4.5.3. Porters Cell Plasma Membrane Transport Proteins Target Nanobody Clone Immunization Biopanning Strategy Pharmacological Activity Reference Channels and pores Mouse P2X7 13A7, bivalent 13A7, half-life extended 13A7 P2X7 transfected HEK cells, cDNA + cell boost Mouse P2X7 transfected HEK cells Antagonist [68] 14D5, bivalent 14D5 P2X7 transfected HEK cells, cDNA + cell boost Mouse P2X7 transfected HEK cells Antagonist [68] Human P2X7 Dano1, Dano1-Fc fusion P2X7 transfected HEK cells, cDNA + cell boost Human P2X7 transfected HEK cells Antagonist [68,102] Mouse and human P2X4 Nb271-Fc fusion, Nb284-Fc fusion cDNA + P2X4 transfected HEK cells Mouse and human P2X4 transfected HEK cells Functional effects have not been investigated [84] Human Kv1.3 channels Monovalent, bivalent, trivalent, half-life and non-half-life extended bivalent nanobodies Unknown Unknown Antagonist [110] ATP-powered pumps Zinc-transporting ATPase ZntA (Shigella sonnei) (SsZntA) Nb9 Puriﬁed SsZntA (membrane extracts + detergent) Biotinylated SsZntA Antagonist [114] BtuCD-F transporter complex (Escherichia coli) Nb9 Vitamin B12-bound BtuF (detergent) Vitamin B12-bound BtuF and apo-BtuF Antagonist [116] Porters Rat vesicular glutamate 1 (VGLUT1) Nb3, Nb9 Truncated and non-glycosylated mutant of the rat VGLUT1 protein (detergent) Rat VGLUT1 mutant protein Antagonist [121] Table 2. Overview of therapeutic nanobodies targeting cell plasma membrane transport proteins. 5. Conclusions and Future Perspectives Cell plasma membrane proteins control biological processes, but equally underlie a wide spectrum of pathologies [2,4]. Compromised trafﬁcking of molecules and ions across the cell plasma membrane may trigger disease or dysregulate signaling pathways towards pathology [4]. The development of drugs that restore cell plasma membrane transport protein function, therefore, is a promising avenue to explore [2,9]. However, small-molecule drug discovery in this research area is complicated since cell plasma membrane transport proteins share common structural properties, resulting in the non-speciﬁc binding of drugs targeting this class of proteins [2,10,11]. In this regard, aptamers or proteinaceous afﬁnity reagents (DARPins, monobodies, afﬁbodies, anticalins, or knob-like structures from cow antibodies) recognizing speciﬁcally membrane transport proteins can be an interesting alternative. In this review, we focused exclusively on nanobodies, as they are easily obtainable from immune libraries and free to use in therapeutic applications. All other formats or afﬁnity reagents are only available from groups specialized in generating good quality, vast and diverse repertoires and handling these libraries and/or processing the techniques and skills for subsequent afﬁnity maturation. The therapeutic use of such afﬁnity reagents will always somehow be restricted. y g y Given their therapeutic potential, the identiﬁcation of nanobodies modulating the activity of cell plasma membrane transport proteins is a relevant approach as this could offer new possibilities for the treatment of a variety of human diseases [18,19]. To date, only Biomolecules 2021, 11, 63 14 of 19 14 of 19 a limited number of examples has been described. However, multiple nanobodies targeting other classes of multi-pass transmembrane proteins, such as G-protein coupled receptors, have been demonstrated on many occasions, making it conceivable to assume that this would also be feasible for a broad diversity of cell-plasma membrane transport proteins. At present, both monovalent and multivalent formats of cell plasma membrane trans- port protein-targeting nanobodies have been described. Interestingly, monovalent nanobod- ies can sufﬁce to modulate the transfer of many substances and ions through the cell plasma membrane. By doing so, there is an interest in nanobodies that exert an agonistic or an- tagonistic effect [68]. Moreover, speciﬁc biopanning strategies and functional assays can be developed to retrieve agents that adapt inﬂux and efﬂux processes mediated by cell plasma membrane transport proteins from nanobody libraries. 5. Conclusions and Future Perspectives Therapeutic nanobodies may here act as orthosteric or allosteric modulators and can overcome a lack of selectivity, speciﬁcity, potency or stability in small-molecule drugs by targeting small and hidden cryptic epitopes of these transport proteins [17–20,68–70]. Nevertheless, the reformatting of monovalent nanobodies to multivalent formats (i.e., bivalent, biparatopic or nanobody-Fc fusions) could be advantageous for therapeutic application [122–126]. It has been sug- gested that such agents are able to improve the envisaged functional effects based on an increase in avidity, potency and/or efﬁcacy [18,127]. Another reason to reformat monova- lent (non-modulating) nanobodies to multivalent would be the possibility of changing the functional characteristics of these nanobodies [127]. Besides changing the functional prop- erties of the nanobodies, the reformatting of therapeutic nanobodies is also recommended for in vivo administration. To keep a constant level of circulating nanobodies in patients, it is necessary to compensate for the low molecular weight and small size of monovalent nanobodies [127–129]. PEGylation and PASylation of nanobodies, or their association with afﬁnity reagents that target serum albumin or the Fc of IgG prolongs the serum half-life of antibody fragments [127–129]. Furthermore, nanobodies intended for therapeutic use can also be humanized, i.e., mutating camelid-speciﬁc amino acid sequences to their human equivalents, to reduce the possible risks of immunogenicity in patients [130]. Besides acting as antigen modulatory therapeutics, nanobodies can also be used as a vehicle to direct effector molecules towards a target antigen. At present, the application of nanobodies for targeted therapy has been mainly demonstrated for several classes of re- ceptor proteins that are overexpressed on cancer cells [18]. In targeted therapy, nanobodies binding extracellular parts of overexpressed proteins are coupled with therapeutic agents via chemical conjugation or gene fusion [131]. In this way, nanobodies redirect molecules to speciﬁc organ systems, cell types or cell compartments [132]. Examples of molecules that have been fused to nanobodies include chemotherapeutics, toxins, radionuclides, pho- tosensitizers, T cell and natural killer cell engagers [18,131,132]. Furthermore, nanobodies can be linked to liposomes, micelles or polymer particles containing hydrophilic or toxic drugs and redirect this payload to cells overexpressing the target receptor protein [18,131]. The speciﬁcity of nanobodies towards cell plasma membrane transport proteins might also be envisaged for these targeted therapy applications. Author Contributions: Conceptualization: R.V.C., S.M., M.V., N.D., T.W.D.G.; Original draft prepa- ration: R.V.C.; Review and editing: S.M., M.V., N.D., T.W.D.G.; Visualization: R.V.C., T.W.D.G.; Supervision: S.M., M.V., N.D., T.W.D.G.; Funding acquisition: M.V. All authors have read and agreed to the published version of the manuscript. 5. Conclusions and Future Perspectives In this light, nanobodies that bind cell plasma membrane transport proteins that are overexpressed in pathological conditions can deliver drugs that lack selectivity, speciﬁcity or stability towards the target cells of interest. In summary, the generation of nanobodies targeting cell plasma membrane transport proteins remain a challenging endeavor due to the nature of these proteins. However, nanobodies targeting these proteins have signiﬁcant therapeutic potential in a wide range of applications, which remains to be explored. Biomolecules 2021, 11, 63 15 of 19 15 of 19 Funding: This work was ﬁnancially supported by the European Research Council (Proof-of-Concept grant 861913), the Research Foundation Flanders-Belgium (FWO grants G012318N, G020018N and G0F7219N) and the University Hospital of the Vrije Universiteit Brussel-Belgium (Scientiﬁc Fund Willy Gepts). Institutional Review Board Statement: Not Applicable. Institutional Review Board Statement: Not Applicable. Informed Consent Statement: Not Applicable. Informed Consent Statement: Not Applicable. Data Availability Statement: Not Applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Dubyak, G.R. Ion homeostasis, channels, and transporters: An update on cellular mechanisms. Am. J. Physiol. Adv. Physiol. Educ. 2004, 28, 143–154. [CrossRef] [PubMed] 2. 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https://openalex.org/W2122698215	https://www.research-collection.ethz.ch/bitstream/20.500.11850/288322/1/elife-07390-v1%281%29.pdf	English	null	Serotonin's many meanings elude simple theories	eLife	2,015	cc-by	2,297	ETH Library Copyright Dayan and Huys. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Journal Article Journal Article Author(s): Dayan, Peter; Huys, Quentin J.M. Publication date: 2015-04 Permanent link: https://doi.org/10.3929/ethz-b-000100596 Rights / license: Creative Commons Attribution 4.0 International Originally published in: eLife 4(e07390), https://doi.org/10.7554/eLife.07390 This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. elifesciences.org INSIGHT elifesciences.org NEUROPHYSIOLOGY Serotonin’s many meanings elude simple theories Neurons that produce serotonin respond in a number of different and complex ways in anticipation and receipt of rewards or punishments. Neurons that produce serotonin respond in a number of different and complex ways in anticipation and receipt of rewards or punishments. PETER DAYAN AND QUENTIN HUYS PETER DAYAN AND QUENTIN HUYS that the phasic activity of dopamine-producing neurons—the brief spikes in electrical activity seen after a stimulus is applied—closely resembles a sophisticated form of ‘prediction error’ that can be used to learn how much reward to expect and then influence the choice of appropriate actions. Interpreting electrophysiological recordings, how- ever, has always been difficult. Related research article Cohen JY, Amoroso MW, Uchida N. 2015. Serotonergic neurons signal reward and punishment on multiple timescales. eLife 4:e06346. doi: 10.7554/eLife.06346 Image Serotonin (cyan) and channelrho- dopsin (magenta) labelled in the midbrain of a mouse Related research article Cohen JY, Neuromodulatory neurons reside in complex nuclei that harbour many different types of neurons, raising doubts about whether any recorded electrophysiological activity can really be related to particular neuromodulators. Such doubts have largely been settled for dopamine by Cohen, Uchida and co-workers at Harvard University (Cohen et al., 2012) using optogenetic tagging: this technique allows the dopamine neurons to be electrophysiologically identified by genetically modifying them so that they can be stimulated with light (Lima et al., 2009). D opamine and serotonin are neuromodula- tors. Produced by small assemblies (or nuclei) of neurons deep in the brain stem, these molecules are projected throughout the brain to regulate the excitability and plasticity of broad neural networks via a fiendishly complex cast of receptor types. The impor- tance of neuromodulators is underscored by their involvement in a wealth of neurological and psychiatric diseases. What has been harder to pin down are the details of their computational roles, particularly the semantics of what they signal. Now, in eLife, Jeremiah Cohen, Mackenzie Amoroso and Naoshige Uchida add much-needed data about the activity of neurons that release serotonin in a task involving predictable rewards and punish- ments (Cohen et al., 2015). These data nicely muddy the theoretical waters. D Serotonin, by comparison, has been more elusive. There is a rather broad, though not completely self-consistent, cluster of electro- physiological, pharmacological, depletion- and lesion-based results suggesting that serotonin might play a critical role in preventing active behaviours or deciding to withdraw from a situa- tion. In this role, it is often associated with the anticipation and/or delivery of a punishment (Deakin and Graeff, 1991; Schweimer et al., 2008; Amo et al., 2014). More recent optoge- netic evidence that serotonin is involved in patience could be at least partially related to this (Miyazaki et al., 2014). Along with more direct findings, these results have collectively, if some- what controversially, been discussed in terms of serotonin (putatively linked with punishment and The past two decades have ascribed dopa- mine a particularly crisp computational role. Seminal electrophysiological recordings suggested Dayan and Huys. eLife 2015;4:e07390. DOI: 10.7554/eLife.07390 1 of 3 Insight Neurophysiology \| Serotonin’s many meanings elude simple theories inhibition) and dopamine (putatively linked with reward and activation) playing opposing roles (Deakin and Graeff, 1991). inhibition) and dopamine (putatively linked with reward and activation) playing opposing roles (Deakin and Graeff, 1991). Related research article Cohen JY, serotonin represents average reward is, however, complicated: though responding more to rewards than losses, serotonin neurons mostly decreased their tonic firing rates as the size of the average reward increased. Nevertheless, the phasic responses of the neurons to reward-predicting odours were more prominent than those to punishment-predicting odours. Conversely, the actual delivery of a punishment produced more pronounced phasic activity than the delivery of a reward. This latter finding is consistent with a class of neurons recorded in anaesthetized animals (Schweimer et al., 2008). (Deakin and Graeff, 1991). However, there is both electrophysiological and optogenetic evidence that serotonin is involved in many other roles, such as rhythmic motor activity (Ranade and Mainen, 2009). There is also recent, direct, evidence for its association with reward (Liu et al., 2014). Indeed, the fact that selective serotonin reuptake inhibi- tors (SSRIs) are the major treatment for de- pression has always hinted at a role for serotonin in the ascription or use of positive values. The mooted explanation for serotonin’s role in this process—that the positive associations arise from adaptions that produce appropriate responses to losses (Dayan and Huys, 2008)—seems unlikely to suffice in the face of all this contrary evidence. Here, Cohen (who is now at Johns Hopkins University), Amoroso and Uchida (who are both at Harvard University) used optogenetic tagging to identify the serotonergic neurons of mice in a brain area called the dorsal raphe nucleus (Cohen et al., 2015). They then studied the activation of these cells in awake animals under a Pavlovian conditioning paradigm. In blocks of trials, particular odours preceded a reward (water), a punishment (bitter-tasting quinine, or an airpuff to the face) or nothing, so that the mice learned to associate an odour with a particular outcome. The first, sobering, finding was that both tagged and untagged neurons show a sub- stantial diversity in their electrical activity and the aspects of the behaviour with which this activity was correlated. This shows the likely impossibility of classifying whether a neuron is serotonergic without some form of molecular proof. In addition, the results add substantially to our knowledge about the complex relationship be- tween the activity of serotonergic neurons and rewards and punishments. Related research article Cohen JY, This shows the likely impossibility of classifying whether a neuron is serotonergic without some form of molecular proof. Peter Dayan is in the Gatsby Computational Neuro- science Unit, University College London, London, United Kingdom Peter Dayan is in the Gatsby Computational Neuro- science Unit, University College London, London, United Kingdom dayan@gatsby.ucl.ac.uk Quentin Huys is in the Translational Neuromodeling Unit, Institute for Biomedical Engineering, University of Zurich & ETH Zurich, Zurich, Switzerland and Department of Psychiatry, Psychotherapy and Psychosomatics, University of Zurich, Zurich, Switzerland qhuys@cantab.net dayan@gatsby.ucl.ac.uk Quentin Huys is in the Translational Neuromodeling Unit, Institute for Biomedical Engineering, University of Zurich & ETH Zurich, Zurich, Switzerland and Department of Psychiatry, Psychotherapy and Psychosomatics, University of Zurich, Zurich, Switzerland qhuys@cantab.net In addition, the results add substantially to our knowledge about the complex relationship be- tween the activity of serotonergic neurons and rewards and punishments. There are three key responses to consider: the baseline activity just before each odour, potentially reflecting the level of reward or punishment of the block; the activity inspired by the odour; and the activity produced by the outcome that the odour predicts. Competing interests: The authors declare that no competing interests exist. Related research article Cohen JY, There are three key responses to consider: the baseline activity just before each odour, potentially reflecting the level However, there is both electrophysiological and optogenetic evidence that serotonin is involved in many other roles, such as rhythmic motor activity (Ranade and Mainen, 2009). There is also recent, direct, evidence for its association with reward (Liu et al., 2014). Indeed, the fact that selective serotonin reuptake inhibi- tors (SSRIs) are the major treatment for de- pression has always hinted at a role for serotonin in the ascription or use of positive values. The mooted explanation for serotonin’s role in this process—that the positive associations arise from adaptions that produce appropriate responses to losses (Dayan and Huys, 2008)—seems unlikely to suffice in the face of all this contrary evidence. This notable paper by Cohen and colleagues is credibly the end of the end of theories of serotonin acting as an aversive counterpart to dopamine. It may also be the end of the beginning of a new wave of results (Schweimer et al., 2008; Amo et al., 2014; Liu et al., 2014; Miyazaki et al., 2014) that have reinforced a richly varied picture of this neuromodulator’s role in motivation and emotion. The beginning of the end of our befuddlement might come through using markers or methods that allow neurons activated during behaviour to be re- activated experimentally (such as the conditional expression of channelrhodopsin in activated serotonergic neurons). This could allow the motley collection of neural subgroups observed in the dorsal and median raphe nuclei (Lowry et al., 2005) to be further resolved. Here, Cohen (who is now at Johns Hopkins University), Amoroso and Uchida (who are both at Harvard University) used optogenetic tagging to identify the serotonergic neurons of mice in a brain area called the dorsal raphe nucleus (Cohen et al., 2015). They then studied the activation of these cells in awake animals under a Pavlovian conditioning paradigm. In blocks of trials, particular odours preceded a reward (water), a punishment (bitter-tasting quinine, or an airpuff to the face) or nothing, so that the mice learned to associate an odour with a particular outcome. The first, sobering, finding was that both tagged and untagged neurons show a sub- stantial diversity in their electrical activity and the aspects of the behaviour with which this activity was correlated. Dayan and Huys. eLife 2015;4:e07390. DOI: 10.7554/eLife.07390 References Very crudely, blocks of rewards elicited greater tonic activity—that is, more sustained firing—between trials in serotonin neurons than blocks of punishments (although the opposite pattern was also apparent). Such a link of tonic activity to the average level of reward had previously been proposed for dopamine rather than serotonin (Niv et al., 2007). Strikingly, when Cohen and colleagues recorded from dopamine neurons they failed to find such a signal. How tonic Amo R, Fredes F, Kinoshita M, Aoki R, Aizawa H, Agetsuma M, Aoki T, Shiraki T, Kakinuma H, Matsuda M, et al. 2014. The habenulo-raphe serotonergic circuit encodes an aversive expectation value essential for adaptive active avoidance of danger. Neuron 84: 1034–1048. doi: 10.1016/j.neuron.2014.10.035. Cohen JY, Amoroso MW, Uchida N. 2015. Serotonergic neurons signal reward and punishment on multiple timescales. eLife 4:e06346. doi: 10.7554/eLife.06346. Cohen JY, Haesler S, Vong L, Lowell BB, Uchida N. 2012. Neuron-type-specific signals for reward and Dayan and Huys. eLife 2015;4:e07390. DOI: 10.7554/eLife.07390 2 of 3 Insight Neurophysiology \| Serotonin’s many meanings elude simple theories punishment in the ventral tegmental area. Nature 482: 85–88. doi: 10.1038/nature10754. Dayan P, Huys QJ. 2008. Serotonin, inhibition, and negative mood. PLOS Computational Biology 4:e4. doi: 10.1371/journal.pcbi.0040004. Deakin JF, Graeff FG. 1991. 5-HT and mechanisms of defence. Journal of Psychopharmacology 5:305–316. doi: 10.1177/026988119100500414. Lima SQ, Hrom´adka T, Znamenskiy P, Zador AM. 2009. Pinp: a new method of tagging neuronal populations for identification during in vivo electrophysiological recording. PLOS ONE 4:e6099. doi: 10.1371/journal. pone.0006099. Liu Z, Zhou J, Li Y, Hu F, Lu Y, Ma M, Feng Q, Zhang JE, Wang D, Zeng J, et al. 2014. Dorsal raphe neurons signal reward through 5-ht and glutamate. Neuron 81: 1360–1374. doi: 10.1016/j.neuron.2014.02.010. Lowry CA, Johnson PL, Hay-Schmidt A, Mikkelsen J, Shekhar A. 2005. Modulation of anxiety circuits by punishment in the ventral tegmental area. Nature 482: 85–88. doi: 10.1038/nature10754. punishment in the ventral tegmental area. Nature 482: 85–88. doi: 10.1038/nature10754. serotonergic systems. Stress 8:233–246. doi: 10.1080/ 10253890500492787. serotonergic systems. Stress 8:233–246. doi: 10.1080/ 10253890500492787. Miyazaki KW, Miyazaki K, Tanaka KF, Yamanaka A, Takahashi A, Tabuchi S, Doya K. 2014. Optogenetic activation of dorsal raphe serotonin neurons enhances patience for future rewards. Current Biology 24: 2033–2040. doi: 10.1016/j.cub.2014.07.041. Miyazaki KW, Miyazaki K, Tanaka KF, Yamanaka A, Takahashi A, Tabuchi S, Doya K. 2014. Optogenetic activation of dorsal raphe serotonin neurons enhances patience for future rewards. Current Biology 24: 2033–2040. doi: 10.1016/j.cub.2014.07.041. References Niv Y, Daw ND, Joel D, Dayan P. 2007. Tonic dopamine: opportunity costs and the control of response vigor. Psychopharmacology 191:507–520. d i 10 1007/ 00213 006 0502 4 Dayan P, Huys QJ. 2008. Serotonin, inhibition, and negative mood. PLOS Computational Biology 4:e4. j p Deakin JF, Graeff FG. 1991. 5-HT and mechanisms of defence. Journal of Psychopharmacology 5:305–316. doi: 10.1177/026988119100500414. j Niv Y, Daw ND, Joel D, Dayan P. 2007. Tonic dopamine: opportunity costs and the control of response vigor. Psychopharmacology 191:507–520. Lima SQ, Hrom´adka T, Znamenskiy P, Zador AM. 2009. Pinp: a new method of tagging neuronal populations for identification during in vivo electrophysiological recording. PLOS ONE 4:e6099. doi: 10.1371/journal. pone.0006099. Ranade SP, Mainen ZF. 2009. Transient firing of dorsal raphe neurons encodes diverse and specific sensory, motor, and reward events. Journal of Neurophysiology 102:3026–3037. doi: 10.1152/jn.00507.2009. p Liu Z, Zhou J, Li Y, Hu F, Lu Y, Ma M, Feng Q, Zhang JE, Wang D, Zeng J, et al. 2014. Dorsal raphe neurons signal reward through 5-ht and glutamate. Neuron 81: 1360–1374. doi: 10.1016/j.neuron.2014.02.010. Lowry CA, Johnson PL, Hay-Schmidt A, Mikkelsen J, Liu Z, Zhou J, Li Y, Hu F, Lu Y, Ma M, Feng Q, Zhang JE, Wang D, Zeng J, et al. 2014. Dorsal raphe neurons Schweimer J, Brierley D, Ungless M. 2008. Phasic nociceptive responses in dorsal raphe serotonin neurons. Fundamental & Clinical Pharmacology 22:119. doi: 10.1111/j.1472-8206.2008.00601.x. signal reward through 5-ht and glutamate. Neuron 81: 1360–1374. doi: 10.1016/j.neuron.2014.02.010. Lowry CA, Johnson PL, Hay-Schmidt A, Mikkelsen J, Shekhar A. 2005. Modulation of anxiety circuits by Lowry CA, Johnson PL, Hay-Schmidt A, Mikkelsen J, Shekhar A. 2005. Modulation of anxiety circuits by 3 of 3
https://openalex.org/W4390906131	https://www.nature.com/articles/s41405-023-00182-4.pdf	English	null	Detection of viable oral bacteria of the patient on the surgical mask of dentists	BDJ open	2,024	cc-by	4,880	Received: 30 August 2023 Revised: 16 November 2023 Accepted: 17 November 2023 1Department of Operative Dentistry, Periodontology and Preventive Dentistry, Saarland University, Homburg, Germany. 2Oral Surgery Clinic, German Armed Forces Central Hospital, Koblenz, Germany. 3Institute of Medical Microbiology and Hygiene, Department of Hospital Hygiene, Saarland University, Homburg, Germany. 4Synoptic Dentistry, Saarland University, Homburg, Germany. ✉email: madline.gund@uks.eu ARTICLE OPEN Detection of viable oral bacteria of the patient on the surgical mask of dentists Madline Priska Gund 1,2✉, Jusef Naim1, Janina Lang1, Matthias Hannig1, Barbara Gärtner3, Alexander Halfmann3, Gabor Boros2 and Stefan Rupf1,4 Madline Priska Gund 1,2✉, Jusef Naim1, Janina Lang1, Matthias Hannig1, Barbara Gärtner3, Alexander Halfmann3, Gabor Boros2 and Stefan Rupf1,4 © The Author(s) 2024 © The Author(s) 2024 © The Author(s) 2024 INTRODUCTION AND AIM: Bioaerosols contaminate the personal protective equipment (PPE), especially masks. The PPE harbors microorganisms from various sources. However, no previous studies have investigated the speciﬁc sources of bacteria found on used masks and their correlation with those from the treated patient. SETTING, DESIGN, MATERIAL AND METHODS: Intraoral samples from the patient were collected prior to dental aerosol- producing treatments using a nylon ﬂock ﬁber swab. After treatment, the practitioner’s mask was imprinted onto agar plates. MAIN OUTCOME METHODS: Following cultivation, colony forming units were counted and identiﬁed using matrix-assisted laser desorption/ionization time-of-ﬂight mass spectrometry (MALDI-TOF MS). After the samples were analyzed, the intraoral samples as well as the mask samples were assessed for the presence of identical species, which were subsequently quantiﬁed. RESULTS: 126 treatments were included. One species match occurred most frequently (26.2%), followed by two (11.9%%) and three or more (3.97%). In the intraoral samples, Neisseria subﬂava occurred most often, within mask samples Staphylococcus epidermidis were detected most. Staphylococcus aureus could be cultivated three times more often in intraoral samples than on the mask. DISCUSSION AND CONCLUSION: Oral microorganisms originating from the patient’s oral cavity can be found on the outside of masks. When using PPE during treatments, it should therefore always be in mind that potentially pathogenic microorganisms may land on the mask becoming a source of for itself. BDJ Open (2024) 10:4 ; https://doi.org/10.1038/s41405-023-00182-4 MATERIAL AND METHODS Setting h d This prospective study was conducted at Saarland University Hospital, Clinic of Operative Dentistry, Periodontology and Preventive Dentistry. All instruments used for treatment were sterile. The treatment unit and surrounding surfaces were routinely disinfected (using Celtex® Wipes, Lotfex, Bremen, Germany; Incidin® 0,25%, Dräger, Lübeck, Germany). The room temperature was 20–22 °C, the relative humidity 40–60%. Contamination of PPE has already been intensively investigated [1–5]. Especially the bacterial contamination rate of the mask [6–9], with and without a protective face shield [10] was examined. Moreover, one research group was able to demonstrate that the mask itself has a contamination potential after aerosol-producing treat- ments [11, 12]. It is assumed that bacteria from aerosols originate from the patient (calculus, bioﬁlm, blood, saliva) [13–15]. However, also patient-independent sources such as contaminated water pipes and general air contamination are known factors [16, 17]. www.nature.com/bdjopen INTRODUCTION So far, it has never been demonstrated from which source the bacteria on the used mask originate. In order to prevent PPE contamination as much as possible, it is important to know and assess the sources correctly. Wearing personal protective equipment (PPE) has been given great importance in dentistry, and not just since the 2019 coronavirus (Covid-19) outbreak. Aerosol-producing dental treatments generate bioaerosols contaminating the practitioner, assistant, patients and environ- ment. Standard PPE is therefore recommended for every dental treatment preventing contamination, transmission and possible infection with or by pathogens. This includes gloves, face masks and protective goggles. Further precautions are advisable (e.g. protective gown, face shield). The aim of this study was therefore to investigate the sources of contamination, starting with the mask by evaluating replication competent bacteria from the mask and the corresponding patient. Statistics Qualitative and quantitative results of bacteria in intraoral samples and bacterial contamination of surgical masks were presented descriptively. Frequencies of detection of bacteria in intraoral samples and surgical masks overall as well as for the treatment modalities were compared using the One-Way ANOVA for repeated measures. For the comparison of the detection frequencies of bacteria between different treatment modalities from intraoral samples and from surgical masks, the One-Way ANOVA for independent measures was used (p < 0.05). Sampling P i t t Prior to treatment, intraoral samples were taken with a nylon ﬂock ﬁber swab (eSwab™universal, Mast Diagnostica, Reinfeld, Germany). For this purpose, the swab was passed retromolar on the terminal lower right molar along the mucogingival border of the lingual surface of the front teeth and buccally back. The swab was then stored in Amies medium. Prior to treatment, intraoral samples were taken with a nylon ﬂock ﬁber swab (eSwab™universal, Mast Diagnostica, Reinfeld, Germany). For this purpose, the swab was passed retromolar on the terminal lower right molar along the mucogingival border of the lingual surface of the front teeth and buccally back. The swab was then stored in Amies medium. After the treatment, the practitioner´s surgical mask was removed by the assistant without touching the exterior surface of the mask. It was then imprinted immediately onto two different agar plates: Columbia (Columbia III Agar with 5% sheep blood, Becton Dickinson GmbH, Heidelberg, Germany) and Chocolate agar (GC II Agar with Hemoglobin and IsoVitaleX; Becton Dickinson GmbH, Heidelberg, Germany) plates for 10 s each. After 48 h of cultivation, no bacterial growth was observed in any of the control samples. Subjects Experiments were performed by 14 specially instructed and supervised second-year clinical dental students. Hygienic hand disinfection was performed before donning PPE. The surgical mask was picked up by the ties while wearing gloves, the assistant knotted it behind the head. All participants were instructed not to touch the exterior surface of their surgical mask. After treatment, masks were removed by the assistant. PPE consisted of pathogenfree (“non-sterile”) medical gloves (nitrile powder- free gloves: Joza®, Hebei Titans Hongsen Medical Technology Co., Ltd., Hebei, China), surgical masks (tie-band medical surgical mask type II, Mölnlycke Health Care, Düsseldorf, Germany), protective eyewear (Safe- view® eyewear, Halyard, Neunkirchen, Germany), hair caps (BARRIER® Nurses Cap, Mölnlycke Health Care AB, Göteborg, Schweden und FarStar® medical GmbH, Barsbüttel, Germany) and protective gown (Simani Industrie s.r.l., Gallicano, Italy). Sampling was conducted from October 2022 to February 2023. Matching species After the samples were analyzed, the intraoral samples as well as the mask samples were assessed for the presence of identical species, which were subsequently quantiﬁed. The concordance was documented regarding a single species, two species, and three or more species. Qualitative results The identiﬁed microorganisms are presented in Supplementary Table 1. S. epidermidis was found most frequently, followed by N. subﬂava, M. luteus, A. oris. Concerning the intraoral samples, most often N. subﬂava occurred, followed by A. oris, H. parainﬂuenzae, R. dentocariosa. With regard to the mask samples S. epidermidis, M. luteus, S. hominis and S. capitis could be detected most frequently in descending order. S. aureus was cultivated three times more often in the intraoral samples than on the mask. It was most frequently detected before ﬁlling therapy (4 times) and profes- sional tooth cleaning (4), but also before periodontal (1) and endodontic treatment (1). On the mask, S. aureus could only be cultivated after professional tooth cleaning (3). Qualitative bacterial analysis All phenotypically distinguishable colony forming units grown on the agar plates were classiﬁed using matrix-assisted laser desorption/ionization time-of-ﬂight mass spectrometry (MALDI-TOF BiotyperTM MBTTM smart, Bruker Daltonik GmbH, Bremen, Germany). Colonies were picked and transferred to a stainless-steel target (MSP 96 spot target, Bruker Daltonik GmbH, Bremen, Germany) and overlayed with 1 μl of formic acid (AppliChem GmbH, Darmstadt, Germany). After it dried, 1 μl of matrix (Bruker HCCA = α-Cyano-4-hydroxycinnamic acid, Bruker Daltonik GmbH, Bremen, Germany) was applied. Measurements were continued until the bacterium was clearly identiﬁed. If this was not the case, the measurement was carried out again. If a spectrum could still not be assigned to a known species, it was noted as “unidentiﬁed”. Controls 126 aerosol-producing treatments were included: professional tooth clean- ings, periodontal treatments, ﬁlling therapies and endodontic treatments. For all types of treatments, water from the dental unit was used for cooling. An evacuation was established by means of conventional dental suction (CDS) using a cannula of 3.3 mm in diameter (suction ﬂow 1.1 l/s) and a high-volume evacuation (HVE) of 8.0 mm in diameter (suction ﬂow 6.0 l/s). The CDS was placed lingual to the lower central incisors. The HVE was held near the aerosol source by an assistant. Professional tooth cleaning included the supragingival cleaning with ultrasonics and manual instruments of the entire dentition and afterwards polishing. Periodontal cleaning included the subgingival cleaning with ultrasonics and manual instruments of all diseased pockets. Filling therapy and endodontic treatment was mainly performed with a rubber dam. The caries excavation was performed without a rubber dam. The ﬁlling was placed with a rubber dam if it was not subgingival. It was removed again for ﬁnishing. Trepanation was performed without a rubber dam in order to properly assess the axis of the tooth. All further steps were performed under rubber dam. In each case, one tooth was treated, rarely several. Five unused surgical masks (n = 5) were worn for 120 min each during simulated aerosol-producing dental treatments (trepanation, cavity pre- paration) on a phantom simulator and used as negative controls. No intraoral swab was taken, as it was a phantom head (Fig. 1). Microbial cultivation In order to cultivate the intraoral sample, 100 μl of the Amies medium was transferred to each agar plate using the triple-streak plating method. For this purpose, bacteria were removed from the bacterial suspension using an inoculation loop (Sarstedt, Nümbrecht, Germany) and the ﬁrst zigzag streak was made on the agar plate. Bacterial density was then reduced by passing a second sterile inoculation loop through the ﬁrst streak- but only two to three times. This procedure was repeated using a third sterile inoculation loop to further reduce bacterial density and facilitate isolation of different species for subsequent analysis. The Columbia agar plates were placed in incubation containers for gas generating systems (AnaeroPack Rectangular Jar, Mitsubishi Gas Chemical Company, ING., Tokyo, Japan). A gas bag (GasPak CO2 Container System, Becton Dickinson GmbH, Heidelberg, Germany) to enrich a carbon dioxide rich environment was inserted and the container was sealed and incubated at 35° +/−2° for 48 h. Quantitative results After the treatment, the practitioner´s surgical mask was removed by the assistant without touching the exterior surface of the mask. It was then imprinted immediately onto two different agar plates: Columbia (Columbia III Agar with 5% sheep blood, Becton Dickinson GmbH, Heidelberg, Germany) and Chocolate agar (GC II Agar with Hemoglobin and IsoVitaleX; Becton Dickinson GmbH, Heidelberg, Germany) plates for 10 s each. 126 aerosol-producing treatments were included: 50 professional tooth cleanings, 33 periodontal treatments, 31 ﬁlling therapies and 12 endodontic treatments. The average duration of treatment was 120 min. One matching species occurred most frequently (in total: 26.2%), followed by two matching species (in total: 11.9%). The rarest, three matching species or more were found (in total: 3.97%). There were only minor differences depending on the treatment modality. Three matching species or more could not be determined for ﬁlling therapy and endodontic therapy (Fig. 2). Patients Only adult patients without known infectious diseases were included in the study. Use of antibiotics in the last 6 months resulted in exclusion from the study. All samples were anonymized. Both verbal and written informed M.P. Gund et al. 2 consent were obtained from all participants. Ethical approval for the study was obtained (Vote No. 195/22). Fig. 1 Flow chart. Flow chart of the study. Fig. 1 Flow chart. Flow chart of the study. Fig. 2 Number matching species (comparison of bacteria of mask and intraoral sample) in percent according to treatment modalit Fig. 2 Number matching species (comparison of bacteria of mask and intraoral sample) in percent according to treatment modality. arison of bacteria of mask and intraoral sample) in percent according to treatment modality. Fig. 2 Number matching species (comparison of bacteria of mask and intraoral sample) in percent according to treatment modality. on the mask, results cannot be directly compared or discussed with literature. Statistical analysis y Statistically signiﬁcant differences were observed between all intraoral and all mask samples (p = 0.021). Statistically signiﬁcant differences were observed between all intraoral and all mask samples (p = 0.021). The bacterial species found are consistent with those described in literature for contamination of PPE [6–9, 11, 18]. When analyzing the individual treatment modalities, statistically signiﬁcant differences were found on masks between ﬁlling therapy (p = 0.03), periodontal therapy (p = 0.004), and endodon- tic therapy (p = 0.01). However, no statistically signiﬁcant differ- ence was observed when comparing intraoral samples to mask samples for professional tooth cleaning (p = 0.1). Moreover, the bacteria detected in the intraoral samples correspond to the commonly described bacteria for the oral cavity [19–23]. The bacteria detected in this study belonged to the natural microbiota of the oral cavity in humans. This is in line with the fact that only healthy patients were included in the study and patients with any risk of infection were excluded. In addition, dental staff adhere strictly to hygiene standards. In the real world, it is therefore possible that obligate pathogens are also part of the spectrum of bacteria that are transmitted and thus pose a signiﬁcant risk for practitioners and others. Summarized, transmission of pathogenic bacteria cannot be ruled out. Moreover, the comparison of the detection frequency of different bacteria between treatment modalities did not yield statistically signiﬁcant results for either the intraoral samples (p = 0.9) or the mask samples (p = 0.9). Quantitative bacterial analysis l f d Colony forming units were counted using a colony counter (schuett-biotec GmbH, Göttingen, Germany). BDJ Open (2024) 10:4 BDJ Open (2024) 10:4 Fig. 2 Number matching species (comparison of bacteria of mask and intraoral sample) in percent according to treatment modality. Fig. 1 Flow chart. Flow chart of the study. M.P. Gund et al. M.P. Gund et al. 3 Fig. 1 Flow chart. Flow chart of the study. Fig. 1 Flow chart. Flow chart of the study. DISCUSSION To our best knowledge, this is the ﬁrst study to compare the intraoral bacteria of the patient before aerosol-producing dental treatments with those on the practitioner’s mask afterwards. Since this is the ﬁrst study to investigate the origin of microorganisms However, even when investigating only healthy individuals, some facultative pathogens were found and should be discussed in more detail. BDJ Open (2024) 10:4 M.P. Gund et al. 4 bacterial composition between intraoral samples and mask samples, no statistically signiﬁcant difference was observed. This is noteworthy considering that the intraoral samples exhibited remarkable similarity despite the inclusion of different patients with diverse treatment requirements, ranging from preventive measures and treatment of periodontitis and dental caries to invasive endodontic therapy. Staphylococcus aureus is a potentially high-risk, sometimes multiresistant, nosocomial pathogen causing a wide variety of diseases from bacteremia to skin abscesses, bone infection, pneumonia, respiratory tract infections, prosthetic joint, surgical site and cardiovascular infections [24]. Attachments to medical implants and host tissues, furthermore formation of mature bioﬁlms contribute signiﬁcantly to the persistence of chronic infections [25]. Although S. aureus was found three times more frequently in oral samples than on the mask, it has a high risk for transmission and infection. y When using personal protective equipment during treatment, it should always be kept in mind that potentially pathogenic microorganisms may contaminate the mask, becoming a source of contamination for the environment itself [11]. The patient’s health status is sometimes unknown and risk factors for the shift of an at present apathogenic microorganism into a pathogenic one (in case of facultative pathogens) is not always apparent. Finally, colonization of a patient with obligate or facultative pathogens cannot be conclusively clariﬁed. Transmission to vulnerable patients, dentists or assistants is possible. Frequency of exposure and virulence of pathogens determine transmission, infection, and clinical manifestation of diseases frequency [32]. Therefore, it is essential following the recommendations for prevention of nosocomial infections [33, 34] including masks [11]. In addition, a detailed medical history of the patient should always be taken in order to better assess possible infection-related risks. Staphylococcus epidermidis was most often detected in all samples and in particular on masks. Like other coagulase- negative staphylococci, it is a facultative and often multiresistant pathogen causing a wide variety of infections in connection with medical and surgical procedures [26, 27]. DATA AVAILABILITY The dataset used and analyzed during the current study is available from the corresponding author upon reasonable request. Sampling may also have reduced the microbial diversity and viable bacteria on masks, since the entire exterior surface can never be brought into contact with the agar plate, but rather a central section. CONCLUSION I l i In conclusion, the presented study pointed out that oral microorganisms, only originating from the patient’s oral cavity, can be found on the exterior surface of the surgical mask. Thus, the route of transmission could be clearly demonstrated. Future studies should investigate the proportion of contamination from other possible sources such as contaminated water pipes and general air contamination. In addition, a similar study design might be used when investigating the role of viruses. Since many genera have a similar appearance and are difﬁcult to distinguish visually, they may not always have been interpreted correctly, which in turn would explain a low number of matching species. Exemplary are Streptococcus vestibularis and Streptococ- cus mitis: both viridans streptococci and both looking very similar. To reduce this problem, more than one colony with the same morphology could have been identiﬁed. However, given the fact that this is a proof of principle study, the additional effort seems to be of little beneﬁt. DISCUSSION As part of the commensal microbiome, the bacteria are harmless to healthy individuals, but immunosuppressed or immunocompromised patients may be at risk [28–31]. Same applies for Streptococcus oralis, Staphylococcus capitis, M. luteus or R. dentocariosa, also often detected. Overall, only a few oral microorganisms could be detected on the mask and the intraoral samples at the same time. This is due to the fact that many species cannot be cultivated, moreover anaerobic bacteria must be excluded from the outset. To cultivate more fastidious bacteria, special culture media would have had to be used. Many microorganisms were detected in the mouth, but not on the surgical mask, indicating that only a small subset of bacteria were transferred. This is in line with the fact that in most sample pairs only one species matched between the oral cavity and the mask. However, the bacterial load of the oral cavity might be underestimated, since only one quadrant was swabbed during the intraoral sampling but treatment covers the entire oral cavity. REFERENCES 1. Kaufmann M, Solderer A, Gubler A, Wegehaupt FJ, Attin T, Schmidlin PR. Quan- titative measurements of aerosols from air-polishing and ultrasonic devices: (How) can we protect ourselves? PLoS One. 2020;15:e0244020. https://doi.org/ 10.1371/journal.pone.0244020. 1. Kaufmann M, Solderer A, Gubler A, Wegehaupt FJ, Attin T, Schmidlin PR. Quan- titative measurements of aerosols from air-polishing and ultrasonic devices: (How) can we protect ourselves? PLoS One. 2020;15:e0244020. https://doi.org/ 10.1371/journal.pone.0244020. In addition, the MALDI-TOF MS analysis refers to colonies identiﬁed as different phenotypes, which may underestimate the bacterial spectrum. In summary, there are probably more viable bacteria on the mask originating from the oral cavity than described in the present study. 2. Chen IH, Lin CH, Liao YS, et al. Assessment of dental personal protective equip- ment (PPE) and the relationship between manual dexterity and dissemination of aerosol and splatter during the COVID-19 pandemic. J Dent Sci. 2022;17:1538–43. https://doi.org/10.1016/j.jds.2022.04.021. 3. Teichert-Filho R, Baldasso CN, Campos MM, Gomes MS. Protective device to reduce aerosol dispersion in dental clinics during the COVID-19 pandemic. Int Endod J. 2020;53:1588–97. https://doi.org/10.1111/iej.13373. An investigation by means of polymerase chain reaction (PCR) would not have been an alternative. In the end, only viable bacteria are of interest as only these can be transmitted representing a potential risk of transmission and infection. Furthermore, a lot of sequencing or species-speciﬁc polymerase chain reactions would have been necessary for the investigation. A methodologically enormous additional effort without advantages. 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Virulence. 2021;12:547–69. https://doi.org/10.1080/21505594.2021.1878688. Reprints and permission information is available at http://www.nature.com/ reprints ACKNOWLEDGEMENTS We would like to thank Sandra Rauch from the Institute of Medical Microbiology and Hygiene for her support in the evaluation of the samples. We would like to thank Sandra Rauch from the Institute of Medical Microbiology and Hygiene for her support in the evaluation of the samples. 13. Zemouri C, Volgenant CMC, Buijs MJ, Crielaard W, Rosema NAM, Brandt BW, et al. Dental aerosols: microbial composition and spatial distribution. J Oral Microbiol. 2020;12:1762040 https://doi.org/10.1080/20002297.2020.1762040. 14. Chatoutsidou SE, Saridaki A, Raisi L, et al. Airborne particles and microorganisms in a dental clinic: variability of indoor concentrations, impact of dental proce- dures, and personal exposure during everyday practice. Indoor Air. 2021;31:1164–77. https://doi.org/10.1111/ina.12820. AUTHOR CONTRIBUTIONS MPG, JN, JL, MH, BG, AH, GB and SR planned the study. JL, MG, JN, AH, SR analyzed and interpreted the data. MG, JN, JL and SR were major contributors to writing the manuscript. All authors edited and reviewed the draft manuscript and read and approved the ﬁnal manuscript. MPG, JN, JL, MH, BG, AH, GB and SR planned the study. JL, MG, JN, AH, SR analyzed and interpreted the data. MG, JN, JL and SR were major contributors to writing the manuscript. All authors edited and reviewed the draft manuscript and read and approved the ﬁnal manuscript. 15. Bennett AM, Fulford MR, Walker JT, Bradshaw DJ, Martin MV, Marsh PD. Microbial aerosols in general dental practice. Br Dent J. 2000;189:664–7. https://doi.org/ 10.1038/sj.bdj.4800859. 16. Zhang Y, Ping Y, Zhou R, Wang J, Zhang G. High throughput sequencing-based analysis of microbial diversity in dental unit waterlines supports the importance of providing safe water for clinical use. 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Staphylococcus aureus bioﬁlms: recent developments in bioﬁlm dispersal. Front Cell Infect Microbiol. 2014;4:178 https://doi.org/10.3389/ fcimb.2014.00178. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 26. Otto M. Staphylococcus epidermidis pathogenesis. Methods Mol Biol. 2014;1106:17–31. https://doi.org/10.1007/978-1-62703-736-5_2. 27. Heilmann C, Ziebuhr W, Becker K. Are coagulase-negative staphylococci virulent? Clin Microbiol Infect. 2019;25:1071–80. https://doi.org/10.1016/j.cmi.2018.11.012. 28. Irlinger F. Safety assessment of dairy microorganisms: coagulase-negative sta- phylococci. Int J Food Microbiol. 2008;126:302–10. https://doi.org/10.1016/ j.ijfoodmicro.2007.08.016. 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https://openalex.org/W4230257025	https://www.researchsquare.com/article/rs-25817/latest.pdf	English	null	High Resolution Computed Tomography Finding in 552 Patients with Symptomatic COVID-19: First Report from North of Iran	Research Square (Research Square)	2,020	cc-by	4,537	Hadi Majidi Hadi Majidi Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Elham-Sadat Bani-Mostafavi Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Zahra Mardanshahi Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Farnaz Godazandeh Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Roya Gasemian Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Keyvan Heydari Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran Reza Alizadeh-Navaei (  reza_nava@yahoo.com ) Gastrointestinal Cancer Research Center, Mazandaran University of Medical Sciences, Sari, Iran https://orcid.org/0000-0003-0580-000X Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Elham-Sadat Bani-Mostafavi Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Zahra Mardanshahi Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Zahra Mardanshahi High Resolution Computed Tomography Finding in 552 Patients with Symptomatic COVID-19: First Report from North of Iran Hadi Majidi Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Elham-Sadat Bani-Mostafavi Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Zahra Mardanshahi Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Farnaz Godazandeh Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Roya Gasemian Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Keyvan Heydari Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran Reza Alizadeh-Navaei (  reza_nava@yahoo.com ) Gastrointestinal Cancer Research Center, Mazandaran University of Medical Sciences, Sari, Iran https://orcid.org/0000-0003-0580-000X Research Article Keywords: Corona virus 2019, Ground glass, Peripheral distribution, Pleural effusion Posted Date: November 6th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-25817/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Emergency Radiology on July 13th, 2020. Abstract bst act Purpose Due to the emergence of the new coronavirus 2019 and the lack of sufficient information about infected patients, this study was conducted to investigate the chest high-resolution computed tomography (HRCT) findings of patients infected with the new coronavirus 2019. Methods This cross-sectional study was performed on COVID-19 patients referred to Medical Imaging Centers of Sari, Mazandaran, Iran, on March 2020 for computed tomography (CT) scan. Symptomatic patients were referred to the Medical Imaging Center for diagnosis confirmation through CT scan. In addition to age and sex, HRCT findings were collected from the picture archiving and communication system (PACS) for further evaluations. p g y Results Out of 552 patients with mean age of 51.2 ± 14.8 years, the male/female ratio was 1.38 to 1. The most common expressive findings in patients were ground-glass opacity (GGO) (87.3%), peripheral distribution (82.4%), and posterior distri- bution (81.5%). The most conflicting findings in patients were pleural effusion (7.6%), peribronchovascular distribution (7.6%), and lymphadenopathy (5.1%). The peripheral distribution (p = 0.034), round opacities (p = 0.02), single lobe (p = 0.003), and pleural effusion (p = 0.037) were significant in people under and over 50 years of age. Conclusion In summary, the present study indicated that in addition to GGO, peripheral distribution findings could be a vital diagnostic choice in COVID-19 patients. Keywords Coronavirus 2019 . Ground glass . Peripheral distribution . Pleural effusion Hadi Majidi1 & Elham-Sadat Bani-Mostafavi1 & Zahra Mardanshahi1 & Farnaz Godazandeh1 & Roya Ghasemian2 & K H d i3 R Ali d h N i4 Received: 14 April 2020 /Accepted: 6 July 2020 # American Society of Emergency Radiology 2020 Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Emergency Radiology on July 13th, 2020. See the published version at https://doi.org/10.1007/s10140-020-01819-9. Emergency Radiology https://doi.org/10.1007/s10140-020-01819-9 ORIGINAL ARTICLE * Reza Alizadeh-Navaei reza_nava@yahoo.com 1 Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran 2 Antimicrobial Resistance Research Center, Mazandaran University of Medical Sciences, Sari, Iran 3 Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran 4 Gastrointestinal Cancer Research Center, Non-communicable Diseases Institute, Mazandaran University of Medical Sciences, Sari, Iran 2 Antimicrobial Resistance Research Center, Mazandaran University of Medical Sciences, Sari, Iran 1 Department of Radiology, Faculty of Medicine, Mazandaran 1 Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran 4 Gastrointestinal Cancer Research Center, Non-communicable Diseases Institute, Mazandaran University of Medical Sciences, Sari, Iran Introduction The COVID-19 is a novel heat-sensitive coronavirus. Based on evolutionary research, it has been found that the virus has originated from animals like bats, rodents, and birds [1]. In early December 2019, the first cases of pneumonia of unknown etiology were observed in Wuhan, the capital of Hubei Province in China [2]. The pathogen, which was a single-stranded enveloped coronavirus RNA [3], was called acute respiratory syn- drome coronavirus 2 (SARS-CoV-2), and the course of the disease caused by the virus is pathologically some- what similar to that of severe acute respiratory syn- drome (SARS) [4]. So far, seven types of coronaviruses have been identified that can infect humans [4–6]. * Reza Alizadeh-Navaei reza_nava@yahoo.com 3 Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran 4 Gastrointestinal Cancer Research Center, Non-communicable Diseases Institute, Mazandaran University of Medical Sciences, Sari, Iran Emerg Radiol Viruses that cause severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and the coronavirus disease (COVID-19) have zoonotic origins [6] and are transmitted from person to person [7]. About 13.9% of patients with COVID-19 died, and about half of those who visited hospitals were discharged after receiving the necessary treatment [8]. According to the study by Guan et al., 1099 patients in 552 hospitals with COVID-19 and an average age of 47 years were examined. Common manifestations of the virus include fever above 37.5 °C (56.2%), cough (67.8%), and lymphocytopenia (83.2%) [9]. According to several studies that examined the imaging findings of COVID-19 patients, 69 to 89% of the patients had positive chest CT findings [10, 11]. Ground-glass opac- ity (GGO) is frequently observed which has been re- ported in most studies [12, 13]. Radiological evaluation often plays a key role in confirming the diagnosis of COVID-19 in suspected cases when test kits are not available. Given the recency of the virus and the lack of sufficient information on patients involved in COVID-19 in Iran, during this study, the imaging find- ings of patients with COVID-19 were examined in Mazandaran province, located in the north of Iran. CT scanner was a Somatom Emotion 16 by Siemens with a 16-slice configuration. The high-resolution com- puted tomography (HRCT) in axial sections was used without the contrast material with section thickness of 2 mm. The low-dose protocol (low-dose computed tomography) with radiation conditions of Kvp = 100– 120, mAs = 50–100, and pitch = 1 mm was used. Results The study was conducted on 552 symptomatic patients with clinical suspicion who visited for HRCT. The mean age of participants was 51.2 ± 14.8 years, in the range of 16 to 99 years, and the age group of 50 to 59 years with the relative frequency of 25.4% showed the highest involvement (Fig. 1). In this study, 317 males (57.4%) and 235 females (42.6%) participated with a male/female sex ratio of 1.38. Introduction The patient was supine in the CT scanner with his/her arm above their head. CT scanning started from the apex to the lowest part of the lungs in a deep inspiration. The required information, including demographic data such as age, gender, and HRCT results, was collected and recorded in a researcher-made checklist by four radiologists using the PACS system. Data were analyzed in SPSS 20 using t test, chi-square, and Fisher’s exact tests, and a p < 0.05 was considered as signifi- cant. Finally, due to the very different sample size in the liter- ature, meta-analysis was undertaken using random effect method and Stata software to better compare the reported percentages. Materials and methods This cross-sectional study was conducted in March 2020 on symptomatic patients who visited the imaging centers of Sari during the coronavirus epidemic. The study pop- ulation were patients with high clinical suspicion who performed a CT scan because of limited laboratory test- ing in Iran to confirm the diagnosis and treatment. The The most common presented findings in patients were ground glass opacity (482 cases, 87.3%), peripheral distribution (455 Fig. 1 Age distribution of patients with coronavirus 2019 Fig. 1 Age distribution of patients with coronavirus 2019 alue 09 65 53 03 34 07 27 62 2 52 86 32 p value Age ≤50 n = 271 Frequency (%) Age > 50 n = 281 Frequency (%) p value 239 (88.2) 243 (86.5) 0.609 0.262 127 (46.9) 134 (47.7) 0.865 0.186 76 (28) 51 (18.1) 0.053 75 (27.7) 77 (27.4) 83 (30.6) 101 (35.9) 17 (6.3) 27 (9.6) 0.655 63 (23.2) 37 (13.2) 0.003 0.735 233 (86) 222 (79) 0.034 0.169 131 (48.3) 149 (53) 0.307 0.658 222 (81.9) 228 (81.1) 0.827 0.727 153 (56.5) 172 (61.2) 0.262 0.243 54 (19.9) 35 (12.5) 0.02 14 (5.2) 27 (9.6) 0.052 0.477 2 (0.7) 6 (2.1) 0.286 6 (2.2) 3 (1.1) 0.332 symptomatic pati symptomatic pati Emerg Radiol Emerg Radiol Fig. 2 a Bilateral and anterior to posterior peripheral ground glass opacities (white arrows). b Ground glass opacity in upper lobe (black arrow) and consolidation in lower lobe of right lung (red arrow). c Bilateral and peripheral ground glass opacities (white arrows). d Consolidation in right lower lobe (black arrow). e Right-sided pleural effusion (red arrow). f Bilateral and anterior to posterior peripheral ground glass opacities (white arrows) with peribronchovascular distribu- tion in right middle lobe (black arrow). g Bilateral and anterior to poste- rior peripheral ground glass opacities (black arrows). h Small lymphade- nopathies in middle mediastinum (white arrow) R involvement in the CT images. The most common presented find- ings in patients were ground glass opacity, peripheral distribution, and posterior distribution. The prevalence of ground glass opacity in the study population was 87.3%, which varied from 41 to 100% in other studies [9, 14, 17–28], and the prevalence of this finding was 67% given the difference in sample size in different studies and conducting the meta-analysis on these studies (Table 3). Peripheral distribution was the second most common finding in 82.4% of the study population. Materials and methods The prevalence of this finding in other studies ranged from 33 to 98% [15–21, 23, 25, 27, 28]. The meta-analysis conducted on the present study and other studies presented the peripheral distribution with 79% prevalence as the most common CT finding in these patients (Table 3). cases, 82.4%), posterior distribution (450 cases, 81.5%), and anterior distribution (325 cases, 58.9%) (Table 1 and Fig. 2). Figure 2 shows the CT scan finding of 5 patients as examples. There was no significant difference between patients of both genders in the distribution of presented findings. Peripheral dis- tribution (86% vs 79%, p = 0.034) along with the findings of round opacity (19.5% vs 12.5%, p = 0.02) and single lobe (23.2% vs 13.2%, p = 0.003) was significantly higher in patients under 50 compared to those over 50 (Table 1). The most common inconsistent findings in patients were pleu- ral effusion, peribronchovascular distribution, and lymphadenop- athy. Pleural effusion was 7.6% in the present study, which ranged between 2 and 9.7% in other studies, and the combination of the results of the present study and other studies estimated a 7% pleural effusion (Table 3). Lymphadenopathy in the CT scan was 5.1% higher than the value reported by other studies, such that this finding was not observed in two studies [15, 27] while it was 2.7% in one study [17] and 6% in another study. The total estimation was 4% (Table 3). The most common inconsistent findings in the study popula- tion were pleural effusion (42 cases, 7.6%), peribronchovascular distribution (42 cases, 7.6%), and lymphadenopathy (28 cases, 5.1%) (Table 2 and Fig. 2). There was no significant difference between patients of both genders in the distribution of inconsis- tent findings. The age distribution of inconsistent findings indi- cated that pleural effusion was significantly higher (p = 0.037) in patients over 50 years of age (28 cases, 10%) compared to those under 50 (14 cases, 5.2%) (Table 2). A halo sign of 1.6% was an uncommon finding in the present study, while other studies reported 11.1 to 64% [15, 20, 29]. The cumulative rate of the present study and other studies estimated a 41% rate of halo sign (Table 3). In addition, a similar situation occurred with crazy-paving as an uncommon finding in the pres- ent study which varied from 1.4% in this study to 12–89% in other studies [18–20, 24]. Materials and methods The meta-analysis of the present study and other studies provided a rate of 32% for this finding. Discussion Most CT findings in the present study showed no significant difference between genders and age groups, except for peripheral distribution, round opacities, single lobe, and pleural effusion, which showed a significant difference between the age groups under and over 50 years old. In a study by Song et al., ground glass opacity and consolidative opacities were significantly dif- ferent between age groups under and over 50 years old [28]. CT images with yielded 98% sensitivity play an important role in the diagnosis and evaluation of COVID-19 and can be used as a standard method for rapid diagnosis of COVID-19 to manage patients [4–16]. Discussion Keyvan Heydari: data collection, writing of the paper, final approval. Reza Alizadeh-Navaei: study design, data analysis, writing of the paper, final approval. Mean age of participants was 51.2 ± 14.8 years. The male/ female sex ratio was 1.38. Similar to the results of the present study, in a study conducted on 1099 patients in China, the mean age of patients was 47 years, and the same sex ratio was observed [9]. Compliance with ethical standards The present study utilized an acceptable sample size which is one of the strengths of the present study. One of the weak- nesses of the study is the lack of diagnostic confirmation by RT-PCR test, which was not possible for all patients due to the epidemic that occurred and the limited number of tests in Iran at the beginning of the epidemic. Other limitations of the study include the lack of a standard reporting format for the chest CT scans in these patients. Ethics declarations The study protocol was approved by the Ethics Committee of Mazandaran University of Medical Sciences (IR.MAZUMS.REC.1398.1425). Conflict of interest The authors declare that they have no conflict of interest. Discussion The present study was conducted on 552 patients with clinical suspicion who referred for HRCT and had signs of Table 2 Inconsistent findings in HR CT scan of symptomatic patients infected with coronavirus 2019 Findings Total n = 552 Frequency (%) Female n = 235 Frequency (%) Male n = 317 Frequency (%) p value Age ≤50 n = 271 Frequency (%) Age > 50 n = 281 Frequency (%) p value Tree-in-bud capacities 7 (1.3) 2 (0.9) 5 (1.6) 0.704 2 (0.7) 5 (1.8) 0.451 Centrilobular distribution 5 (0.9) 2 (0.9) 3 (0.9) 1 1 (0.4) 4 (1.4) 0.373 Peribronchovascular distribution 42 (7.6) 22 (9.4) 20 (6.3) 0.196 19 (7) 23 (8.2) 0.633 Predominantly nodular opacities 12 (2.2) 7 (3) 5 (1.6) 0.377 5 (1.8) 7 (2.5) 0.772 Cavitation 6 (1.1) 2 (0.9) 4 (1.3) 1 1 (0.4) 5 (1.8) 0.217 Lymphadenopathy 28 (5.1) 15 (6.4) 13 (4.1) 0.244 11 (4.1) 17 (6) 0.335 Pleural effusion 42 (7.6) 21 (8.9) 21 (6.6) 0.333 14 (5.2) 28 (10) 0.037 Table 2 Inconsistent findings in HR CT scan of symptomatic patients infected with coronavirus 2019 Emerg Radiol Table 3 CT scan findings in different study and pooled estimation of these findings Author Sample size Ground- glass opacity Peripheral distribution Pleural effusion Lymphadenopathy Halo sign Crazy- paving Guan, WJ [9] 975 56.4 Wu, J [14] 80 91.0 Li, Y [15] 51 90.2 2.0 0.0 21.5 Bai, HX [17] 424 91.0 80.0 4.1 2.7 Chung, M [18] 21 87.0 33.0 19.0 Guan, CS [19] 47 100.0 93.6 89.4 Han, R [20] 108 60.0 90.0 64.0 40.0 Liu, H [21] 59 72.0 98.0 Liu, KC [22] 73 59.0 3.4 Shi, H [23] 81 65.0 54.0 Xu, X [24] 90 72.0 12.0 Zhao, W [25] 101 86.1 87.1 Zhu, W [26] 32 47.0 Li, K [27] 56 80.4 87.5 8.9 0.0 Song, F [28] 51 77 8 6 Zhou, S [29] 62 77.4 9.7 Yoon, SH [30] 9 11.1 Majidi, H (Present) 552 87.3 82.4 7.6 5.1 1.6 1.4 Pooled estimation (CI95%) with meta-analysis 67 (58–76) 79 (66–92) 7 (−24–39) 4 (−26–35) 41 (11–72) 32 (4–59) Table 3 CT scan findings in different study and pooled estimation of these findings collection, writing of the paper, final approval. Fanaz Godazandeh: data collection, writing of the paper, final approval. Roya Gasemian: data collection, writing of the paper, final approval. Conclusion Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W, Lu R, Niu P, Zhan F, Ma X, Wang D, Xu W, Wu G, Gao GF, Tan W, China Novel Coronavirus Investigating and Research Team (2020) A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med 382(8):727–733. https://doi.org/10. 1056/NEJMoa2001017 18. Chung M (2020) CT imaging features of 2019 novel coronavirus (2019-nCoV). Radiology. 295(1):202–207. https://doi.org/10. 1148/radiol.2020200463 5. Su S, Wong G, Shi W, Liu J, Lai AC, Zhou J et al (2016) Epidemiology, genetic recombination, and pathogenesis of coronaviruses. Trends Microbiol 24(6):490–502. https://doi.org/ 10.1016/j.tim.2016.03.003 19. Guan CS, Lv ZB, Yan S, Du YN, Chen H, Wei LG, et al (2020) Imaging features of coronavirus disease 2019 (COVID-19): evalu- ation on thin-section CT. 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Liu KC, Xu P, Lv WF, Qiu XH, Yao JL, Gu JF, Wei W (2020) CT manifestations of coronavirus disease-2019: a retrospective analysis of 73 cases by disease severity. Eur J Radiol 126:108941. https:// doi.org/10.1016/j.ejrad.2020.108941 8. Rodriguez-Morales AJ, Cardona-Ospina JA, Gutiérrez-Ocampo E, Villamizar-Peña R, Holguin-Rivera Y, Escalera-Antezana JP, Alvarado-Arnez LE, Bonilla-Aldana DK, Franco-Paredes C, Henao-Martinez AF, Paniz-Mondolfi A, Lagos-Grisales GJ, Ramírez-Vallejo E, Suárez JA, Zambrano LI, Villamil-Gómez WE, Balbin-Ramon GJ, Rabaan AA, Harapan H, Dhama K, Nishiura H, Kataoka H, Ahmad T, Sah R, Latin American Network of Coronavirus Disease 2019-COVID-19 Research (LANCOVID-19) (2020) Clinical, laboratory and imaging features of COVID-19: a systematic review and meta-analysis. Travel Med Infect Dis 101623:101623. https://doi.org/10.1016/j.tmaid.2020. 101623 23. Conclusion 1. Li H, Zhou Y, Zhang M, Wang H, Zhao Q, Liu J (2020) Updated approaches against SARS-CoV-2. Antimicrob Agents Chemother. https://doi.org/10.1128/AAC.00483-20 Compared to other studies, the findings of the present study reported the peripheral distribution and ground glass opacity as important findings in the study population and by summa- rizing the results of studies, it can be concluded that peripheral distribution is the most common finding in these patients. 2. Chung M, Bernheim A, Mei X, Zhang N, Huang M, Zeng X, Cui J, Xu W, Yang Y, Fayad ZA, Jacobi A, Li K, Li S, Shan H (2020) CT imaging features of 2019 novel coronavirus (2019-nCoV). Radiology. 295(1):202–207. https://doi.org/10.1148/radiol. 2020200230 3. Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, Wang W, Song H, Huang B, Zhu N, Bi Y, Ma X, Zhan F, Wang L, Hu T, Zhou H, Hu Z, Zhou W, Zhao L, Chen J, Meng Y, Wang J, Lin Y, Yuan J, Xie Z, Ma J, Liu WJ, Wang D, Xu W, Holmes EC, Gao GF, Wu G, 3. Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, Wang W, Song H, Huang B, Zhu N, Bi Y, Ma X, Zhan F, Wang L, Hu T, Zhou H, Hu Z, Zhou W, Zhao L, Chen J, Meng Y, Wang J, Lin Y, Yuan J, Xie Z, Ma J, Liu WJ, Wang D, Xu W, Holmes EC, Gao GF, Wu G, Author contributions Hadi Majidi: study design, data collection, writing of the paper, final approval. Elham-Sadat Bani-Mostafavi: data collec- tion, writing of the paper, final approval. Zahra Mardanshahi: data Emerg Radiol pictorial review. Eur Radiol. https://doi.org/10.1007/s00330-020- 06801-0 Chen W, Shi W, Tan W (2020) Genomic characterisation and ep- idemiology of 2019 novel coronavirus: implications for virus ori- gins and receptor binding. Lancet 395(10224):565–574. https://doi. org/10.1016/S0140-6736(20)30251-8 Chen W, Shi W, Tan W (2020) Genomic characterisation and ep- idemiology of 2019 novel coronavirus: implications for virus ori- gins and receptor binding. Lancet 395(10224):565–574. https://doi. org/10.1016/S0140-6736(20)30251-8 17. Bai HX, Hsieh B, Xiong Z, Halsey K, Choi JW, Tran TML, Pan I, Shi LB, Wang DC, Mei J, Jiang XL, Zeng QH, Egglin TK, Hu PF, Agarwal S, Xie F, Li S, Healey T, Atalay MK, Liao WH (2020) Performance of radiologists in differentiating COVID-19 from viral pneumonia on chest CT. Radiology. 200823:200823. https://doi. org/10.1148/radiol.2020200823 4. Conclusion Shi H, Han X, Jiang N, Cao Y, Alwalid O, Gu J, Fan Y, Zheng C (2020) Radiological findings from 81 patients with COVID-19 pneumonia in Wuhan, China: a descriptive study. Lancet Infect Dis 20(4):425–434. https://doi.org/10.1016/S1473-3099(20) 30086-4 24. Xu X, Yu C, Qu J, Zhang L, Jiang S, Huang D, Chen B, Zhang Z, Guan W, Ling Z, Jiang R, Hu T, Ding Y, Lin L, Gan Q, Luo L, Tang X, Liu J (2020) Imaging and clinical features of patients with 2019 novel coronavirus SARS-CoV-2. Eur J Nucl Med Mol Imaging 47(5):1275–1280. https://doi.org/10.1007/s00259-020- 04735-9 9. 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Radiology. 295(1):210–217. https://doi.org/10.1148/radiol. 2020200274 13. Conclusion Sun D, Li H, Lu XX, Xiao H, Ren J, Zhang FR, Liu ZS (2020) Clinical features of severe pediatric patients with coronavirus dis- ease 2019 in Wuhan: a single center's observational study. World J Pediatr 16:251–259. https://doi.org/10.1007/s12519-020-00354-4 29. Zhou S, Wang Y, Zhu T, Xia LCT features of coronavirus disease 2019 (COVID-19) pneumonia in 62 patients in Wuhan, China. AJR Am J Roentgenol 2020:1–8. https://doi.org/10.2214/AJR.20.22975 g 14. Wu J, Wu X, Zeng W, Guo D, Fang Z, Chen L, Huang H, Li C (2020) Chest CT findings in patients with corona virus disease 2019 and its relationship with clinical features. Investig Radiol 55:257– 261. https://doi.org/10.1097/rli.0000000000000670 g p g 30. Yoon SH, Lee KH, Kim JY, Lee YK, Ko H, Kim KH et al (2020) Chest radiographic and CT findings of the 2019 novel coronavirus disease (COVID-19): analysis of nine patients treated in Korea. Korean J Radiol 21(4):494–500. https://doi.org/10.3348/kjr.2020. 0132 15. Li Y, Xia L (2020) Coronavirus disease 2019 (COVID-19): role of chest CT in diagnosis and management. AJR Am J Roentgenol 214:1280–1286. https://doi.org/10.2214/AJR.20.22954 Publisher’s note Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional affiliations. 16. Ye Z, Zhang Y, Wang Y, Huang Z, Song B (2020) Chest CT manifestations of new coronavirus disease 2019 (COVID-19): a Figures Figure 1 Age distribution of patients with coronavirus 2019 Figures Figure 1 Age distribution of patients with coronavirus 2019 Fi 2 Figure 2 a Bilateral and anterior to posterior peripheral ground glass opacities (white arrows). b Ground glass opacity in upper lobe (black arrow) and consolidation in lower lobe of right lung (red arrow). c Bilateral and peripheral ground glass opacities (white arrows). d Consolidation in right lower lobe (black arrow). e Right-sided pleural effusion (red arrow). f Bilateral and anterior to posterior peripheral ground glass opacities (white arrows) with peribronchovascular distribu- tion in right middle lobe (black arrow). g Figure 2 Bilateral and anterior to poste- rior peripheral ground glass opacities (black arrows). h Small lymphade- nopathies in middle mediastinum (white arrow) Figure 2 a Bilateral and anterior to posterior peripheral ground glass opacities (white arrows). b Ground glass opacity in upper lobe (black arrow) and consolidation in lower lobe of right lung (red arrow). c Bilateral and peripheral ground glass opacities (white arrows). d Consolidation in right lower lobe (black arrow). e Right-sided pleural effusion (red arrow). f Bilateral and anterior to posterior peripheral ground glass opacities (white arrows) with peribronchovascular distribu- tion in right middle lobe (black arrow). g Bilateral and anterior to poste- rior peripheral ground glass opacities (black arrows). h Small lymphade- nopathies in middle mediastinum (white arrow) Bilateral and anterior to poste- rior peripheral ground glass opacities (black arrows). h Small lymphade- nopathies in middle mediastinum (white arrow) Bilateral and anterior to poste- rior peripheral ground glass opacities (black arrows). h Small lymphade- nopathies in middle mediastinum (white arrow) Bilateral and anterior to poste- rior peripheral ground glass opacities (black arrows). h Small lymphade- nopathies in middle mediastinum (white arrow)
https://openalex.org/W1548518775	https://www.intechopen.com/citation-pdf-url/43265	English	null	The Application of Piezoelectric Materials in Machining Processes	InTech eBooks	2,013	cc-by	4,302	Additional information is available at the end of the chapter http://dx.doi.org/10.5772/54579 Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact book.department@intechopen.com Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com Open access books available Countries delivered to Contributors from top 500 universities International authors and editors Our authors are among the most cited scientists Downloads We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 14% 191,000 210M TOP 1% 154 7,200 Chapter 3 © 2013 Assarzadeh and Ghoreishi; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Application of Piezoelectric Materials in Machining Processes Saeed Assarzadeh and Majid Ghoreishi Additional information is available at the end of the chapter Additional information is available at the end of the chapter 1. Introduction In 1880, Jacques and Pierre Curie discovered that pressure generates electrical charges in a number of crystals such as Quartz and Tourmaline, calling this phenomenon the “piezoelectric effect”. Later, they noticed that electrical fields can also deform piezoelectric materials, showing an inverse effect comparing to their first observation. Nowadays, the effect has gained considerable practical attractions in miscellaneous industrial applications including machin‐ ing processes as a broad range within manufacturing methods in our competitively global technologies. This chapter focuses mainly on the usages of piezoelectric materials in two differently common practiced machining operations adopted by both manufacturers and research scholars. The first study pays attention to the role of piezoelectric transducers as the core system in producing ultrasonic waves in ultrasonic machines. Ultrasonic machining (USM) process, as one of the popular nontraditional machining processes, is capable of chip removal from every brittle material, whether conductive or nonconductive, susceptible to failure under mechanical loads in conventional machining processes of whatsoever hardness, such as ceramics, glass, porcelains, etc. The viability and effectiveness of piezoelectric transformers with high rate of electro-mechanical conversion compared to their old magnetostrictive counterparts are described and analyzed. The USM process capabilities and applications are also succinctly introduced. The second is the application of quartz as a piezoelectric material in dynamometers used to measure forces and torques during conventional machining processes, like turning, milling, drilling, and so on. The basic principles and features of how a piezoelectric-based dynamome‐ ter works are discussed along with the need to measure forces and torques through dynamometry. Piezoelectric Materials and Devices- Practice and Applications 58 2. Ultrasonic machining (USM) of materials Ultrasonic machining is an economically viable operation by which a hole or a cavity can be pierced in hard and brittle materials, whether electric conductive or not, using an axially oscillating tool. The tool oscillates with small amplitude of 10-15 µm at high frequencies of 18-40 KHz to avoid unnecessary noise and being above the upper frequency limit of the human ear, justifying the tem “ultrasonic” [1, 2]. During tool oscillation, abrasive slurry (B4C and SiC) is continuously fed into the working gap between the oscillating tool and the stationary WP. The abrasive particles are, therefore, hammered by the tool into the WP surface, and consequently abrading the WP into a conjugate image of the tool form. Moreover, the tool imposes a static pressure ranging from 1N to some kilograms depending on the size of the tool tip, see Fig. 1. This static pressure is necessary to sustain the tool feed during machining. Owing to the fact that the tool oscillates and moves axially, USM is not limited to the production of circular holes. The tool can be made to the shape required, and hence extremely complicated shapes can be produced in hard materials. Beside machining domain, US techniques are applied in nondestructive testing (NDT), welding, and surface cleaning, as well as diagnostic and medical applications. Figure 1. Characteristics of the USM process [1] Figure 1. Characteristics of the USM process [1] 2.1. Elements of process The USM equipment shown in Fig. 2 has a table capable of orthogonal displacement in X and Y directions, and a tool spindle and carrying the oscillating system moving in direction Z The Application of Piezoelectric Materials in Machining Processes http://dx.doi.org/10.5772/54579 59 perpendicular to the X-Y plane. The machine is equipped with a HF generator of a rating power of 600 W, and a two-channel recording facility to monitor important machining variables (tool displacement Z and oscillation amplitude ξ). A centrifugal pump is used to supplement the abrasive slurry into the working zone. Fig. 3 shows schematically the main elements of the equipment, which consist of the oscillating system, the tool feeding mechanism, and the slurry system. equipment, which consist of the oscillating system, the tool feeding mechanism, and the slurry system. Figure 2. USM equipment [1] Figure 3. The schematic of complete vertical USM equipment [1] 2 1 1 O ill i d i i ff y Figure 2. USM equipment [1] Figure 3. The schematic of complete vertical USM equipment [1] Figure 3. The schematic of complete vertical USM equipment [1] 2.1.1. Oscillating system and magnetostriction effect 2.1.1. Oscillating system and magnetostriction effect The core element of each US machine, the oscillating system, includes a transducer in the acoustic head, a primary horn, and a secondary acoustic horn (see Fig. 4). Piezoelectric Materials and Devices- Practice and Applications 60 Figure 4. The oscillating system of USM equipment [1] Figure 4. The oscillating system of USM equipment [1] Figure 4. The oscillating system of USM equipment [1] 2.1.2. Acoustic transducer This transforms electrical energy to mechanical energy in the form of oscillations. Magneto‐ strictive transducers are generally employed in USM, but piezoelectric ones may also be used. The magnetostriction effect was first discovered by Joule in 1874. According to this effect, in the presence of an applied magnetic field, ferromagnetic metals and alloys change in length. The deformation can be positive or negative, depending on the ferromagnetic material. An electric signal of US-frequency fr is fed in to a coil that is wrapped around a stack made of magnetostrictive material (iron-nickel alloy). This stack is made of laminates to minimize eddy current and hysteresis losses; moreover, it must be cooled to dissipate the generated heat (Fig. 3a). The alternating magnetic field produced by the HF-ac generator causes the stack to expand and contract at the same frequency. To achieve the maximum magnetostriction effect, the HF-ac current i must be superimposed on an appropriate dc premagnetizing current Ip that must be exactly adjusted to attain an optimum or working point. This point corresponds to the inflection point (d2ε/dl2=0) of the magnetostriction curve, (Fig 3b). Without the application of premagnetizing direct current I p, it is evident that the magnetostriction effect occurs in the same direction for a given ferro‐ magnetic material irrespective of the field polarity, and hence the deformation will vary at twice the frequency 2fr of the oscillating current providing the magnetic field (Fig. 3b). Therefore, the premagnetizing direct current Ip has the following functions: To achieve the maximum magnetostriction effect, the HF-ac current i must be superimposed on an appropriate dc premagnetizing current Ip that must be exactly adjusted to attain an optimum or working point. This point corresponds to the inflection point (d2ε/dl2=0) of the magnetostriction curve, (Fig 3b). Without the application of premagnetizing direct current I magnetostriction curve, (Fig 3b). Without the application of premagnetizing direct current I p, it is evident that the magnetostriction effect occurs in the same direction for a given ferro‐ magnetic material irrespective of the field polarity, and hence the deformation will vary at twice the frequency 2fr of the oscillating current providing the magnetic field (Fig. 3b). Therefore, l = n 2 λ = λ 2 , if n =1 and λ = c f r = 1 f r E ρ where l = n 2 λ = λ 2 , if n =1 and and where where c = acoustic speed in magnetostrictive materials (m/s) c = acoustic speed in magnetostrictive materials (m/s) fr = resonant frequency (1/s) fr = resonant frequency (1/s) E, ρ = Young’s modulus (MPa) and density (kg/m3) of the magnetostrictive material Hence, Hence, l = c 2 f r = 1 2 f r E ρ l = c 2 f r = 1 2 f r E ρ 2.1.4. Piezoelectric transducers 2.1.2. Acoustic transducer Therefore, the premagnetizing direct current Ip has the following functions: • When precisely adjusted, it provides the maximum magnetostriction effect (maximum oscillating amplitude) • When precisely adjusted, it provides the maximum magnetostriction effect (maximum oscillating amplitude) • It prevents the frequency doubling phenomenon If the frequency of the ac signal, and hence that of the magnetic field, is tuned to be the same as the natural frequency of the transducer (and the whole oscillating system), so that it will be at mechanical resonance, then the resulting oscillation amplitude becomes quite large and the exciting power attains its maximum value. The Application of Piezoelectric Materials in Machining Processes http://dx.doi.org/10.5772/54579 61 The Application of Piezoelectric Materials in Machining Processes http://dx.doi.org/10.5772/54579 61 2.1.3. Transducer length 2.1.3. Transducer length The resonance condition is realized if the transducer length is l, which is equal to half of the wave length, λ (or positive integer number n of it). Therefore, 2.1.4. Piezoelectric transducers A main drawback of magnetostriction transducers is the high power loss (η = 55%). The power loss is converted into heat, which necessitates the cooling of the transducer. In contrast, piezoelectric transducers are more efficient (η = 90%), even at higher frequencies (f = 25-40 KHz). Piezoelectric transducers utilize crystals like quartz and lead titanate-zirconate that undergoe dimensional changes proportional to the voltage applied. Similar to magnetostric‐ tors, the length of crystal should be equal to half the wavelength of the sound in the crystal to produce resonant condition. At a frequency of 40 KHz, the resonant length l of the quartz crystal (E = 5.2 ×104 MPa, ρ = 2.6 × 103 kg/m3) is equal to 57 mm. Sometimes a polycrystalline ceramic like barium titanate is used. Piezoelectric transformers were first introduced into modern ultrasonic machines in the late 1960s. In 1970, Tyrrell [3] has described such a system. In essence, piezoelectric transducers are composed of small particles bound together by sintering; undergoing polarization by heating above the Curie point and placing it in an electric field such that orientation is preserved on cooling. A disc of the piezoelectric material which has a very high electrome‐ chanical conversion rating is sandwiched between two thick metal plates to form the ultrasonic horn. When a current of fixed frequency is fed to the horn the whole system is found to vibrate at some resonant frequency along the longitudinal axis; acoustically the motion is equal to one half a wavelength. Piezoelectric Materials and Devices- Practice and Applications 2 Advantages Some of the special priorities can be mentioned as follow: Some of the special priorities can be mentioned as follow: • Intricate and complex shapes and cavities in both electric and nonelectric materials can be readily machined ultrasonically • As the tool exhibits no rotational movement, the process is not limited to produce circular holes • High dimensional accuracy and surface quality • Especially, in the sector of electrically nonconductive materials, the USM process is not in competition with other nontraditional machining processes regarding accuracy and removal rates • Since there is no temperature rise of the WP, no changes in physical properties or micro‐ structure whatsoever can be expected However, the USM process has some disadvantages listing below. Disadvantages: • The USM is not capable of machining holes and cavities with a lateral extension of more than 25-30 mm with a limited depth of cut • The tool suffers excessive frontal and side wear when machining conductive materials such as steels and carbides. The side wear destroys the accuracy of holes and cavities, leading to a considerable conicity error. • Every job needs a special high-cost tool, which adds to the machining cost • High rate of power consumption • In case of blind holes, the designer should not allow sharp corners, because these cannot be produced by the USM. 2.3. The applications of USM It should be understood that the USM is generally applied to machining shallow cavities and forms in hard and brittle materials having a surface area not more than 10 cm2. Some typical applications of USM are as follow: It should be understood that the USM is generally applied to machining shallow cavities and forms in hard and brittle materials having a surface area not more than 10 cm2. Some typical applications of USM are as follow: • Manufacturing forming dies in hardened steel and sintered carbides • Manufacturing wire drawing dies, cutting nozzles for jet machining applications in sapphire, and sintered carbides • Slicing hard brittle materials such as glass, ceramics, and carbides • Coining and engraving applications The Application of Piezoelectric Materials in Machining Processes http://dx.doi.org/10.5772/54579 63 The Application of Piezoelectric Materials in Machining Processes http://dx.doi.org/10.5772/54579 63 • Boring, sinking, blanking,, and trepanning • Thread cutting in ceramics by rotating the tool or the WP Figure 5 illustrates some of the products produced ultrasonically. a. Engraving a medal made of agate b. Piercing and blanking of glass c. Producing a fragile graphite electrode for EDM d. Sinking a shearing die in hardened steel or WC e. Production of outside contour and holes of master cutters made of zirconium oxide (ZrO2) of a textile machine • Boring, sinking, blanking,, and trepanning Figure 5 illustrates some of the products produced ultrasonically. e. Production of outside contour and holes of master cutters made of zirconium oxide (ZrO2) of a textile machine f. Drilling fine holes Ф = 0.4 mm in glass Figure 5. Some typical products by USM [1] Figure 5. Some typical products by USM [1] Figure 5. Some typical products by USM [1] 3. Dynamometry in conventional machining processes During machining, the cutting tool exerts a force on the WP as it removes the machining allowance in the form of chips. Empirical values for estimating the cutting forces are no longer sufficient to reliably establish the optimum machining conditions. Depth of cut, feed rate, cutting speed, WP materials, tool material and geometry, and cutting fluid are just a few of the machining parameters governing the amplitude and direction of the cutting force. The optimization of a machining process necessitates accurate measurement of the cutting force by a special device called a machine tool dynamometer, capable of measuring the components of the cutting force in a given coordinate system. It is a useful and powerful tool employed in a variety of applications in engineering research and manufacturing. A few examples of these applications are [1]: Piezoelectric Materials and Devices- Practice and Applications 64 • Investigating into the machinability of materials • Comparing similar materials from different sources • Comparing and selecting cutting tools • Determining optimum machining conditions • Analyzing causes of tool failure • Investigating the most suitable cutting fluids • Determining the conditions that yield the best surface quality • Establishing the effect og fluctuating cutting forces on tool wear and tool life The machine tool dynamometer is not standard equipment or a device that can be used on every machine. Rather, it is equipment especially designed to fulfill some desired requirements that adapt a specific machine type operating at a specific range of machining conditions. • High rigidity, hence high resonant frequency 3.1. Piezoelectric (Quartz) dynamometers The outstanding features of quartz dynamometers are [1, 6]: • High rigidity, hence high resonant frequency The Application of Piezoelectric Materials in Machining Processes http://dx.doi.org/10.5772/54579 65 • Minimal deflections (few micrometers at full load) • Wide measuring range • Linear characteristics, free of hysteresis Linear characteristics, free of hysteresis • Lowest cross talk (typically under 1%) • Simple in operation and without need for bridge balancing • Compact design • Unlimited life expectancy • Lowest cross talk (typically under 1%) • Simple in operation and without need for bridge balancing Figure 6. Disks of quartz crystals. (a) Pressure-sensitive, (b) shear-sensitive, (c) multi-components in reference to a Car‐ tesian coordinate system [6] Figure 6. Disks of quartz crystals. (a) Pressure-sensitive, (b) shear-sensitive, (c) multi-components in reference to a Car‐ tesian coordinate system [6] 3.1. Piezoelectric (Quartz) dynamometers Of the numerous piezoelectric materials, quartz is by far the most suitable one for force measurement, because it is stable material with constant properties. In its crystalline form, quartz is anisotropic, in that its material properties are not identical in all directions. Depending on the position in which they are cut out of the crystal, disks are obtained that are: 1. Sensitive only to pressure (longitudinal effect), Fig. 6a, which measure the main force component Fz (brown) 1. Sensitive only to pressure (longitudinal effect), Fig. 6a, which measure the main force component Fz (brown) 2. Sensitive only to shear in one particular direction (shear effect), Fig. 6b, which measures components Fx (blue) and Fy (green), perpendicular to Fz, as well as the torque Mz (red). Figure 6c illustrates the generalized multi-components with reference to a Cartesian coordinate system. 2. Sensitive only to shear in one particular direction (shear effect), Fig. 6b, which measures components Fx (blue) and Fy (green), perpendicular to Fz, as well as the torque Mz (red). Figure 6c illustrates the generalized multi-components with reference to a Cartesian coordinate system. The piezoelectric force measuring principle differs fundamentally from their old traditional counterparts, the strain and displacement based dynamometers, in that it is an active system. When a force acts on a quartz element, a proportional electric charge appears on the loaded surfaces, meaning that it is not necessary to measure the actual deformation. In piezoelectric dynamometers, the deflection is not more than a few micrometers at full load, whereas with conventional systems, several tenths of a millimeter may be needed. Thus, piezoelectric dynamometers are very stiff systems and their resonant frequency is high, so that even rapid events can be measured satisfactorily. Moreover, the individual components of the cutting force can be measured directly, eliminating any interference between measuring channels. Quartz dynamometers require no zero adjustment or balancing of the bridge circuit. It is just a matter of pressing a button, being ready for duty. 3.2. Typical piezoelectric dynamometers Piezoelectric dynamometers are efficiently used on the majority of machine tools. Three application examples are described below with their corresponding setup. 1. Two-component piezoelectric drilling dynamometers.Figure 7 illustrates a two-component drilling dynamometer in which shear-sensitive discs are organized in a circle with their shear-sensitive axes oriented to respond to the torque Mz (red), whereas pressure- sensitive disks are arranged and oriented to measure the trust load Fz (brown). A high Piezoelectric Materials and Devices- Practice and Applications 66 preload is necessary because the shear forces must be transmitted by friction to measure the torque. The two-component dynamometer, shown in Fig. 7, is suited for operations including drilling, thread cutting, countersinking, reaming, and so on. Torques and forces acting when machining holes from less than 1 mm to over 20 mm diameter can be measured satisfactorily by this dynamometer. A record of Mz and Fz is illustrated in Fig. 7, from which it is clearly seen that Fz rises steeply at the beginning (entry of tool chisel), followed by the gradual rise of the Mz component, as the latter is more affected by the force acting on the two drill lips. 2. Three-component piezoelectric turning dynamometer. This model includes several shear- sensitive quartz, with their shear-sensitive axes oriented to measure Fx (blue ring) and Fy (green ring), respectively. Their shear sensitive-axes are inclined to each other at an angle of 900, and both are contained in a housing to form a two-component force measurement element for Fx and Fy (Figure 8). Pressure-sensitive quartz disks are contained in a single housing to form a single-component force-measuring element for Fz (brown ring). Anoth‐ er alternative is illustrated in the construction shown in Figure 8, where three separate elements for measuring Fx, Fy, and Fz are sandwiched under high preload between a base plate and a top plate. The dynamometer is mounted on the lathe slide in place of a cross- slide. A record of the three components is shown also in the same figure, from which it is clear that Fx=Fy, meaning that the cut is preferred at an approach angle χ=450. Three-component piezoelectric milling or grinding dynamometer. Whole quartz rings may be employed. Two-shear-sensitive quartz pairs, for Fx (blue) and Fy (green), and a pressure- sensitive pair for Fz (brown), can be assembled in a common housing to form a three- component force-measuring element (Figure 9). 3.2. Typical piezoelectric dynamometers The pressure-sensitive quartz are arranged in the middle so that they lie in the neutral axis under bending. During milling and grinding, the application point of the force varies a great deal. Consequently, dynamometers having four piece three-component force-measuring elements are employed. All the x, y, and z channels respectively are paralleled electrically. This makes the measurement independent of the momentary force application point. For bigger work, two dynamometers paralleled electrically and mechanically may be employed together. This system measures correctly independent of the point of force application. A typical output of the three-component milling dynamometer is shown in Fig. 9. The milling process has been performed under the following conditions: Three-component piezoelectric milling or grinding dynamometer. Whole quartz rings may be employed. Two-shear-sensitive quartz pairs, for Fx (blue) and Fy (green), and a pressure- sensitive pair for Fz (brown), can be assembled in a common housing to form a three- component force-measuring element (Figure 9). The pressure-sensitive quartz are arranged in the middle so that they lie in the neutral axis under bending. During milling and grinding, the application point of the force varies a great deal. Consequently, dynamometers having four piece three-component force-measuring elements are employed. All the x, y, and z channels respectively are paralleled electrically. This makes the measurement independent of the momentary force application point. For bigger work, two dynamometers paralleled electrically and mechanically may be employed together. This system measures correctly independent of the point of force application. A typical output of the three-component milling dynamometer is shown in Fig. 9. The milling process has been performed under the following conditions: 3. • Status: Up milling • Cutter diameter = 63 mm, helix β = 300, n = 90 rpm, Z = 12 teeth • Cutter diameter = 63 mm, helix β = 300, n = 90 rpm, Z = 12 teeth • Feed u = 53 mm/min • Feed u = 53 mm/min • Depth of cut t = 3.5 mm The severe periodic fluctuation in the measured forces is attributed to an eccentric motion of the cutter shaft. Superimposed are vibrations due to gearing of the machine. It is perfectly clear from the record that the setup shown is far from ideal. The force measure, therefore, sheds light on the machine tool behavior as well, and not just on the actual cutting operation. 4. Conclusion In this chapter, attempt has been made to cover the basic details of utilizing piezoelectric materials in two different fields of machining processes. The first focused on the effectiveness of a piezoelectric transducer in producing ultrasonic waves as a cutting tool in ultrasonic machining processes. Compared to their old magnetostriction counterparts, the piezo ones demonstrate higher electro-mechanical efficiency. Besides, they are more compact as well as being simpler in design and operation. The second emphasizes the unique suitability of piezoelectric dynamometers in measuring various components of forces and torques gener‐ ated during different kinds of conventional machining processes. The importance of measur‐ ing forces and torques in an on-line manner during every traditional machining operation for the purposes of modeling and optimization makes the use of a precise piezoelectric dyna‐ mometer an inseparable part in manufacturing industries as well as academic domains. 3.2. Typical piezoelectric dynamometers The Application of Piezoelectric Materials in Machining Processes http://dx.doi.org/10.5772/54579 67 Figure 7. Two-component piezo drilling dynamometer [6] Figure 7. Two-component piezo drilling dynamometer [6] Figure 8. Three-component piezo turning dynamometer [6] Figure 9. Three-component piezo milling dynamometer [6] Figure 7. Two-component piezo drilling dynamometer [6] Figure 8. Three-component piezo turning dynamometer [6] Figure 8. Three-component piezo turning dynamometer [6] Figure 8. Three-component piezo turning dynamometer [6] Figure 9. Three-component piezo milling dynamometer [6] Figure 9. Three-component piezo milling dynamometer [6] Piezoelectric Materials and Devices- Practice and Applications 68 Address all correspondence to: saeed_assarzadeh@yahoo.com Department of Mechanical Engineering, K. N. Toosi University of Technology, Tehran, Iran Department of Mechanical Engineering, K. N. Toosi University of Technology, Tehran, Iran Author details Saeed Assarzadeh and Majid Ghoreishi *Address all correspondence to: saeed_assarzadeh@yahoo.com References [1] Youssef Helmi A., El-Hofy, H., Machining technology: Machine tools and operations, CRC Press, (2008). 978-1-42004-339-6 [1] Youssef Helmi A., El-Hofy, H., Machining technology: Machine tools and operations, CRC Press, (2008). 978-1-42004-339-6 [2] Mcgeough, J. A. Advanced methods of machining, Chapman and Hall, London and New York, (1988). 0-41231-970-5 [2] Mcgeough, J. A. Advanced methods of machining, Chapman and Hall, London and New York, (1988). 0-41231-970-5 [3] Tyrrell, W. R. Rotary ultrasonic machining, Soc. Manufacturing Eng., USA, Paper (1970). (MR70-516), 1-10. [3] Tyrrell, W. R. Rotary ultrasonic machining, Soc. Manufacturing Eng., USA, Paper (1970). (MR70-516), 1-10. [4] El-Hofy, H. Advanced machining processes: Nontraditional and hybrid machining processes, McGraw Hill Book Com., (2005). 0-07145-334-2 [4] El-Hofy, H. Advanced machining processes: Nontraditional and hybrid machining processes, McGraw Hill Book Com., (2005). 0-07145-334-2 [5] Pandey, P. C, & Shan, H. S. Modern machining processes, Tata McGraw Hill Book Com., (2004). 0-07096-553-6 [5] Pandey, P. C, & Shan, H. S. Modern machining processes, Tata McGraw Hill Book Com., (2004). 0-07096-553-6 [6] http://www.Kistler.com
https://openalex.org/W2062673203	http://cds.cern.ch/record/1321592/files/JINST5.C12044.pdf	English	null	First experiences with the LHC BLM sanity checks	Journal of instrumentation	2,010	cc-by	2,791	Home Search Collections Journals About Contact us My IOPscience 1Corresponding author. First experiences with the LHC BLM sanity checks This article has been downloaded from IOPscience. Please scroll down to see the full text article. 2010 JINST 5 C12044 (http://iopscience.iop.org/1748-0221/5/12/C12044) View the table of contents for this issue or go to the journal homepage for more Please note that terms and conditions apply. RECEIVED: October 15, 2010 ACCEPTED: November 27, 2010 PUBLISHED: December 20, 2010 RECEIVED: October 15, 2010 ACCEPTED: November 27, 2010 PUBLISHED: December 20, 2010 TOPICAL WORKSHOP ON ELECTRONICS FOR PARTICLE PHYSICS 2010, 20–24 SEPTEMBER 2010, AACHEN, GERMANY 2010 JINST 5 C1204 c⃝2010 IOP Publishing Ltd and SISSA First experiences with the LHC BLM sanity checks JINST 5 C12044 J. Emery,1 B. Dehning, E. Efﬁnger, A. Nordt, M.G. Sapinski and C. Zamantzas CERN, CH-1211 Gen`eve 23, Switzerland E-mail: Jonathan.emery@cern.ch 3 Non-conformities 3 Non-conformities JINST 5 C12044 1 LHC BLM overview The Beam Loss Monitoring (BLM) system for the Large Hardron Collider (LHC) has been de- signed to protect the machine equipments against unintended energy deposition by losses espe- cially on the superconducting magnets. When a partial loss of the proton beam around the ring exceeds a predetermined threshold, the system sends a beam abort request, which will result in a complete dump of both beams. In addition to the protective functionality, the system is used as measurement instrument for various studies and ﬁne tuning of the accelerator. E-mail: Jonathan.emery@cern.ch ABSTRACT: The reliability concerns have driven the design of the Large Hardron Collider (LHC) Beam Loss Monitoring (BLM) system from the early stage of the studies up to the present com- missioning and the latest development of diagnostic tools. To protect the system against non- conformities, new ways of automatic checking have been developed and implemented. These checks are regularly and systematically executed by the LHC operation team to ensure that the system status is after each test ”as good as new”. The sanity checks are part of this strategy. They are testing the electrical part of the detectors (ionisation chamber or secondary emission detector), their cable connections to the front-end electronics, further connections to the back-end electronics and their ability to request a beam abort. During the installation and in the early commissioning phase, these checks have shown their ability to ﬁnd also non-conformities caused by unexpected failure event scenarios. In every day operation, a non-conformity discovered by this check inhibits any further injections into the LHC until the check conﬁrms the absence of non-conformities. KEYWORDS: Control and monitor systems online; Beam-line instrumentation (beam position and proﬁle monitors; beam-intensity monitors; bunch length monitors) doi:10.1088/1748-0221/5/12/C12044 Contents Contents 1 LHC BLM overview 1 1.1 Hardware 1 1.2 Reliability 2 2 Sanity checks 2 2.1 Internal and external beam permit check 3 2.2 Connectivity check 3 2.3 Optimization of the connectivity check 3 2.4 Reproducibility of measurements 4 3 Non-conformities 4 4 Summary 6 1 1 2 2 3 3 3 4 4 6 2010 JINST 5 C120 1.1 Hardware There are about 4000 monitors around the 27km of the LHC tunnel ring. The most wildly used detector type is the ionisation chamber, which is supplied by high voltage and produces a current proportional to the received particles with a high dynamic range (10e9). The signal of 8 of these detectors is integrated and digitalized every 40µs by the Current to Frequency card (BLECF) [1] with a dynamic range of about 10e8 (10pA to 1mA). The resulting data are transmitted to the surface through a redundant optical link to the Threshold Comparators card (BLETC) [2]. This card produces longer integration windows up to 80s and continuously compares these values to predeﬁned thresholds. Additionally, this card processes the maximum of these running sums every second for display and long term logging. In the case of losses above the thresholds, the card inhibits the beam permit signal, which is sent to the Combiner and Survey card (BLECS) and transmitted further to other systems to abort the beam. This last card also takes care of various checks sequences including the sanity checks. The ﬁgure 1 gives a complete hardware overview from the ionisation chambers placed in the tunnel (left) to the external systems (right). Users of the produced information like databases – 1 – Figure 1. Overview of the LHC BLM hardware system. 2010 JINST 5 C1204 Figure 1. Overview of the LHC BLM hardware system. or operational applications are connected to the front-end CPU (top-right). The beam interlock interfaces and the beam energy receiver are connected to the BLECS (bottom-right). JINST 5 C12044 1.2 Reliability The reliability concerns have driven the design and implementation of the LHC BLM system. The PhD thesis of G. Guaglio [3] has introduced the calculation of the failure rate of the main compo- nents. Based on this ﬁrst results, failure scenarios have been identiﬁed and strategies to minimize them have been developed. The component reception procedures, installation and yearly mainte- nance, regular checks and continuous checks are the areas where various actions have been taken. The focus of this paper is on the regular checks of the hardware, combined under the name sanity checks. The idea is to reduce as much as possible the probability of a failure event leading to an incident by detecting non conformities on the system before a problematic situation occurs. To achieve this goal, the system should be checked as often as possible without reducing signiﬁcantly the availability (the system is unavailible when being checked). The regular check of the settings, called online check [4], which ensures the consistency between settings held inside the front-end and in the LHC Software Architecture (LSA) database will not be covered. To enforce the trigger- ing of these procedures, hardware timers are implemented. If not triggered at least every 24h, the next injection in the LHC will be blocked through the beam permit lines connecting to the Beam Interlock System (BIS). 2 Sanity checks As previously introduced, the sanity checks are the procedures which are regularly running to ensure that the hardware is in a deﬁned state, i.e. “as good as new”. In case of a detected non- conformity, the beam permit is not given back and no injections in the LHC can be performed. The internal beam permit check and the connectivity check [5] are the different parts of this procedure. – 2 – Figure 2. The Internal Beam Permit Check (IBPC) tests the beam permit links of the BLM system; the External Beam Permit Check (EBPC) tests the connections from the BLM to the interlock system. Figure 2. The Internal Beam Permit Check (IBPC) tests the beam permit links of the BLM system; the External Beam Permit Check (EBPC) tests the connections from the BLM to the interlock system. 2010 JINST 5 C120 2.2 Connectivity check The primary purpose of this check is to ensure the integrity of the cabling of each beam loss detec- tor. By adding a small harmonic modulation signal (0.06Hz, 30V) on the high voltage supply of the detector (1500V), it is possible to detect a small current on the measurement side. If anywhere in the signal chain a cable is missing, disconnected or discontinued for any reason, the measurement will not show any harmonic variation of the current. The second goal is to survey the integrity of the components. The measured amplitude and phase of every channel (detector or spare) is compared to a predeﬁned threshold measured for every channel. If one of them is outside the limits, the beam will not be permitted in the LHC. This procedure takes place in the Combiner card FPGA. For this purpose the acquisitions data are written by the front-end CPU onto the memory of the FPGA. In the FPGA digital ﬁltering is applied, as well as amplitude and phase recognition and ﬁnally threshold comparisons against the predeﬁned reference database (LSA) settings. The entire signal period of each channel is also saved into the volatile memory of the card. A dedicated application has been developed to download, save and analyze the information and display channel signals. It also generates warnings when signals are getting close to the accepted limit. In addition it is possible to display individual or groups of channels for observation. 2.1 Internal and external beam permit check The Internal Beam Permit Check (IBPC) ensures that all Thresholds Comparators cards are able to request the beam dump by checking if the signal is correctly passed through the daisy chain interconnecting the cards. To perform this check, the processing has been introduced in the Field- Programmable Gate Array (FPGA) of the Combiner card. All the Threshold Comparators are requesting the beam dump one after each other and the FPGA checks that these signals are prop- agated to all the interconnected BLM crates (ﬁgure 2). The External Beam Permit Check (EBPC) tests the connection between the BLM system and the machine interlock interface; this function is under the responsibility of the Beam Interlock System (BIS). 0 JINST 5 C12044 2.4 Reproducibility of measurements To measure the reproducibility of the connectivity check, 100 executions were performed in a row. Root and python scripts were used to extract the measured data from the databases to perform statistical calculation. Figure 4 shows the results for the IC (top) and the SEM (bottom) by two di- mensional representations. The standard deviation over the 100 checks is plotted versus amplitude (left) and phase (right). As expected, the amplitude repeatability of the IC is better than for the SEM since the signal is about 10 times higher. The difference in amplitude can be explained by the differences in the equivalent circuit capacitance, 312pF (IC) and 22pF (SEM) [5]. Structures can be observed on the IC phase plot (right-top) which are due to sampling frequency (resulting by 1.4 [deg] of resolution). 2.3 Optimization of the connectivity check Since the ﬁrst presentation of this technique [5], optimisation has been applied to minimize the inﬂuence of the check to the system and improve the measurement repeatability. – 3 – Figure 3. Volatile memory data for IC (left) and SEM (right). The raw data received from the CPU (stairs) are smoothed by a low pass ﬁlter (FIR) before amplitude and phase determination. 2010 JINST 5 C120 Figure 3. Volatile memory data for IC (left) and SEM (right). The raw data received from the CPU (stairs) are smoothed by a low pass ﬁlter (FIR) before amplitude and phase determination. The harmonic amplitude excitation has been doubled to increase the signal to noise ratio on the secondary emission monitors (10 times smaller than the IC). To avoid a too long lasting neg- ative induced current on the IC (the lower part of the sinus is now truncated) the frequency of the modulation has been doubled. Systematic measurements have shown very good reproducibility even with a truncated signal and an improved signal to noise ratio with the SEM. JINST 5 C12044 The second part of the improvements concerns the digital processing. The parameters of the low pass ﬁlter were optimized to improve the smoothing of the “stairs” introduced by the signal quantiﬁcation and the rejection of unwanted parasitical frequencies (ﬁgure 3). The base offset subtraction to the calculated peak to peak amplitude has been introduced to minimize the error introduced by the variation of this base offset. C12044 3 Non-conformities During the commissioning of the LHC BLM installation in 2009 and during the early stage of the operation, multiple types of non-conformities have been detected by the connectivity check. The table 1 highlights the occurrences of these events and the resulting signal deviation from the expected behaviour. Comparing these values with the reproducibility presented in 2.4, we can conclude that they can easily be detected by this technique. – 4 – Figure 4. Connectivity check measurements for the IC (top) and SEM (Bottom). 2010 JINST 5 C1204 0 JINST 5 C12044 Figure 4. Connectivity check measurements for the IC (top) and SEM (Bottom). Table 1. Non-conformities detected with the connectivity check since spring 2009. Type of non-conformity Occurrence Modiﬁed parameter Deviation from the expected behavior IC parameters sensitivity Chamber ﬁlter badly sol- dered or disconnected 27 Phase and amplitude 9%-33% 3% and 2% Tunnel card (BLECF) non- conform behavior on one or more channel 4 Phase 10%-30% 3% Monitor not supplied with high voltage 4 Phase and amplitude Large 3% and 2% Connection of monitor on the wrong channel 3 Phase and amplitude Large 3% and 2% High voltage distribution box 1 Phase and amplitude Large 3% and 2% Table 1. Non-conformities detected with the connectivity check since spring 2009. The most frequent non-conformities are related to the chamber high voltage input ﬁlter. The soldering on the ground pin was not done as expected. Environmental condition stresses this weak point which then fails randomly. Figure 5 shows an example of this non-conformity. Following this, all the 459 spares in stock were opened for inspection and about 10% showed various soldering non- conformities. By knowing the parallel capacitor value (470nF) and the measurement repeatability (1-3%), the measure sensitivity for this particular component has been estimated to be around 10% of its initial value (i.e. 50nF). – 5 – Figure 5. Modulated signal (left-top) with a non-conformity on the HV input ﬁlter shown on the detector (left-bottom). Detector sensitivity loss due to non-conformity of the tunnel card discovered by measurements with beam (right-top) and conﬁrmed by the connectivity check (right-bottom). 2010 JINST 5 C120 JINST 5 C12044 Figure 5. Modulated signal (left-top) with a non-conformity on the HV input ﬁlter shown on the detector (left-bottom). Detector sensitivity loss due to non-conformity of the tunnel card discovered by measurements with beam (right-top) and conﬁrmed by the connectivity check (right-bottom). 3 Non-conformities A second interesting case has been identiﬁed during a beam test. A detector was identiﬁed to have lost sensitivity. Figure 5 (right-top) shows the beam loss signal for different detectors versus their physical position. The detector with a smaller signal than expected is identiﬁed with its name. Below, the modulation of this detector shows a clear difference with the others (10% phase difference). This was due to non-conformity on the tunnel board analog circuit. Such changes can accurately be measured and monitored by the connectivity check. C12044 4 Summary The sanity checks are parts of the global strategy of high reliability of the LHC BLM system. They are checking regularly the hardware and searching for non-conformities on the detector cabling and its internal parts. The link to the beam interlock system to inhibit the next injection in case of non-conformity and the integration of their executions into the LHC sequencer has been com- pleted during the shutdown 2009-2010. It ensures the regularity of their executions mandatory for the luminosity increase toward the nominal operation of the LHC. After few months of operating them on a daily basis, the stability of the procedures and the reproducibility of the measurements have been conﬁrmed. The accuracy of the connectivity check results obtained by the optimization of the parameters and the signal processing has enabled the detection of unexpected variation of components values. It extended the use of this check to the detection of non-conformity on the high voltage ﬁlter of individual ionization chambers as well as speciﬁc decrease of the sensitivity of the current to frequency card. In addition to these unexpected features, the detection of disconnected – 6 – detector has been conﬁrmed by real case in the LHC tunnel. The fast execution time of about 6 minutes has allowed these checks to become a convenient way of regularly checking the conformity of the cabling as well as multiple hardware parts of the LHC BLM system. References [1] E. Efﬁnger et al., The LHC beam loss monitoring system’s data acquisition card, talk given at the 12th Workshop on Electronics for LHC and future Experiments (LECC06), September 25–29, Valencia, Spain (2006), online at http://cdsweb.cern.ch/record/1027422/ﬁles/p108.pdf. [2] C. Zamantzas et al., An FPGA based implementation for real-time processing of the LHC beam loss monitoring system’s data, IEEE Nucl. Sci. Symp. Conf. Rec. 2 (2006) 950. 2010 JINST 5 C1204 [3] G. Guaglio, Reliability of the beam loss monitor system for the Large Hadron Collider at CERN, Ph.D. Thesis, University Clermont-Ferrand II-Blaise Pascal, Clermont-Ferrand, France (2005). [4] C. Zamantzas et al., Conﬁguration and validation of the LHC beam loss monitoring system, in the proceedings of the 9th European Workshop on Beam Diagnostics and Instrumentation for Particle Accelerators (DIPAC09), May 25–27, Basel, Switzerland (2009), online at http://dipac09.web.psi.ch/ Proceedings/papers/tupb31.pdf. 0 JINST 5 C12044 [5] J. Emery et al., LHC BLM single channel connectivity test using the standard installation, in the proceedings of the 9th European Workshop on Beam Diagnostics and Instrumentation for Particle Accelerators (DIPAC09), May 25–27, Basel, Switzerland (2009), online at http://dipac09.web.psi.ch/ Proceedings/papers/tupd26.pdf. – 7 –
https://openalex.org/W3197185438	https://www.researchsquare.com/article/rs-853188/latest.pdf	English	null	Underweight is a major risk factor for atrial fibrillation in Asian people with type 2 diabetes mellitus	Cardiovascular diabetology	2,021	cc-by	6,450	Underweight Is A Major Risk Factor of Atrial Fibrillation In Asian People With Type 2 Diabetes Mellitus Jung-Chi Hsu (  cactus146@gmail.com ) Saint Mary's Hospital Luodong Yen-Yun Yang National Taiwan University Hospital Shu-Lin Chuang National Taiwan University Hospital Yi-Wei Chung Shuang Ho Hospital Lian-Yu Lin National Taiwan University Hospital Research Article ed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License icense:   This work is licensed under a Creative Commons Attribution 4.0 Internation Version of Record: A version of this preprint was published at Cardiovascular Diabetology on November 24th, 2021. See the published version at https://doi.org/10.1186/s12933-021-01415-2. Page 1/16 Abstract Background: Atrial fibrillation (AF) is prevalent in patients with type 2 diabetes mellitus (T2DM). Obesity commonly accompanies with T2DM and increases AF incidence. However, the dose-relationship of body mass index (BMI) and the risk of AF has seldom been studied in diabetes patients. Methods: This cohort study was conducted utilizing a database from National Taiwan University Hospital, a tertiary medical center in Taiwan. Between 2014 and 2019, a total of 64339 adult patients with T2DM were enrolled for analysis. The BMI was measured and categorized into several groups including underweight (BMI <18.5), normal range (18.5≤BMI <24), overweight (24≤BMI <27), obesity class 1 (27≤BMI <30), obesity class 2 (30≤BMI <35), and obesity class 3 (BMI≥35). Multivariate Cox regression models and spline regression model were employed to estimate the relationship between BMI and the risk of AF in patients with T2DM. Results: The incidence rate of AF was 1.97 per 1000 person-year during a median follow-up period of 70.7 months. In multivariate Cox regression model, by using normal BMI as reference group, individuals with underweight (HR 1.47, 95%CI 1.19-1.81, p<0.001) was significantly associated with increased risk of AF while overweight was significantly associated with reduced risk of AF (HR 0.83, 95%CI 0.76-0.91, p<0.001). The Kaplan-Meier analysis showed that the risk of AF was highest in the underweight group, followed by obesity class 3, while the overweight group had the least incidence of AF (log-rank test, p<0.001). Cubic restrictive spline model showed a “J-shaped” or “L-shape” relationship between BMI and the risk AF. Conclusions: We found that underweight carries the highest risk of AF in Asian patients with T2DM. Study Population We evaluated the longitudinal data of Taiwanese who were diagnosed with T2DM and aged 50 years or older in a tertiary medical center between January 1, 2014 and December 31, 2019. The detailed medical records were from the National Taiwan University Hospital integrated Medical Database (NTUH-iMD) which were well-established based on the International Classification of Diseases, tenth revision codes and ATC (Anatomical Therapeutic Chemical Classification) drug codes, and regulated examination codes in Taiwan. The study was approved by the Institutional Review Board (IRB) of National Taiwan University Hospital. Patients with previous AF since the inception of the T2DM or lost to follow-up (defined as an absence of follow-up at the outpatient clinics more than three months) were excluded. Baseline characteristics including hypertension (HTN), hyperlipidemia, gout, heart failure, coronary artery disease (CAD), valvular heart disease (VHD), chronic kidney disease (CKD), chronic obstructive pulmonary disease (COPD), peripheral arterial occlusive disease (PAOD) were obtained from the electronic health records (EHRs). Estimated glomerular filtration rate (eGFR) was calculated by modification of diet in renal disease (MDRD) equation. History of transient ischemic accident (TIA) or ischemic stroke was defined as the occurrence of TIA or ischemic stroke before the diagnosis of DM, and history of heart failure was constrained to those patients who had been hospitalized because of acute decompensated heart failure. Prescription information were categorized into antiarrhythmic agents, calcium channel blocker (CCB), beta-blocker, angiotensin converting enzyme inhibitor (ACEI), angiotensin receptor blocker (ARB), mineralocorticoid-receptor antagonist (MRA), anticoagulants including direct oral anticoagulant (DOAC) and warfarin, and anti-diabetic medications including insulin, metformin, sodium-glucose co-transporter–2 (SGLT2) inhibitor, dipeptidyl peptidase 4 (DPP4) inhibitor, sulphonylurea, repaglinide, acarbose, thiazolidinedione (TZD), glucagon like peptide–1 (GLP–1) agonist. Echocardiographic studies were performed with Phillips iE33 (Phillips, Bothell, WA, USA) and two‐dimensional‐guided M‐mode measurements with a 3.0‐ or 3.5‐MHz transducer. Left atrium (LA) size, left ventricular internal dimension in end‐diastole (LVIDd) and systole (LVIDs), and left ventricular ejection function (LVEF) were collected in the parasternal long-axis view with M-mode cursor. LA size was anterior-posterior diameter measured at the end-ventricular systolic phase. Left ventricular mass (LVM) was calculated by using the Devereux formula. All the echocardiographic data were assessed from the EHRs. Introduction There is lack of Page 2/16 Page 2/16 compelling evidence about the obesity paradox for AF in diabetes patients. Thus, we plan to explore the dose-relationship between BMI and AF in patients with T2DM. Introduction Type 2 diabetes mellitus (T2DM) and atrial fibrillation (AF) are worldwide public health challenges and important causes of death and cardiovascular events. [1] A multitude of studies have indicated DM as an independent risk for AF, in conjunction with coexisting precipitating environment for AF. [2, 3] Serving as one of the notorious risk factors, obesity has been closely mirrored trends in prevalence of T2DM. In United States, 61% to 85% of people with T2DM are overweight or obese. [4, 5] Moreover, obesity is also an established risk factor for AF. Compared to nonobese individual, obesity increased the risk of developing AF by 49% in the general population, and the risk parallelly escalated with increased body mass index (BMI). [6, 7] In the Framingham Heart Study, every unit increase in BMI correlated with a 4–5% increase in AF risk. [8] Besides, a population-based study showed that each BMI reduction of 1 and 5 kg/m2 were associated with a 7% and 12% reduction in the risk of new-onset AF. [9] BMI is the most commonly used parameter to determine the degree of obesity. However, the data for BMI and AF in T2DM population was relatively scarce. A synergistic effect on the risk of new-onset AF was ever observed between body weight and DM. [10] Although DM is usually associated with overweight and obesity, its prevalence among normal-weight individuals has been noticed. Of concern, Asians are more likely to be overweight but less likely to be obese, and 30%– 50% more likely to develop DM than their white counterparts despite having a lower BMI. [11] Significant racial and ethnic disparities in the definition of obese with BMI cutoffs continued to persist. In a recent study, the prevalence of T2DM was 5.4% and 23.5% in underweight and normal weight Asian men and 0.0% and 6.1% in the BMI counterparts of White men. The prevalence of T2DM was 5.6% and 13.6% in underweight and normal weight Asian women and 2.3% and 2.8% in underweight and normal weight White women. [12] In addition, the intriguing observation that J- and U-shaped distributions of BMI aligns with cardiovascular complications and mortality implies that the impact of being underweight may be overlooked. The risk is rather difficult to address possibly because underweight comprises a relatively small proportion in Caucasians than Asians. Data Measurement Body height was measured using a stadiometer against the wall. Subjects stood in an upright position on the flat surface of the stadiometer without shoes, with the back of the heels and the occiput on the stadiometer. The body height was recorded with a unit of centimeter (cm) round off to the first decimal place. Body weight was measured using an electronic digital scale and recorded with a unit of kilogram (kg) nearest 0.1 kg. BMI was calculated by dividing weight in kilograms by height in meters squared (kg/m2). Subjects were categorized into 5 groups according to BMI following the recommendations from Health Promotion Administration, Ministry of Health and Welfare, Taiwan: underweight, BMI <18.5; normal range, 18.5≤BMI <24; overweight, 24≤BMI <27; obese class 1 (mild), 27≤BMI <30; obese class 2 (moderate), 30≤BMI <35; obese class 3 (severe), BMI≥35. New-onset AF and its occurrence time were identified by the diagnosis code from either the EHRs or the standard 12-lead electrocardiogram. The end point of study was AF occurrence, last clinical visit or death. Statistical Analysis Statistical Analysis Page 3/16 Page 3/16 Page 3/16 Continuous variables were described as mean (SD) and categorical variables were presented as frequency (percentage). Differences among groups were tested by using chi square test for categorical variables and analysis of variance (one- way ANOVA) test for continuous variables. The relationship between BMI and the AF was assessed by Multivariate Cox’s regression models from which hazard ratios (HRs) and 95% confidence intervals (CIs) were derived. We adjusted the confounders step by step to make sure the association was consistent through increasingly complex models. In the basic model 1, we adjusted for baseline characteristics including age, gender (male as reference group), hyperlipidemia, gout, history of heart failure, VHD, CAD, COPD, PAOD, prior TIA/ischemic stroke, baseline HbA1C, baseline FG, and baseline eGFR. Subsequently, we further adjusted model 2 with three echocardiogram parameters including baseline LA size, LVEF, and LVM. The estimated cumulative incidences for AF were derived by using Kaplan–Meier approach and the significance of difference between curves were examined by a log-rank test. Continuous variables were described as mean (SD) and categorical variables were presented as frequency (percentage). Differences among groups were tested by using chi square test for categorical variables and analysis of variance (one- way ANOVA) test for continuous variables. The relationship between BMI and the AF was assessed by Multivariate Cox’s regression models from which hazard ratios (HRs) and 95% confidence intervals (CIs) were derived. We adjusted the confounders step by step to make sure the association was consistent through increasingly complex models. In the basic model 1, we adjusted for baseline characteristics including age, gender (male as reference group), hyperlipidemia, gout, history of heart failure, VHD, CAD, COPD, PAOD, prior TIA/ischemic stroke, baseline HbA1C, baseline FG, and baseline eGFR. Subsequently, we further adjusted model 2 with three echocardiogram parameters including baseline LA size, LVEF, and LVM. The estimated cumulative incidences for AF were derived by using Kaplan–Meier approach and the significance of difference between curves were examined by a log-rank test. Since non-linear dose-response associations were expected, restricted cubic splines with five knots located at the 5th, 27.5th, 50th, 72.5th and 95th percentiles of the BMI distribution were used to determine the relationship of BMI and AF. [13] We also conducted the subgroup analyses stratified by gender and age. Missing values were handled by discard. Statistical Analysis The forest plot was displayed for subgroup analyses with adjusted hazard ratios (aHR) along with confidence intervals and p- values are plotted for each variable. A two-tailed p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using R version 3.6.2 (University of Auckland, Auckland, New Zealand) and SPSS statistical software 25.0 (SPSS Inc., Chicago, IL, USA). Discussion Our study demonstrated a “L-shape” relationship between BMI and the risk of AF in patients with T2DM. To our knowledge, this is the first dose-response analysis to survey the non-linear trend of BMI with the development of AF in T2DM in Asian people. There is mounting epidemiological evidence for the link of obesity and AF. The proposed mechanisms of facilitating AF by obesity are multifactorial including structural remodeling caused by increased atrial stretch, atrial fibrosis affected by endothelial dysfunction, increased systemic inflammation, impaired diastolic function, enlarged LA volume and increased pericardial fat. [14, 15] Increased LA pressure and volume, and shortened effective refractory period (ERP) in the left atrium and pulmonary vein are potential factors predisposing and perpetuating AF in obese patients. [16] In this study, the relationship between BMI and the risk of AF in obesity groups became insignificant after adjusting the echocardiography parameters (LA, LVEF, LVM) suggesting that the effects of obesity on AF were mainly mediated through obesity-mediated cardiac structural changes. By contrast, sustained weight loss is associated with reverse remodeling of the AF substrate and reduction in AF burden in conjunction with favorable changes in the coexisting cardiometabolic risk factors. [17, 18] In an ovine model, weight loss is actually associated with structural and electrophysiological reverse remodeling and a reduced propensity for AF. [19] However, without direct conflict, several studies have noticed that being underweight is also a risk factor for new-onset AF and worse cardiovascular outcomes. [20, 21] In general population, underweight is an independent risk factor for AF, just secondary to obesity class 2. One study demonstrated that 1-unit increase of BMI is associated with a 6%–7% increased risk of AF while 1-unit decrease of BMI is associated with a 13% increase in AF risk. [22] Besides, not only being obese but also underweight was reported to have higher AF recurrence after catheter ablation. [23] Data from the ORIGIN trial revealed that obesity and weight loss were inversely related to mortality and cardiovascular outcomes in T2DM. [24] A recent meta-analysis also showed that obesity paradox existed with respect to all-cause and cardiovascular mortality. [25] The obesity paradox for heart failure and cardiovascular mortality has been explained by several aspects. Obesity-related paradoxically increased mobilization of endothelial progenitor cells, increased ghrelin sensitivity, decreased thromboxane production and decreased TNF levels all have potential contributions to the enhancement of myocardial function. Baseline Characteristics The log-rank test was significant (Log-rank test, p <0.001). The relationship between AF risk and BMI was demonstrated in Figure 3 by using cubic spline models and by using normal BMI group as reference. As depicted in Figure 3, the relationship between AF risk and BMI was “L-shape” (Figure 3a) initially and became “J-shape” (Figure 3b and Figure 3c) after the adjustment of age and gender and comorbidities (model 1). The relationship was back to “L-shape” with the underweight group carrying the highest risk of AF after further adjustment of echocardiographic parameters (Figure 3D). A forest plot of HRs for subgroup analyses were demonstrated in Figure 4. Covariates including age above 65, hypertension, gout, VHD, COPD, CAD, PAOD, CKD, history of TIA/old ischemic stroke, LA size >4.0cm, LVEF <50%, LVM more than 200 mg were associated with higher incidence of new-onset AF. Baseline Characteristics The flowchart of patient selection was demonstrated in Figure 1. A total of 74835 with T2DM diagnosis code between 2014 and 2019 were enrolled. Among them, 121 patients without firm evidence of T2DM (only one blood test or without DM medications) and 1607 patients aged below 50 years were excluded. We excluded 1745 patients with pre-existing AF, and 7023 patients with missing BMI values. Finally, a total of 64339 subjects were enrolled for the DM-BMI cohort analysis. Among the study subjects, 2.9% (n = 1862) were classified as underweight, 35.6% (n = 22933) as normal range, 30.6% (n = 19713) as overweight, 18.5% (n = 11875) as obesity class 1, 10.0% (n = 6456) as obesity class 2, 2.33% (n = 1500) as obesity class 3. The baseline characteristics were shown in Table 1. The underweight patients were older and had a worse baseline eGFR while the severely obese patients were more likely to have cardiovascular risk factors such as hypertension, hyperlipidemia, gout, and CKD. After a median follow-up period of 70.7 months, 5692 individuals (8.8% of total population, 1.97 per 1000 person-year) developed AF. The incidence rates of AF were 3.60, 2.03, 1.86, 1.95, 1.88, and 2.25 per 1000 person-year for groups of underweight, normal range, overweight, obesity class 1, obesity class 2, and obesity class 3, respectively. The risk of AF according to the BMI classification in univariable and multivariable Cox regression models was demonstrated in Table 2. In univariate analysis, by using normal BMI group as reference, underweight group was associated with significantly increased risk of AF (HR 1.60, 95%CI 1.39–1.83, p<0.001) while overweight group was significantly associated with reduced risk of AF (HR 0.91, 95%CI 0.85–0.97, p = 0.003). The risk of AF for underweight group remained significantly increased after multivariable adjustment for baseline risk factors and echocardiographic parameters (HR 1.47, 95%CI 1.19–1.81, p<0.001). Also, the risk of AF for overweight group remained significantly decreased after full adjustment (HR 0.83, 95%CI 0.76–0.91, p<0.001). For obesity groups (class 1–3), the risks of AF were not significantly different to that of normal BMI group. Page 4/16 Page 4/16 The cumulative incidence of AF curves of different groups were illustrated in Figure 2. As shown in Figure 2, the cumulative incidence of AF was highest in the underweight group, followed by obesity class 3, and normal group. The overweight group had the least cumulative incidence of AF. Discussion Besides, several adipokines produced by adipose tissue have shown to be cardiovascular protective. [26] Sympathetic nervous system activation has been supposed to be less toxic in obesity- related hypertension. [27] Although the mechanisms of “L-shape” phenomenon in our study is unknown, it is likely that both the inflammatory cytokines and autonomic nervous system may play a role. It is well-known that those who have related hypertension. [27] Although the mechanisms of “L-shape” phenomenon in our study is unknown, it is likely that both the inflammatory cytokines and autonomic nervous system may play a role. It is well-known that those who have Page 5/16 Page 5/16 normal BMI but higher waist circumference and worse metabolic profiles are more prevalent in nonwhites and is characterized as “metabolically unhealthy normal-weight”. [28, 29] There were debates on the existence of obesity paradox. The major doubt was whether the utility of BMI could reflect the true nutrition status such as body composition and body fat distribution. [30] Evidence has shown that abdominal obesity might be more accurate in predicting AF in nonobese Asian. [31] Nevertheless, BMI is still considered as important as or even more important than the total adiposity measures assessed by using complex and expensive methods, and is consistently proved to be a stronger predictor of cardiovascular outcomes. [32] The WHO has proposed lower BMI cutoff values for defining overweight BMI ≥23 kg/m2 and obesity BMI≥ 25 kg/m2 in Asian populations but most of the evidence were from cross-sectional studies. In line with previous studies which do not support the use of lower BMI cutoff values for overweight and obesity in Asian populations, [33] our study adopted the BMI criteria from the suggestion of Taiwan government. Furthermore, in our study, underweight (BMI <18.5) is associated with the highest risk for AF which surpassed the obesity class 3 (BMI≥ 35) in full adjustment model. Because obesity is one of the potential targets for lifestyle modification to reduce AF occurrence, the interpretation of our study should be cautious and further studies are needed. Ethics approval and consent to participate The study protocol complies with the Declaration of Helsinki and was approved by the Institutional Review Board of National Taiwan University Hospital. Limitations This study has some limitations. First, since we did not measure body weight in every outpatient clinic visits, the influence of temporal changes of BMI was not addressed. Second, other obesity-related parameters such as waist circumference or physical activity were not assessed in this study. Third, some potential confounders such as smoking status, alcohol intake, and obstructive sleep apnea were not also not evaluated in this study. Abbreviations ACEI, angiotensin converting enzyme inhibitor; AF, atrial fibrillation; ARB, angiotensin receptor blocker; BMI, body mass index; HTN, hypertension; CAD, coronary artery disease; CCB, calcium channel blocker; CKD, chronic kidney disease; COPD, chronic obstructive pulmonary disease; DOAC, direct oral anticoagulant; DPP4, dipeptidyl peptidase 4; eGFR, estimated glomerular filtration rate; EHRs, electronic health records; GLP–1, glucagon like peptide–1; LA, left atrium; LVEF, left ventricular ejection function; LVIDd, left ventricular internal dimension in end‐diastole; LVIDs, left ventricular internal dimension in systole; LVM, left ventricular mass; MDRD, modification of diet in renal disease; MRA, mineralocorticoid- receptor antagonist; PAOD, peripheral arterial occlusive disease; SGLT2, sodium-glucose co-transporter–2; T2DM, type 2 diabetes mellitus; TIA, transient ischemic accident; TZD, thiazolidinedione; VHD, valvular heart disease We found that in patients with T2DM, underweight is a major risk factor for AF development. We found that in patients with T2DM, underweight is a major risk factor for AF development. Consent for publication Not applicable. Page 6/16 Page 6/16 Authors’ contributions LY-L contributed to the conception or design of the work. YY-Y, SL-C, and YW-C contributed to the acquisition of data for the work. JC-H analyzed data and drafted the manuscript. LY-L critically revised the manuscript. All gave final approval and agreed to be accountable for all aspects of work ensuring integrity and accuracy. Availability of data and materials The datasets used in this study were only available in the National Taiwan University Hospital. The R programs (codes) involved for this study are available from the corresponding author on reasonable request. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Acknowledgements The authors would like to express their thanks to the staff of Department of Medical Research for providing clinical data from National Taiwan University Hospital-integrated Medical Database (NTUH-iMD). Competing interests The authors declare that they have no competing interests. References 1. Lidia Staszewsky, Laura Cortesi, Marta Baviera, Mauro Tettamanti, Irene Marzona, Alessandro Nobili, et al. Diabetes mellitus as risk factor for atrial fibrillation hospitalization: Incidence and outcomes over nine years in a region of Northern Italy. Diabetes Res Clin Pract. 2015;109(3):476-84. 2. Marie J Béland, Kevin C Harris, Ariane J Marelli, Lucile Houyel, Frédérique Bailliard, Frédéric Dallaire, et al. Improving Quality of Congenital Heart Disease Research in Canada: Standardizing Nomenclature Across Canada. Can J Cardiol. 2018;34(12):1674-1676. 2. Marie J Béland, Kevin C Harris, Ariane J Marelli, Lucile Houyel, Frédérique Bailliard, Frédéric Dallaire, et al. 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Tables SU Abbreviations: BMI: body mass index; VHD, valvular heart disease; COPD, chronic obstructive pulmonary disease; CAD, coronary artery disease; PAOD, peripheral arterial occlusive disease; FPG, fasting glucose; eGFR, estimated glomerular filtration rate; LA, left atrium; DT, deceleration time; E/A, early diastolic transmitral flow velocity/ late diastolic transmitral flow velocity; E’, early diastolic mitral annular velocity; LVEF, left ventricular ejection fraction; LVIDd, left ventricular internal diameter in diastole; LVIDs, left ventricular internal diameter in systole; LV mass, left ventricle mass; CCB, calcium channel blocker; ACEI/ARB, angiotensin converting enzyme inhibitor/angiotensin receptor blocker; SGLT-2 inhibitor, sodium-glucose co-transporter-2 inhibitor; DPP4 inhibitor, dipeptidyl peptidase 4 inhibitor; TZD, thiazolidinediones; GLP-1 agonist, glucagon like peptide-1 agonist. Abbreviations: BMI: body mass index; VHD, valvular heart disease; COPD, chronic obstructive pulmonary disease; CAD, coronary artery disease; PAOD, peripheral arterial occlusive disease; FPG, fasting glucose; eGFR, estimated glomerular filtration rate; LA, left atrium; DT, deceleration time; E/A, early diastolic transmitral flow velocity/ late diastolic transmitral flow velocity; E’, early diastolic mitral annular velocity; LVEF, left ventricular ejection fraction; LVIDd, left ventricular internal diameter in diastole; LVIDs, left ventricular internal diameter in systole; LV mass, left ventricle mass; CCB, calcium channel blocker; ACEI/ARB, angiotensin converting enzyme inhibitor/angiotensin receptor blocker; SGLT-2 inhibitor, sodium-glucose co-transporter-2 inhibitor; DPP4 inhibitor, dipeptidyl peptidase 4 inhibitor; TZD, thiazolidinediones; GLP-1 agonist, glucagon like peptide-1 agonist. Table 2: Adjusted hazard ratio for AF incidence across category of BMI Crude Model 1 Model 2 BMI HR p HR p HR p Continuous per unit increase 0.99 (0.98-0.99) <0.001 1.03 (1.02-1.03) <0.001 0.99 (099-1.00) 0.435 Underweight 1.60 (1.39-1.83) <0.001 1.33 (1.12-1.58) 0.001 1.47 (1.19-1.81) <0.001 Normal range 1 1 1 Overweight 0.91 (0.85-0.97) 0.003 0.92 (0.85-0.99) 0.034 0.83 (0.76-0.91) <0.001 Obesity class 1 0.98 (0.91-1.06) 0.663 1.05 (0.96-1.14) 0.299 0.88 (0.80-0.98) 0.016 Obesity class 2 0.95(0.87-1.05) 0.325 1.15 (1.03-1.28) 0.014 0.89 (0.79-1.02) 0.088 Obesity class 3 1.15 (0.98-1.36) 0.084 1.50 (1.25-1.82) <0.001 0.97 (0.78-1.21) 0.783 Table 2: Adjusted hazard ratio for AF incidence across category of BMI Model 1 (*): adjusting age (<65, 65-74, ≥75), gender, hypertension, hyperlipidemia, gout, history of heart failure, VHD, CAD, COPD, PAOD, prior TIA/ischemic stroke, baseline HbA1C, baseline FG, CKD. Model 2 (): adjusting model 1, plus baseline LA size, baseline LVEF, baseline LVM. Model 2 (): adjusting model 1, plus baseline LA size, baseline LVEF, baseline LVM. Tables Table 1: Baseline Characteristics According to BMI Categories Page 9/16 Total N=64339 Underweight BMI <18.5 N=1862 Normal range 18.5≤BMI <24 N=22933 Overweight 24≤BMI <27 N=19713 Obesity class 1 27≤BMI <30 N=11875 Obesity class 2 30≤BMI <35 N=6456 Obesity class 3 BMI≥35 N=1500 P- value Age (yr) 67.7 ± 9.9 71.6 ± 11.6 68.9 ±10.1 67.6 ± 9.7 66.7 ± 9.6 65.1 ± 9.3 63.0 ± 8.6 <0.001 <65 26723 (41.6) 559 (30.0) 8443 (36.8) 8112 (41.2) 5352 (45.1) 3360 (52.0) 897 (59.8) 65-74 20989 (32.6) 564 (30.3) 7596 (33.1) 6632 (33.6) 3826 (32.2) 1933 (29.9) 438 (29.2) ≥75 16627 (25.8) 739 (39.7) 6894 (30.1) 4969 (25.2) 2697 (22.7) 1163 (18.1) 165 (11.0) Male 33930 (52.7) 813 (43.7) 11486 (50.1) 11241 (57.0) 6565 (55.2) 3199 (49.6) 626 (41.7) <0.001 Body height (cm) 160.8 ± 8.5 159.8 ± 8.3 160.6 ± 8.2 161.4 ± 8.4 161.0 ± 8.7 160.2 ± 8.9 158.8 ± 9.7 <0.001 Body weight (kg) 65.9 ± 12.8 44.1 ± 5.4 56.8 ± 7.1 66.4 ± 7.3 73.6 ± 8.2 81.9 ± 9.6 96.6 ± 14.8 <0.001 BMI (kg/m2) 25.4 ± 4.2 17.2 ± 1.1 21.9 ± 1.4 25.4 ± 0.9 28.3 ± 0.8 31.8 ± 1.3 38.3 ± 6.1 <0.001 Hypertension 42088 (65.4) 946 (50.8) 13244 (57.8) 13079 (66.3) 8634 (72.7) 4939 (76.5) 1246 (83.1) <0.001 Hyperlipidemia 30339 (47.2) 541 (29.1) 9795 (42.7) 9640 (48.9) 6075 (51.2) 3389 (52.5) 789 (66.7) <0.001 Gout 5831 (9.1) 93 (5.0) 1539 (6.7) 1861 (9.4) 1312 (11.0) 833 (12.9) 193 (12.9) <0.001 History of heart failure 61 (0.1) 5 (0.3) 19 (0.1) 16 (0.1) 9 (0.1) 11 (0.2) 1 (0.1) 0.049 VHD 569 (0.9) 22 (1.2) 212 (0.9) 182 (0.9) 100 (0.8) 45 (0.7) 8 (0.5) 0.182 COPD 4076 (6.3) 210 (11.3) 1406 (6.1) 1157 (5.9) 746 (6.3) 443 (6.9) 114 (7.6) <0.001 CAD 4163 (6.4) 64 (3.4) 1080 (4.7) 1360 (6.9) 977 (8.2) 555 (8.6) 127 (8.5) <0.001 CKD 8460 (13.1%) 212 (11.4) 2950 (12.8) 2598 (13.2) 1599 (13.5) 887 (13.7) 214 (14.3) 0.044 PAOD 695 (1.1) 18 (1.0) 263 (1.1) 204 (1.0) 138 (1.2) 63 (1.0) 9 (0.6) 0.288 History of TIA/old stroke 692 (1.1) 31 (1.6) 240 (1.0) 201 (1.0) 144 (1.2) 65 (1.0) 11 (0.7) 0.054 Baseline FG (mg/dL) 136.8 ± 53.4 136.0 ± 62.9 136.8 ± 59.4 136.1 ± 49.1 136.7 ± 46.6 138.7 ± 50.8 141.2 ± 62.7 0.006 Baseline HbA1C (%) 7.2 ± 1.5 7.2 ± 1.7 7.2 ± 1.5 7.2 ± 1.4 7.3 ± 1.4 7.3 ± 1.5 7.3 ± 1.4 <0.001 B li GFR 72 1 64 9 45 2 66 6 72 0 29 7 75 7 82 3 95 5 0 001 1.73m2) 1.73m2) CHA2DS2- VASc score 2.5 ± 1.1 2.8 ± 1.1 2.6 ± 1.1 2.5 ± 1.1 2.5 ± 1.2 2.4 ± 1.2 2.4 ± 1.1 <0.001 UCG LA size (cm) 3.9 ± 0.7 3.4 ± 0.8 3.7 ± 0.7 3.9 ± 0.6 4.0 ± 0.6 4.1 ± 0.6 4.2 ± 0.7 <0.001 DT (sec) 0.22 ± 0.07 0.21 ± 0.07 0.22 ± 0.07 0.22 ± 0.07 0.23 ± 0.07 0.22 ± 0.06 0.23 ± 0.06 <0.001 E (mm/sec) 80.7 ± 27.4 81.7 ± 29.7 81.6 ± 28.5 79.5 ± 26.8 80.2 ± 26.4 81.8 ± 27.1 82.9 ± 26.7 <0.001 A (mm/sec) 97.3 ± 24.9 94.5 ± 26.8 96.9 ± 26.1 97.5 ± 24.4 97.7 ± 23.7 97.6 ± 23.5 98.6 ± 25.2 0.110 E/A 0.9 ± 7.6 1.5 ± 12.2 1.1 ± 12.6 0.9 ± 2.3 0.8 ± 0.5 0.8 ± 0.4 0.8 ± 0.4 0.387 E/E’ 21.5 ± 123.1 12.6 ± 6.5 20.0 ± 107.9 24.9 ± 147.7 25.6 ± 152.7 15.0 ± 54.4 11.9 ± 6.0 0.466 LVEF (%) 64.8 ± 12.4 63.3 ± 14.9 64.4 ± 13.3 65.2 ± 12.1 64.9 ± 11.8 65.3 ± 11.0 65.3 ± 10.3 <0.001 LVIDs (cm) 3.0 ± 0.7 2.8 ± 0.8 3.0 ± 0.7 3.0 ± 0.7 3.1 ± 0.7 3.1 ± 0.6 3.2 ± 0.6 <0.001 LVIDd (cm) 4.7 ± 0.6 4.3 ± 0.7 4.6 ± 0.7 4.8 ± 0.6 4.8 ± 0.6 4.9 ± 0.6 5.0 ± 0.6 <0.001 LV mass (gm) 201.4 ± 61.7 158.1 ± 56.8 186.1 ± 58.8 201.4 ± 57.4 213.9 ± 60.9 225.8 ± 63.9 238.5 ± 61.1 <0.001 Medication Antiplatelet 20584 (32.0) 439 (23.6) 6524 (28.4) 6445 (32.7) 4289 (36.1) 2338 (36.2) 549 (36.6) <0.001 Anticoagulant 2774 (4.3) 73 (3.9) 906 (4.0) 807 (4.1) 559 (4.7) 325 (5.0) 104 (6.9) <0.001 CCB 27767 (43.2) 719 (38.6) 8892 (38.8) 8531 (43.3) 5592 (47.1) 3254 (50.4) 779 (51.9) <0.001 Beta-blocker 19073 (29.6) 424 (22.8) 6082 (26.5) 5873 (29.8) 3922 (33.0) 2249 (34.8) 534 (35.6) <0.001 ACEI/ARB 27703 (43.1) 578 (31.0) 8570 (37.4) 8675 (44.0) 5768 (48.6) 3313 (51.3) 799 (53.3) <0.001 Diuretics 16060 (25.0) 509 (27.3) 5241 (22.9) 4525 (23.0) 3202 (27.0) 2028 (31.4) 555 (37.0) <0.001 Statin 23732 (36.9) 372 (20.0) 7572 (33.0) 7509 (38.1) 4937 (41.6) 2723 (42.2) 618 (41.2) <0.001 Metformin 32407 (50.4) 799 (42.9) 11174 (48.7) 10173 (51.6) 6163 (51.9) 3344 (51.8) 754 (50.3) <0.001 SGLT2i 5385 (8.4) 62 (3.3) 1324 (5.8) 1641 (8.3) 1269 (10.7) 851 (13.2) 238 (15.9) <0.001 DDP4i 22464 631 (33.9) 7793 6844 4283 2380 533 <0.001 SU 21744 (33.8) 576 (30.9) 7641 (33.3) 6667 (33.8) 4118 (34.7) 2231 (34.6) 511 (34.1) 0.010 TZD 6075 (9.4) 131 (7.0) 1956 (8.5) 1794 (9.1) 1229 (10.3) 775 (12.0) 190 (12.7) <0.001 Repaglinide 4712 (7.3) 206 (11.1) 1990 (8.7) 1306 (6.6) 749 (6.3) 384 (5.9) 77 (5.1) <0.001 Acarbose 6206 (9.6) 157 (8.4) 2180 (9.5) 1855 (9.4) 1204 (10.1) 657 (10.2) 153 (10.2) 0.054 GLP1 agonist 797 (1.2) 4 (0.2) 134 (0.6) 224 (1.1) 172 (1.4) 183 (2.8) 80 (5.3) <0.001 Insulin 17769 (27.6) 828 (44.5) 6794 (29.6) 5029 (25.5) 3017 (25.4) 1691 (26.2) 410 (27.3) <0.001 Abbreviations: BMI: body mass index; VHD, valvular heart disease; COPD, chronic obstructive pulmonary disease; CAD, coronary artery disease; PAOD, peripheral arterial occlusive disease; FPG, fasting glucose; eGFR, estimated glomerular filtration rate; LA, left atrium; DT, deceleration time; E/A, early diastolic transmitral flow velocity/ late diastolic transmitral flow velocity; E’, early diastolic mitral annular velocity; LVEF, left ventricular ejection fraction; LVIDd, left ventricular internal diameter in diastole; LVIDs, left ventricular internal diameter in systole; LV mass, left ventricle mass; CCB, calcium channel blocker; ACEI/ARB, angiotensin converting enzyme inhibitor/angiotensin receptor blocker; SGLT-2 inhibitor, sodium-glucose co-transporter-2 inhibitor; DPP4 inhibitor, dipeptidyl peptidase 4 inhibitor; TZD, thiazolidinediones; GLP-1 agonist, glucagon like peptide-1 agonist. Model 2 (): adjusting model 1, plus baseline LA size, baseline LVEF, baseline LVM. Model 2 (**): adjusting model 1, plus baseline LA size, baseline LVEF, baseline LVM. Figures Page 12/16 Figure 1 Page 13/16 Figure 2 Figure 2 Cumulative incidence for AF by BMI categories Cumulative incidence for AF by BMI categories Page 14/16 Page 14/16 Figure 3 The association between BMI and AF among overall and adjustment: Crude (3a), adjust age and gender (3b), Model 1 (3c or Model 2 (3d) Figure 3 The association between BMI and AF among overall and adjustment: Crude (3a), adjust age and gender (3b), Model 1 (3c) or Model 2 (3d) The association between BMI and AF among overall and adjustment: Crude (3a), adjust age and gender (3b), Model 1 (3c) r Model 2 (3d) Page 15/16 Page 15/16 Figure 4 Subgroup analyses for AF Page 16/16 Page 16/16
https://openalex.org/W3039424313	https://hal.science/hal-02919709/file/fnins-14-00696.pdf	English	null	Short-Term Sensorimotor Deprivation Impacts Feedforward and Feedback Processes of Motor Control	Frontiers in neuroscience	2,020	cc-by	7,176	Edited by: Giulia Cappagli, Neurological Institute Foundation Casimiro Mondino (IRCCS), Italy Reviewed by: Jacopo Zenzeri, Italian Institute of Technology (IIT), Italy Dalia De Santis, Italian Institute of Technology (IIT), Italy Correspondence: Cécile R. Scotto cecile.scotto@univ-poitiers.fr †ORCID: Cécile R. Scotto orcid.org/0000-0002-7139-2502 Lucette Toussaint orcid.org/0000-0001-8804-9121 Reviewed by: Jacopo Zenzeri, Italian Institute of Technology (IIT), Italy Dalia De Santis, Italian Institute of Technology (IIT), Italy Correspondence: Cécile R. Scotto cecile.scotto@univ-poitiers.fr †ORCID: Specialty section: This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience Received: 12 March 2020 Accepted: 09 June 2020 Published: 01 July 2020 Citation: Scotto CR, Meugnot A, Casiez G and Toussaint L (2020) Short-Term Sensorimotor Deprivation Impacts Feedforward and Feedback Processes of Motor Control. Front. Neurosci. 14:696. doi: 10 3389/fnins 2020 00696 Specialty section: This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience Received: 12 March 2020 Accepted: 09 June 2020 Published: 01 July 2020 Short-Term Sensorimotor Deprivation Impacts Feedforward and Feedback Processes of Motor Control Cécile R. Scotto †, Aurore Meugnot , , Géry Casiez , and Lucette Toussaint † 1 Université de Poitiers, Université de Tours, Centre National de la Recherche Scientiﬁque, Centre de Recherches sur la Cognition et l’Apprentissage, Poitiers, France, 2 Université Paris-Saclay CIAMS, Orsay, France, 3 CIAMS, Université d’Orléans, Orléans, France, 4 Univ. Lille, CNRS, Centrale Lille, UMR 9189 – CRIStAL – Centre de Recherche en Informatique, Signal et Automatique de Lille, Lille, France, 5 Institut Universitaire de France (IUF), Paris, France Sensory loss involves irreversible behavioral and neural changes. Paradigms of short-term limb immobilization mimic deprivation of proprioceptive inputs and motor commands, which occur after the loss of limb use. While several studies have shown that short-term immobilization induced motor control impairments, the origin of such modiﬁcations is an open question. A Fitts’ pointing task was conducted, and kinematic analyses were performed to assess whether the feedforward and/or feedback processes of motor control were impacted. The Fitts’ pointing task speciﬁcally required dealing with spatial and temporal aspects (speed-accuracy trade-off) to be as fast and as accurate as possible. Forty trials were performed on two consecutive days by Control and Immobilized participants who wore a splint on the right arm during this 24 h period. The immobilization modiﬁed the motor control in a way that the full spatiotemporal structure of the pointing movements differed: A global slowdown appeared. The acceleration and deceleration phases were both longer, suggesting that immobilization impacted both the early impulse phase based on sensorimotor expectations and the later online correction phase based on feedback use. First, the feedforward control may have been less efﬁcient, probably because the internal model of the immobilized limb would have been incorrectly updated relative to internal and environmental constraints. Second, immobilized participants may have taken more time to correct their movements and precisely reach the target, as the processing of proprioceptive feedback might have been altered. ORIGINAL RESEARCH published: 01 July 2020 doi: 10.3389/fnins.2020.00696 Keywords: immobilization, sensorimotor deprivation, Fitts’ task, speed-accuracy trade-off, motor control Citation: Overall, studies investigating the anatomical cerebral changes following limb immobilization agree on reductions in cortical excitability of the sensorimotor representation linked to the decrease in sensory input and motor output (Facchini et al., 2002; Huber et al., 2006; Avanzino et al., 2011; Burianová et al., 2016). In the same vein, behavioral studies have highlighted the negative immobilization-induced eﬀects on the cognitive level of action. Alterations at the sensorimotor representation level evaluated by means of an implicit motor imagery task were reported following a few hours of arm non-use (Toussaint and Meugnot, 2013; Debarnot et al., 2018). The authors showed that motor imagery processes used to identify the laterality of hand images were slowed down for stimuli corresponding to the immobilized hand. Other studies reported changes in the peripersonal space representation (Bassolino et al., 2012; Toussaint et al., 2018). Using a reachability judgment task, Toussaint et al. reported that the maximum distance at which objects are perceived as reachable was reduced in subjects forced into arm and hand non-use for 24 h. Overall, these studies have shown that representations in the brain are modiﬁed with immobilization. Apparatus The pointing task was performed on a MacBook Pro Retina (OS X 10.11.6 El Capitan 2.5 GH Core i5) with a screen of 13.3 inches (900 × 1440 pixels) refreshed at 60 Hz. This laptop included an 8.6 cm × 10.5 cm trackpad with a resolution of 400 CPI sampling at 125 Hz. Instructions, stimuli and data from the pointing device were handled using a custom-built application written in C++ using Qt and Libpointing (Casiez et al., 2011). The gain between the trackpad and the visual cursor was set to 1: what was seen on the screen corresponded to what was done on the trackpad. Citation: Scotto CR, Meugnot A, Casiez G and Toussaint L (2020) Short-Term Sensorimotor Deprivation Impacts Feedforward and Feedback Processes of Motor Control. Front. Neurosci. 14:696. doi: 10.3389/fnins.2020.00696 The impact of sensory deprivation has been largely studied to identify behavioral and neural changes following irreversible sensory loss. Over the past decade, such changes have been studied for sensorimotor loss through paradigms involving short-term limb immobilization. Such paradigms mimic the deprivation of motor inputs and outputs that induce maladaptive neural plasticity without compromising brain function (disease-free model; Furlan et al., 2016). Short- term immobilization consists of preventing a body part (often ﬁngers, hand, and/or arm) from July 2020 \| Volume 14 \| Article 696 Frontiers in Neuroscience \| www.frontiersin.org Immobilization Impacts Motor Planning/Control Scotto et al. moving by means of a splint or a bandage for a period ranging from a few hours to a few days. While several studies have shown that short-term immobilization induced motor control impairments, the origin of such modiﬁcations is an open question. Here, a short-term limb immobilization paradigm was used to specify the impact of this sensorimotor deprivation on performance in a Fitts’ task. This pointing task speciﬁcally required dealing with spatial and temporal aspects (speed-accuracy trade-oﬀ) to be as fast and as accurate as possible. The associated kinematic analysis allowed us to assess whether the feedforward and/or the feedback processes of motor control were impacted. contrary to previously used paradigms with immobilization, the Fitts’ task necessitates dealing with speed as well as accuracy (i.e., speed-accuracy trade-oﬀ) to reach the target. A kinematic analysis was provided to determine whether the feedforward and/or feedback processes of motor control of the pointing movement were aﬀected. The feedforward model refers to the initiation of early adjustments based on the comparison between the motor commands and the expected outputs (eﬀerence copy; Miall and Wolpert, 1996). This feedforward process would be associated with early kinematic parameters (i.e., before peak velocity; Meyer et al., 1982; Elliott et al., 2010). The feedback process corresponds to the correction phase, with an online sensory processing comparing the intended to the current state, and would be associated with later kinematic parameters (i.e., after peak velocity; Meyer et al., 1982; Elliott et al., 2010). Participants Forty-nine right-handed participants (29 men and 20 women; mean age ± SE: 20.0 ± 0.28 years) gave written informed consent prior to the study, in accordance with the 1964 Declaration of Helsinki. The experimental protocol was approved by the ethics committee for research in sciences of physical and sports activities (n◦2017250114). All participants reported having normal or corrected-to-normal vision and no neurological or sensorimotor disorders. As we expected, the immobilization eﬀects would disappear within a few trials (Bassolino et al., 2012), and we used a between-subject design. The participants were assigned into one of the two groups (e.g., Control or Immobilized) and performed either the task with an Index of Diﬃculty (ID) of 3 or 7 (see section “Procedure”). Fifteen participants constituted the Control-ID3 group (seven men and eight women; 20.4 ± 0.49 years), twelve were in the Control- ID7 group (seven men and ﬁve women; 20.6 ± 0.49 years), ten were in the Immobilized-ID3 group (six men and four women; 19.4 ± 0.33 years), and twelve were in the Immobilized-ID7 group (nine men and three women; 19.6 ± 0.41 years). Although functional consequences of immobilization have been demonstrated, these studies did not identify which mechanisms of action were altered. The majority of studies have tested how short-term immobilization impacted out-and- back uncorrected movements toward visual targets (Huber et al., 2006; Moisello et al., 2008; Debarnot et al., 2018). The hand path trajectory of such movements deviated after immobilization, showing that spatial parameters were impacted (Huber et al., 2006; Moisello et al., 2008). Debarnot et al. (2018) added that temporal parameters were also modiﬁed during this out-and- back movement following immobilization. They showed that movement time and reaction time were longer than those of Control participants. While these studies demonstrated the impact of immobilization on spatial parameters on the one hand and temporal parameters on the other hand, the associated kinematic analysis was not provided. This analysis was provided in Bassolino et al. (2012) with a reach-to-grasp objects task. The authors showed that the transport phase was impaired following 10 h of immobilization but not the grasping component (Bassolino et al., 2012). To suppress the possible interaction of the grasping component on the transportation phase, we investigated how short-term immobilization impacts the kinematics of a pointing movement (i.e., without a grasping component). A Fitts’ task was used to speciﬁcally assess how immobilization may modify spatiotemporal aspects of motor control. Therefore, Frontiers in Neuroscience \| www.frontiersin.org Data Processing For the Immobilized group, a quantitative check of the activity amount was performed through the recording of both arms with actimeters. During the 24 h immobilization period, 638 ± 59 counts/min were recorded for the right immobilized hand and 2795 ± 115 counts/min were recorded for the left non- immobilized hand (see Toussaint and Meugnot, 2013 for a similar procedure). ANOVA performed on the actimeter values conﬁrmed that the level of manual activity was higher for the left hand than for the right hand [F(1,21) = 525.5; p < 0.001]. screen height and a width (W), which was manipulated with the task’s ID. The ID integrates both the W and the Distance (D) from the starting point to the target’s center as follows: ID = Log2(2D/W). Here, D was set to 8 cm, and W was either 2 or 0.125 cm, deﬁning an ID of 3 or 7, respectively. The participants in the two groups (Control and Immobilized) were assigned to either ID3 or ID7 conditions (between-subject design; see section “Participants”). p ) In an illuminated room, the participants sat in a chair adjustable in elevation. They were approximately at 50 cm faced to a laptop. The experimenter placed their forearm on the table perpendicular to the laptop, in a comfortable position. The joints were not restrained, and the pointing mainly consisted of a wrist movement (i.e., abduction when pointing from the left and adduction when pointing from the right). Talcum was applied on the participant’s ﬁnger before the experiment to reduce dampness and allowed an easy ﬁnger slide on the trackpad. This talcum was reapplied whenever the participants needed it. For each trial, the participants were instructed to explore the trackpad with the right ﬁnger to ﬁnd the starting position on the trackpad (left or right border). When the position was reached, the word “calibration” was displayed on the screen. The trial was launched if the ﬁnger was static at this speciﬁc location for 0.5 s. The trial started with the simultaneous appearance of the cursor and the visual target: The participants could then start the pointing whenever they were ready. The required movement was to point the visual target as precisely and as accurately as possible with a smooth and continuous movement on the trackpad. The participants had to avoid stopping before or after the target. Procedure The task consisted of horizontal 2D pointing (either left to right or right to left; Figure 1) using Fitts’ paradigm (Fitts, 1954). The cursor corresponded to a vertical line of 1 pixel width (0.2 mm), and the target was a rectangle of a length corresponding to the July 2020 \| Volume 14 \| Article 696 2 Immobilization Impacts Motor Planning/Control Scotto et al. FIGURE 1 \| Experimental setup. Subjects were asked to perform a pointing task: They slided their right index on the laptop trackpad to move the visual cursor (white line) into the target (green rectangle). The experiment was composed of two sessions of 40 trials on 2 consecutive days (Pre and Post tests). The ﬁrst session (Pre) also included a training of 20 trials to familiarize the participants with the task prior to data recording. The Pre session lasted 40 min. Immediately after this ﬁrst session, the participants in the Immobilized group had their right arm immobilized with a rigid splint (DONJOY “Comfort Digit”; DJO, Surrey, United Kingdom) that ﬁrmly maintained the wrist and three ﬁngers (index, middle, and ring). An immobilization vest (model DONJOY "Immo Axmed") restraining right shoulder, arm and forearm movements was also used to ensure that the participants kept their arm at rest as much as possible during the 24 h of immobilization. The Immobilized group also wore actimeters (ActiGraph wGT3X-BT) on the wrist of both hands to verify if they had complied with these instructions. The actimeters recorded the activity level (in counts/min) with ActiLife software (ActiLife v6.11.8, Pensacola, FL, United States). Twenty-four hours after the ﬁrst session, both groups returned and performed the second session of 40 trials (Post). For the Immobilized group, the Post test was performed immediately after splint removal by the experimenter. The Post session lasted 15 min. FIGURE 1 \| Experimental setup. Subjects were asked to perform a pointing task: They slided their right index on the laptop trackpad to move the visual cursor (white line) into the target (green rectangle). Frontiers in Neuroscience \| www.frontiersin.org Data Processing After a period of 0.5 s static in the target, the visual stimuli disappeared, and the trial stopped. The trial direction alternated between rightward and leftward. The vision of the arm was not restrained. Position data from the trackpad were low-pass ﬁltered with a dual-pass, no-lag Butterworth ﬁlter (cutoﬀfrequency: 10 Hz; order: 2). The data were then derivated to compute the ﬁnger velocity used to determine the Movement Time (MT) of the pointing. The MT corresponds to the period between the movement onset and oﬀset, which were deﬁned when the velocity reached above and below 5% of Peak Velocity (PV), respectively. We further assessed the impact of immobilization with the analysis of pointing corrections. Although the participants were instructed to point the target with a “smooth and continuous movement,” some movements were stopped (velocity below 5% of PV) before or after the target. We computed the percentage of trials where corrections appeared (i.e., the correction rate). Movement kinematics were also analyzed to further determine the impact of immobilization on the motor impulse phase and online correction phase, associated to feedforward and feedback processes, respectively. Modiﬁcations in the impulse phase were assessed through the analysis of the time of acceleration (AT; time from movement onset to PV). In addition, the time of deceleration (DT; time between PV and movement oﬀset) was associated with the correction phase with online corrections (Meyer et al., 1982; Elliott et al., 2010). DT includes the deceleration period of the ﬁrst submovement (from PV) as well as the period lasting for all potential additional submovements. July 2020 \| Volume 14 \| Article 696 Frontiers in Neuroscience \| www.frontiersin.org Frontiers in Neuroscience \| www.frontiersin.org 3 Immobilization Impacts Motor Planning/Control Scotto et al. FIGURE 3 \| Movement Time (MT) relative to the Session (Pre vs. Post) for the Control and Immobilized (Immo) groups. p < 0.01. Error bars denote standard error. Bassolino et al. (2012) found that the inﬂuence of immobilization in a reach-to-grasp task did not last more than a few trials. Therefore, we ﬁrst assessed whether diﬀerences appeared over trials by comparing the means of each eight successive blocks of ﬁve trials. Repeated-measures ANOVAs were then conducted using a mixed design with two between- subjects factors: Group (Control vs. Immobilized) and ID (3 vs. 7) and two within-subjects factors: Session (Pre vs. Post) and Block (1–8). RESULTS FIGURE 3 \| Movement Time (MT) relative to the Session (Pre vs. Post) for the Control and Immobilized (Immo) groups. p < 0.01. Error bars denote standard error. Data Processing A simple eﬀect of Block appeared but no signiﬁcant interactions were revealed between the Block and the Group or the Session on all the dependent variables. The analyses were then conducted on the mean of the 40 trials. Post-hoc tests (Newman-Keuls) were performed when necessary, and the level of signiﬁcance was set at 0.05 for all statistical analyses. Kinematic Proﬁles Figure 2 depicts velocity proﬁles for representative trials in the Control and Immobilized groups. As classically shown, the ID FIGURE 2 \| Velocity proﬁles from representative trials for the Control and Immobilized (Immo) participants who performed in either the ID3 (A) or ID7 (B) condition. Dashed lines represent session Pre, and full lines represent session Post. Regardless of the ID, the Immobilized participants exhibited longer MT and lower PV in the Post session than in the Pre session. seemed to modify pointing kinematics: ID7 was associated with a lower PV, longer MT and more corrections than ID3. In addition, Figure 1 suggests that the Immobilized participants exhibited a longer MT and a lower PV in the Post session than in the Pre session, regardless of the ID. These observations were statistically tested with mean comparisons of selected kinematic parameters. Movement Time and Corrections The repeated-measures ANOVA Group × ID × Session for MT revealed an eﬀect of ID [F(1,45) = 349.0; p < 0.001] as well as an interaction Group × Session [F(1,45) = 10.4; p < 0.001]. Overall, the MT was shorter in the ID3 than in the ID7 condition (367 ± 12 ms vs. 970 ± 32 ms, respectively). No signiﬁcant eﬀect of Group [F(1,45) = 1.6; p = 0.21], Session [F(1,45) = 2.4; p = 0.13], Group × ID [F(1,45) = 0.0; p = 1.00], Session × ID [F(1,45) = 0.2; p = 0.62] nor Group × ID × Session [F(1,45) = 2.0; p = 0.17] appeared. Figure 3 depicts the Group × Session interaction. The Control and Immobilized groups diﬀered between the Pre and Post sessions. For the Immobilized group, the MT increased between the Pre and Post sessions (661 ± 70 ms vs. 710 ± 73 ms, respectively; p < 0.01). In contrast, for the Control group, the MT did not increase between the Pre and Post sessions and either exhibited a trend toward a decrease (666 ± 64 ms vs. 639 ± 62 ms; p = 0.06). The analysis of corrections (under- and overshoots) revealed an eﬀect of ID [F(1,45) = 26.9; p < 0.001] with a higher correction rate for ID7 than ID3 (2.0 ± 0.6% vs. 14.1 ± 2.5%). No signiﬁcant eﬀect of Group [F(1,45) = 0.9; p = 0.35], Session [F(1,45) = 0.1; p = 0.70], Group × ID [F(1,45) = 0.5; p = 0.50], Session × ID [F(1,45) = 0.0; p = 0.95], Session × Group [F(1,45) = 0.0; p = 0.87] nor Group × ID × Session [F(1,45) = 0.7; p = 0.42] appeared. Therefore, the analysis failed to show an eﬀect of immobilization on corrections. FIGURE 2 \| Velocity proﬁles from representative trials for the Control and Immobilized (Immo) participants who performed in either the ID3 (A) or ID7 (B) condition. Dashed lines represent session Pre, and full lines represent session Post. Regardless of the ID, the Immobilized participants exhibited longer MT and lower PV in the Post session than in the Pre session. FIGURE 2 \| Velocity proﬁles from representative trials for the Control and Immobilized (Immo) participants who performed in either the ID3 (A) or ID7 (B) condition. Dashed lines represent session Pre, and full lines represent session Post. Peak Velocity, Peak Acceleration, and Peak Deceleration We analyzed how fast the pointing movements of the participants were. The analysis of Peak Velocity (PV) revealed an eﬀect of ID [F(1,45) = 66.3; p < 0.001] and a Group × Session interaction [F(1,45) = 13.3; p < 0.01; Figure 5A]. No signiﬁcant eﬀect of Group [F(1,45) = 0.1; p = 0.77], Session [F(1,45) = 3.0; p = 0.09], Group × ID [F(1,45) = 0.4; p = 0.53], Session × ID [F(1,45) = 0.5; p = 0.50] nor Group × ID × Session [F(1,45) = 0.3; p = 0.57] appeared. First, the PV was higher at ID3 (49.5 ± 2.0 cm.s−1) than at ID7 (28.4 ± 1.9 cm.s−1). In addition, the post-hoc analysis of the interaction showed that the PV in the Immobilized group decreased between the Pre and Post sessions (41.5 ± 3.2 cm.s−1 vs. 35.6 ± 2.7 cm.s−1, respectively; p < 0.01). No diﬀerence was found for the Control group between the Pre and Post sessions (514 ± 2.7 cm.s−1 vs. 492 ± 3.0 cm.s−1; p = 0.06). A trend appeared in the Post session between the two groups (p = 0.07). p The Deceleration Time (DT) corresponds to the absolute period between the PV and the end of the movement. This parameter is associated with the homing phase of motor control reﬂecting online movement corrections (e.g., Woodworth, 1899; Meyer et al., 1982). The analysis showed an eﬀect of ID [F(1,45) = 553.1; p < 0.001] and a Group × Session interaction [F(1,45) = 9.3; p < 0.01]. No signiﬁcant eﬀect of Group [F(1,45) = 1.2; p = 0.27], Session [F(1,45) = 0.0; p = 0.83], Group × ID [F(1,45) = 0.6; p = 0.43], Session × ID [F(1,45) = 0.0; p = 0.88] nor Group × ID × Session [F(1, 45) = 0.7; p = 0.42] appeared. Again, the DT was shorter at ID3 (242 ± 9 ms) than at ID7 (790 ± 23 ms), and post-hoc analysis of the interaction showed that the Control and Immobilized groups diﬀered between the Pre and Post sessions (Figure 4B). For the Immobilized group, the DT increased between the Pre and Post sessions (516 ± 65 ms vs. 542 ± 65 ms, respectively; p < 0.05). Acceleration and Deceleration Time and exhibited a trend toward a reduction (514 ± 54 ms vs. 492 ± 53 ms; p = 0.06). The Acceleration Time (AT) corresponds to the absolute period between the movement onset and the PV. This parameter is associated with the impulse phase of motor control reﬂecting the planning process of the movement. The analysis showed an eﬀect of ID [F(1,45) = 34.6; p < 0.001], Session [F(1,45) = 9.7; p < 0.01] and a Group × Session interaction [F(1,45) = 11.5; p < 0.01]. No signiﬁcant eﬀect of Group [F(1,45) = 2.5; p = 0.19], Group × ID [F(1,45) = 0.1; p = 0.73], Session × ID [F(1,45) = 0.9; p = 0.35] nor Group × ID × Session [F(1,45) = 1.4; p = 0.25] appeared. Therefore, the AT was shorter at ID3 (113 ± 4 ms) than at ID7 (172 ± 11 ms). Moreover, the post-hoc analysis of the interaction showed that the Control and Immobilized groups diﬀered between the Pre and Post sessions (Figure 4A). For the Immobilized group, the AT increased between the Pre and Post sessions (138 ± 9 ms vs. 164 ± 11 ms, respectively; p < 0.001). For the Control group, the AT did not increase between the Pre and Post sessions (136 ± 10 ms vs. 134 ± 9 ms; p = 0.86). Further analyses were conducted to determine what caused the modiﬁcations in the temporal parameters (i.e., MT, AT, DT). More precisely, we computed the peak acceleration, velocity and deceleration to determine whether those modiﬁcations occurred at an early or late stage. Movement Time and Corrections Regardless of the ID, the Immobilized participants exhibited longer MT and lower PV in the Post session than in the Pre session. July 2020 \| Volume 14 \| Article 696 Frontiers in Neuroscience \| www.frontiersin.org 4 Immobilization Impacts Motor Planning/Control Scotto et al. FIGURE 4 \| (A) Acceleration Time (AT) and (B) Deceleration Time (DT) relative to the Session (Pre vs. Post) for the Control and Immobilized (Immo) groups. p < 0.05; p < 0.01; *p < 0.001. Error bars denote standard error. e to the Session (Pre vs. Post) for the Control and Immobilized (Immo) groups. FIGURE 4 \| (A) Acceleration Time (AT) and (B) Deceleration Time (DT) relative to the Session (Pre vs. Post) for the Control and Immobilized (Immo) groups. p < 0.05; p < 0.01; p < 0.001. Error bars denote standard error. Peak Velocity, Peak Acceleration, and Peak Deceleration For the Control group, the DT did not increase between the Pre and Post sessions The analysis of Peak Acceleration (PA) revealed an eﬀect of ID [F(1,45) = 52.6; p < 0.001] and a Group × Session interaction [F(1,45) = 10.3; p < 0.01; Figure 5B]. No signiﬁcant eﬀect of Group [F(1,45) = 1.6; p = 0.21], Session [F(1,45) = 2.2; p = 0.15], Group × ID [F(1,45) = 2.0; p = 0.16], Session × ID [F(1,45) = 1.6; p = 0.21] nor Group × ID × Session [F(1,45) = 0.0; p = 0.90] appeared. As with the PV, the PA was higher at ID3 (634 ± 40 cm.s−2) than at ID7 (306 ± 31 cm.s−2). In addition, the post-hoc analysis of the interaction showed that the PA in the July 2020 \| Volume 14 \| Article 696 Frontiers in Neuroscience \| www.frontiersin.org 5 Immobilization Impacts Motor Planning/Control Scotto et al. FIGURE 5 \| (A) Peak Velocity (PV), (B) Peak Acceleration (PA), and (C) Peak Deceleration (PD) relative to the Session (Pre vs. Post) for the Control and Immobilized (Immo) groups. p < 0.05; *p < 0.01. Error bars denote standard error. FIGURE 5 \| (A) Peak Velocity (PV), (B) Peak Acceleration (PA), and (C) Peak Deceleration (PD) relative to the Session (Pre vs. Post) for the Control and Immobilized (Immo) groups. p < 0.05; *p < 0.01. Error bars denote standard error. FIGURE 5 \| (A) Peak Velocity (PV), (B) Peak Acceleration (PA), and (C) Peak Deceleration (PD) relative to the Session (Pre vs. Post) for the Control and Immobilized (Immo) groups. p < 0.05; **p < 0.01. Error bars denote standard error. DISCUSSION Immobilized group decreased between the Pre and Post sessions (489 ± 44 cm.s−2 vs. 393 ± 36 cm.s−2, respectively; p < 0.01). No diﬀerence was found for the Control group between the Pre and Post sessions (481 ± 48 cm.s−2 vs. 517 ± 58 cm.s−2; p = 0.19). Finally, the Pre and Post sessions diﬀered between the two groups (p < 0.01). Here, we tested how short-term immobilization modiﬁed behavioral responses. More precisely, we tested whether the feedforward and/or feedback processes of pointing movements were aﬀected by 24 h of arm non-use. We ﬁrst showed that immobilization had an impact on sensorimotor control with lengthened movement time without damaging accuracy. This decrease in movement time seemed to result from a global slowdown: The acceleration and deceleration phases were both longer and were associated with lower peak acceleration, velocity, and deceleration. Therefore, immobilization appeared to modify sensorimotor control in such a way that the full spatiotemporal structure of the pointing movements diﬀered. The analysis of Peak Deceleration (PD) revealed an eﬀect of ID [F(1,45) = 57.6; p < 0.001] and a Group × Session interaction [F(1,45) = 6.4; p < 0.01; Figure 5C]. No signiﬁcant eﬀect of Group [F(1,45) = 0.3; p = 0.61], Session [F(1,45) = 3.6; p = 0.06], Group × ID [F(1,45) = 0.8; p = 0.37], Session × ID [F(1,45) = 1.0; p = 0.31] nor Group × ID × Session [F(1,45) = 0.1; p = 0.72] appeared. As PA, the PD was also higher at ID3 (−438 ± 29 cm.s−2) than at ID7 (−188 ± 21 cm.s−2). In addition, the post-hoc analysis of the interaction showed that the PD in the Immobilized group decreased between the Pre and Post sessions (−339 ± 37 cm.s−2 vs. −270 ± 35 cm.s−2, respectively; p < 0.05). No diﬀerence was found for the Control group between the Pre and Post sessions (−317 ± 35 cm.s−2 vs. −326 ± 37 cm.s−2; p = 0.60) or between the Pre and Post sessions between the two groups (p = 0.14). First, our data conﬁrmed that immobilization leads to a decrease in motor performance (Huber et al., 2006; Moisello et al., 2008; Bassolino et al., 2012; Bolzoni et al., 2012). Such declines in pointing performance have been shown to arise from changes in joint coordination around the deprived segment (Moisello et al., 2008; Bassolino et al., 2012). DISCUSSION When the immobilized July 2020 \| Volume 14 \| Article 696 Frontiers in Neuroscience \| www.frontiersin.org 6 Immobilization Impacts Motor Planning/Control Scotto et al. participants were instructed to make out-and-back straight movements without correction, an increase in the hand-path area amplitude and variability appeared (Huber et al., 2006; Moisello et al., 2008; Bassolino et al., 2012). When the task integrated spatial constraints for trial validation, immobilization rather induced temporal impairments, such as an increase in movement time. Therefore, Bassolino et al. (2012) showed an increase in movement time for a reach-to-grasp task where spatial constraints were deﬁned (i.e., the object reaching movement to perform the grasping) during the ﬁve ﬁrst trials. Here we found that this increase in movement duration could last longer for a pointing task as we did not ﬁnd an interaction between the immobilization and the trial repetitions. Therefore, no reactivation of the process of proprioceptive inputs would appear contrary to the results of Bassolino et al. (2012). Albeit, here the movement amplitude was reduced and required a less complex motor control than a reach-to-grasp task involving multiple joints from the arm, the hand and the digits. In addition, spatial corrections (stops before or after the target) were not ampliﬁed with immobilization, as Fitts’ paradigm requires ﬁnishing the movement in the target position, and we instructed the participants to perform a “smooth movement.” Therefore, the participants would lengthen their movement rather than doing several sub-movements to reach the target. In addition, we hypothesized that the behavioral consequences of immobilization would be modulated by task diﬃculty. Therefore, we expected a higher impact with ID7 than ID3 because motor planning and control is more complex. While we found the classic eﬀect of ID on the kinematic parameters, no statistical interaction appeared with the immobilization factor. The lack of proprioceptive cues would be suﬃciently strong to aﬀect any movement, as also suggested by the work of Medina et al. (2009) on a deaﬀerented patient (JDY). In this study, the diﬀerence in movement time between controls and JDY would not appear to be modulated by the tested ID (i.e., 4, 4.5, and 5.5) of the pointing task. and its sensory consequences (e.g., future states of the arm at the end of pointing; Wolpert et al., 1995). DISCUSSION Based on these feedforward inputs of the limb, predictions of the future states are compared to the current state which allow for early corrections (Wolpert et al., 1995; Desmurget and Grafton, 2000; Wolpert and Ghahramani, 2000). Our results suggested that the feedforward control was impacted, probably because the internal model of the immobilized limb would be incorrectly updated. In daily life, the internal model of the limb is continuously updated through motion (see Wolpert and Ghahramani, 2000). During the 24 h of immobilization, motor commands of the limb were largely reduced. Consequently, eﬀerence copy as well as dynamical proprioceptive cues could not have been used to maintain or calibrate the internal model with the limb dynamics relative to the environment. Such a decrease in feedback would lead to an altered prediction of the sensory consequences of the action before its execution. Studies with deaﬀerented patients have previously shown that proprioception was critical to update internal models of limb dynamics (Sainburg et al., 1995; Sarlegna et al., 2006; Medina et al., 2009). On the other hand, our results suggested that immobilization also modiﬁes the feedback control of the pointing: A lengthened deceleration duration as well as decreased peak deceleration were observed. These results suggested disruptions in the process of proprioceptive cues correcting the movement online. This is in line with recent studies (Huber et al., 2006; Weibull et al., 2011; Ngomo et al., 2012; Rosenkranz et al., 2014; Opie et al., 2016), which found a decrease in excitability in the somatosensory areas representing the previously immobilized arm: The proprioceptive cues were less processed, as well as the tactile cues (i.e., decrease of tactile discrimination; Weibull et al., 2011). Therefore, immobilized participants would take more time to correct their movement to precisely reach the target, as the processing of proprioceptive cues might be altered. Visual cues would be particularly used to compensate for this deﬁcit, notably with the online visual comparison between the cursor and the target position. This feedback control of the pointing movement throughout vision was shown to start later than the proprioceptive one (Sarlegna et al., 2004; Saunders and Knill, 2004), which could explain the increase of the correction phase duration. This hypothesis is supported by neurophysiological data which showed that the decrease in cortical excitability of the somatosensory areas of the immobilized limb is associated with a sensitivity increase of the other sensory inputs (Rosenkranz et al., 2014). Frontiers in Neuroscience \| www.frontiersin.org DISCUSSION Further experiments would be necessary to speciﬁcally isolate how visual cues impact sensorimotor control after immobilization. Before debating what immobilization changed for feedforward and/or feedback processing, we have to discard the possibility that changes arising from peripheral structures declined. Indeed, immobilization leads to modiﬁcations in muscle contractile properties (from slow to fast ﬁber type) and motor units. However, such transformations appeared after several weeks of immobilization (Desaphy, 2001; Seki et al., 2001a,b; Zanette et al., 2004). In contrast, short-term immobilization (less than 3–4 days) do not impact muscle and nerve properties (Facchini et al., 2002; Huber et al., 2006; Moisello et al., 2008). Therefore, modiﬁcations of motor behavior in the present study cannot be attributed to changes in muscle structure following short- term immobilization. Although functional consequences of immobilization have been demonstrated in the past, the impact of immobilization on the motor control processes has not been fully elucidated. Contrary to previously used paradigms with immobilization, we used a Fitts’ task which necessitates dealing with spatiotemporal constraints (i.e., speed-accuracy trade-oﬀ). Thanks to a spatiotemporal kinematic analysis, we speciﬁcally assessed the impact of sensorimotor deprivation on the motor control processes. For the ﬁrst time, we showed early and late kinematic changes following a short period of limb non-use, which Here, we showed that immobilization impacted both early and late movement kinematics. Since Woodworth’s two-component model, kinematic parameters before the peak velocity are associated with feedforward control and those after are associated with the feedback control (Meyer et al., 1982; Elliott et al., 2010). On the one hand, we showed that immobilization lengthened acceleration duration as well as decreased peak acceleration. Early kinematics modiﬁcations have been shown to reﬂect the use of internal models, i.e., a representation of the action July 2020 \| Volume 14 \| Article 696 7 Immobilization Impacts Motor Planning/Control Scotto et al. sciences of physical and sports activities (n◦2017250114). The patients/participants provided their written informed consent to participate in this study. may be caused by the modiﬁcation of feedforward as well as feedback processes. Even if these results would have to be extended to a broader population, such as the elderly people, they may have implications in rehabilitation and health care. Everybody has been or will be immobilized during his/her own lifetime due to an accident (e.g., broken limb) or for external reasons (e.g., long travel, prolonged bed rest). REFERENCES Huber, R., Ghilardi, M. F., Massimini, M., Ferrarelli, F., Riedner, B. A., Peterson, M. J., et al. (2006). Arm immobilization causes cortical plastic changes and locally decreases sleep slow wave activity. Nat. Neurosci. 9, 1169–1176. doi: 10.1038/nn1758 Avanzino, L., Bassolino, M., Pozzo, T., and Bove, M. (2011). Use-dependent hemispheric balance. J. Neurosci. 31, 3423–3428. doi: 10.1523/JNEUROSCI. 4893-10.2011 Medina, J., Jax, S. A., and Coslett, H. B. (2009). Two-component models of reaching: Evidence from deaﬀerentation in a Fitts’ law task. Neurosci. 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Neuroscience 330, 247–256. doi: 10.1016/j.neuroscience.2016.06. 002 Debarnot, U., Huber, C., Guillot, A., and Schwartz, S. (2018). Sensorimotor representation and functional motor changes following short-term arm immobilization. Behav. Neurosci. 132, 595–603. doi: 10.1037/bne0000274 Desaphy, J.-F. (2001). Skeletal muscle disuse induces ﬁbre type-dependent enhancement of Na+ channel expression. Brain 124, 1100–1113. DATA AVAILABILITY STATEMENT The datasets generated for this study are available on request to the corresponding author. AUTHOR CONTRIBUTIONS CS, AM, and LT conceived and designed the research. CS and GC set up the experiments. LT performed the experiments. CS analyzed the data and prepared the ﬁgures. CS and LT interpreted the results of the experiments and drafted the manuscript. CS, AM, LT, and GC edited and revised the manuscript. All authors contributed to the article and approved the submitted version. DISCUSSION The understanding of the sensorimotor consequences of such short- term immobilization thus appeared of particular interest. We thank Florian Petitcollin for his help in testing participants. We thank Florian Petitcollin for his help in testing participants. ACKNOWLEDGMENTS The studies involving human participants were reviewed and approved by the ethics committee for research in The studies involving human participants were reviewed and approved by the ethics committee for research in REFERENCES doi: 10.1093/ brain/124.6.1100 Rosenkranz, K., Seibel, J., Kacar, A., and Rothwell, J. (2014). Sensorimotor deprivation induces interdependent changes in excitability and plasticity of the human hand motor cortex. J. Neurosci. 34, 7375–7382. doi: 10.1523/ JNEUROSCI.5139-13.2014 Desmurget, M., and Grafton, S. (2000). Forward modeling allows feedback control for fast reaching movements. Trends Cogn. Sci. 4, 423–431. doi: 10.1016/S1364- 6613(00)01537-0 Sainburg, R. L., Ghilardi, M. 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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. Wolpert, D., Ghahramani, Z., and Jordan, M. (1995). An internal model for sensorimotor integration. Science 269, 1880–1882. doi: 10.1126/science. 7569931 July 2020 \| Volume 14 \| Article 696 Frontiers in Neuroscience \| www.frontiersin.org 9
https://openalex.org/W2791374749	https://research.rug.nl/files/56664392/Roman_cooking_vessels_ollae_a_preliminary_study_of_the_material_from_the_Pontine_region_Central_Italy.pdf	English	null	Roman cooking vessels (ollae): a preliminary study of the material from the Pontine region, Central Italy	Science and technology of archaeological research	2,017	cc-by	8,990	University of Groningen Roman cooking vessels (ollae) Borgers, B.; Tol, G.; de Haas, T. Published in: Science and Technology of Archaeological Research DOI: 10.1080/20548923.2018.1445824 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Early version, also known as pre-print Publication date: 2017 Link to publication in University of Groningen/UMCG research database University of Groningen IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Early version, also known as pre-print Publication date: 2017 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Borgers, B., Tol, G., & de Haas, T. (2017). Roman cooking vessels (ollae): A preliminary study of the material from the Pontine region, Central Italy. Science and Technology of Archaeological Research, 3(2), 314-325. https://doi.org/10.1080/20548923.2018.1445824 Citation for published version (APA): Borgers, B., Tol, G., & de Haas, T. (2017). Roman cooking vessels (ollae): A preliminary study of the material from the Pontine region, Central Italy. 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For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 24-10-2024 Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ysta20 ABSTRACT This paper presents the petrographic analysis of cooking vessels (ollae) from the Pontine region, Central Italy, dated between the 4th and the 1st centuries BC. Cooking vessels of three surveys in different parts of the Pontine Plain and around Norba, in the Lepine foothills, are considered. The considered time-span covers the period in which the Pontine region became integrated in the Roman state until the end of the Republic, and cooking vessels have much to tell us about the region’s integration in production and distribution systems, and whether changes occurred therein. The petrographic study shows that the cooking pots were produced and distributed at regional and supra-regional scales. The production and distribution systems that are tentatively inferred show aspects of continuity and change during the time-span considered. Roman cooking vessels that circulated in the Pontine region between the 4th and the 3rd centuries BC had a supra-regional and regional provenance. During the 2nd and the 1st centuries BC, the region continued to have access to these products, as well as to other ones that were produced within and outside the region. Furthermore, the distribution of supra- regional products increased, whereas the importance of existing regional centres decreased in favour of others. Roman cooking vessels (ollae): a preliminary study of the material from the Pontine region, Central Italy B. Borgers, G. Tol & T. de Haas To cite this article: B. Borgers, G. Tol & T. de Haas (2018): Roman cooking vessels (ollae): a preliminary study of the material from the Pontine region, Central Italy, STAR: Science & Technology of Archaeological Research, DOI: 10.1080/20548923.2018.1445824 To link to this article: https://doi.org/10.1080/20548923.2018.1445824 © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 01 Mar 2018. Submit your article to this journal Article views: 76 View related articles View Crossmark data © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 01 Mar 2018. Submit your article to this journal Article views: 76 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ysta20 STAR: SCIENCE & TECHNOLOGY OF ARCHAEOLOGICAL RESEARCH, 2018 https://doi.org/10.1080/20548923.2018.1445824 © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group 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 cited. CONTACT B. Borgers barbara.borgers@sbg.ac.at Roman cooking vessels (ollae): a preliminary study of the material from the Pontine region, Central Italy B. Borgersa, G. Tol b and T. de Haasc aDepartment of Geography and Geology, University of Salzburg, Hellbrunner Straβe 34, 5020 Salzburg, Austria; bSchool of Historical and Philosophical Studies, University of Melbourne, Victoria 3010, Australia; cArchäologisches Institut, Universität zu Köln, Albertus-Magnus- Platz, 50923 Cologne, Germany KEYWORDS Roman cooking vessels; Petrography; Pontine region 1. Introduction cookwares (Leitch, 2011), Pantellerian ware (Santoro Bianchi, 2002), and Pompeian Red Ware (Peña, 1990; Peacock, 1977), to name a few. However, relatively little research to date has been undertaken on Mid- and Late Republican cooking vessels from Central Italy: some studies, using petrographic or chemical analyses, have been carried out on cooking vessels from a number of sites in and around Rome (Bertoldi, 2011; Olcese, 2006; Thierrin-Michael, 2003; Schuring, 1987; Schur- ing, 1986), and Ostia (Capelli, 2016), whereas other work focused on aspects of production and distri- bution, demonstrating that such vessels could be dis- tributed as far as Northern Italy, some 600 km away (Peña, 1992; Olcese, 1990). Studies that use ceramic evidence to investigate the Roman economy have traditionally focused on materials that were traded internationally, such as amphorae and fine ware, highlighting the interconnec- tivity of the Roman world. Only recently, more mun- dane products, such as cooking vessels, have received due attention. Cooking wares are the most commonly encountered group of ceramics in archaeological deposits related to residential sites, given their gener- ally short life-span due to their daily use and frequent handling (Peña, 2007: 57). Moreover, their generally coarse nature makes them suitable for petrographic analysis, providing important indications on how and where these vessels were produced. As a consequence, cooking wares are very suitable to study dominant exchange networks that individual sites and larger regions participated in, and provide vital data for understanding the wider economic phenomena that underlie the manufacture and distribution of these vessels (Spataro & Villing, 2015). Recent research on cooking vessels, dated to between the Late Republican and Imperial periods, highlights their potential in understanding key aspects of ancient economic relations, trade and craft specialisation – North African This article aims to add to the growing corpus of petrographic studies on Roman cooking vessels. It pre- sents the results of a pilot study of two types of cooking jars, dated between the early 4th and the 3rd centuries, and the 2nd and the 1st centuries BC respectively. One is considered to be the typological successor of the other (Olcese, 2003, olla types 2 and 3a). As the ana- lysed fragments were selected from consumption sites only, they are not suited to distil direct relationships between production and consumption. However, understanding the range of fabrics present among the 2 2 B. BORGERS ET AL. 1. Introduction 2 studied material allows to provide information on the complexity of the organization of production, and helps to assess whether production was confined to a single site or to multiple sites in a larger area. The rel- evant fragments derive from sites in the Lepine foot- hills, south of ancient Norba, and from different parts of the Pontine Plain in Southern Lazio, Central Italy. They were collected during three field surveys of the Pontine Region Project (PRP), a long-term research initiative of the Groningen Institute of Archaeology (GIA, University of Groningen, the Neth- erlands), which is currently carried out in close collab- oration with the universities of Melbourne and Cologne. The present study builds upon earlier work by the PRP, which focused on 6th and 5th centuries BC pottery from the Latin town of Satricum (Attema, et al., 2001/2002). morphological features are an ovoid body and a short neck with marked almond-shaped rim. The most rel- evant difference between the two types of vessels is in the typology of rims. Olla type 2 cooking vessels are characterised by a high collared rim with a short convex shaped thicken- ing just below the lip and their diameter varies between 14 and 20 cm (Figure 1). Although the type is known to occur in Southern Etruria and Rome from the 6th cen- tury BC onwards, it does not become common in the Pontine region before the beginning of the 4th century BC (Tol, 2012; de Haas, 2011; Bouma, 1996). In the late 3rd or early 2nd century BC, olla type 3a vessels succeed olla type 2 vessels. They are character- ised by an equally high rim, but have a much more pro- nounced almond shaped thickening below the lip. Their diameter varies generally between 18 and 24 cm (Figure 2) (Bertoldi 2011, 90-91, 95-97; Olcese 2003, type 3a, tav. VIII). Although all fragments from the Pontine region are fairly standardised in shape, a regional variant with a more flattened rim occurs as well (Tol & Borgers, 2016). This paper has three aims: first, to characterise the range of ceramic fabrics present among the studied samples, using thin section petrography. Second, to identify their provenance (or broad area of production) on the basis of the fabric descriptions. 1. Introduction And third, to gain insight in the production technology and distri- bution of Roman cooking vessels in the Pontine region, and whether these systems changed during the time- span covered. Our samples include 46 rim fragments; 16 of olla type 2 vessels, and 30 of olla type 3a vessels (Table 1). The relevant fragments derive from sites and off-site contexts south of the ancient town of Norba on the edge of the Lepine Mountains (de Haas, 2011), around the modern town of Pontinia (de Haas, 2011), and in the surroundings of Forum Appii and Ad Medias, two road stations in the Pontine Plain (Tol, et al., 2014; fig. 3; table 1). Section 2 describes the context of the sample materials and the method used, section 3 presents the different fabrics identified in this study and their poss- ible origin, based on the comparison with published cer- amic fabrics and geological samples. Section 4 puts the results in a wider perspective: it focuses on the technol- ogy of the Roman cooking vessels, highlighting the pres- ence ofpersistent technologicaltraditions inthe Pontine region, and it discusses the preliminary results on the production and distribution patterns of the cooking ware samples that have been analysed in this study. All ceramic rim fragments were prepared as stan- dard 30 µm sections. They were analysed with a polar- ising light microscope, and grouped in petrographic fabrics according to the nature of their inclusions, clay matrix and voids (Quinn, 2013: 73-79). Compo- sitional, textural and shape criteria were used to detect the presence of specific practices, such as clay mixing and the addition of temper (Quinn, 2013: 156-171). 3. Results The samples in this fabric were made from a volcanic clay. Volcanic temper, consisting of a mixture of volca- nic mineral inclusions, has been added. All the vessels were wheel thrown, as suggested by the alignment of thecoarseinclusions andvoids,andfired ata hightemp- erature. Some samples were well fired in an oxidising atmosphere, but the atmospheric conditions appear to have varied within most sherds, and several samples were incompletely oxidised. Both olla type 2 and olla type 3a cooking jars occur in Fabric 1. This fabric is clo- sely related to Fabric 4 in this study. 3.1 Range of ceramic fabric groups present among the cooking vessels 3.1.1 Fabric Group 1: Coarse Sanidine and Augite Fabric (Figure 4.A) This large heterogeneous fabric is defined by the pres- ence of generally moderately to poorly-sorted inclusions of sanidine (3.2 mm), augite and iron (> 2 mm), set in a red base-clay with fine sanidine, biotite and iron inclusions (< 0.2 mm). The coarse sanidine inclusions 3.1 Range of ceramic fabric groups present among the cooking vessels Four main fabric groups were identified among the Roman cooking vessels (Table 2). A summary of the fab- ric groups is given below, and the detailed descriptions of the features of these groups can be found online (http:// minorcenters.gia-mediterranean.nl/petrographic-data/). 3.1.1 Fabric Group 1: Coarse Sanidine and Augite Fabric (Figure 4.A) 2. Archaeological Samples and Method The fabrics of 4th to 1st centuries BC cooking vessels identified in this study were compared with published fabrics of cooking vessels from various sites in and around Rome (Bertoldi, 2011; Thierrin-Michael, 2003), and Ostia (Capelli, 2016), as well as with the GIA’s archive of 6th and 5th centuries BC cooking vessels from the site of Satricum (Attema, et al., 2001/2), with the aim to offer a better consideration of similarities or Our petrographic study focuses on Roman cooking wares that were selected from three surveys in the Pon- tine region. The selected fragments incorporate two types of jars, usually referred to as olla type 2 and olla type 3a (Olcese, 2003). Both types of vessels are common throughout Western Central Italy, and form part of the highly standardized ceramic repertoire characteristic of Mid- and Late Republican sites in the Pontine region (see Tol, 2012, chapter 5, site 15106 for such a typical Republican context). Common p the Pontine region (see Tol, 2012, chapter 5, site 15106 for such a typical Republican context). Common Figure 1. Olla type 2 vessel. Figure 2. Olla type 3a vessel. Figure 2. Olla type 3a vessel. Figure 1. Olla type 2 vessel. STAR: SCIENCE & TECHNOLOGY OF ARCHAEOLOGICAL RESEARCH 3 3 Figure 3. Map of the Pontine region, Central Italy, with the location of the three surveys and sites from which the Roman cooking vessels were selected. Figure 3. Map of the Pontine region, Central Italy, with the location of the three surveys and sites from which the Roman cooking vessels were selected. differences in production and distribution systems between the Late Archaic and Republican periods. Fur- thermore, the petrographic fabrics identified in this study were compared with reference collections of geo- logical samples that are available at the GIA’s Laboratory for Conservation and Material Studies (www.lcm.rug. nl), and in the literature (Peña, 1992; Sevink, et al., 1984), in order to shed light on their provenance. are sub-angular to sub-rounded, and show cracks. The principal mineral inclusions include sanidine and augite, plus occasionally plagioclase and partially bent biotite, and very rarely weathered igneous rock, composed of fine plagioclase inclusions in an opaque matrix, as well as zeolite. The coarse fraction, consisting predominantly of volcanic mineral inclusions, is about 15%, and includes some compositional variation, with a number of samples that are characterised by a variable pro- portion of augite and/or biotite inclusions. 3.1.2 Fabric Group 2: Coarse Augite Fabric Group (Figure 4.B) Fabric 1 Olla type 2 Site 14053 Forum Appii 1 2 Olla type 2 Site 14003 Ad Medias 1 3 Olla type 2 Site 14016 Ad Medias 1 4 Olla type 2 Site 14053 Forum Appii 1 5 Olla type 2 Site 14062 Forum Appii 1 6 Olla type 2 Site 13433 Pontinia 1 7 Olla type 2 Site 14026 Ad Medias 2 8 Olla type 2 Site 14017 Ad Medias 2 9 Olla type 2 Site 14053 Forum Appii 2 10 Olla type 2 Site 14041 Ad Medias 2 11 Olla type 2 Site 14019 Ad Medias 2 12 Olla type 2 Site 12309 Pontinia 2 13 Olla type 2 Site 12408 Norba 2 14 Olla type 2 Site 10952 Norba 2 15 Olla type 2 Site 12408 Norba 2 16 Olla type 2 Site 13433 Pontina 2 17 Olla type 3a Unit ID 101233/01/ 01 Ad Medias 1 18 Olla type 3a Site 14003 Ad Medias 1 19 Olla type 3a Ad Medias Ad Medias 1 20 Olla type 3a Site 14044 Ad Medias 1 21 Olla type 3a Site 14003 Ad Medias 1 22 Olla type 3a Unit ID 101724/01/ 03 Ad Medias 1 23 Olla type 3a Ad Medias Ad Medias 1 24 Olla type 3a Site 14033 Ad Medias 1 25 Olla type 3a Site 14053 Forum Appii 1 26 Olla type 3a Forum Appii Forum Appii 1 27 Olla type 3a Unit ID 100140/2/8 Ad Medias 1 28 Olla type 3a Site 12307 Pontinia 1 29 Olla type 3a Unit ID 1433/01/01 Pontinia 1 30 Olla type 3a Forum Appii Forum Appii 2 31 Olla type 3a Site 14006 Ad Medias 2 32 Olla type 3a Unit ID 101590 Ad Medias 3 33 Olla type 3a Unit ID 101590 Ad Medias 3 34 Olla type 3a Site 14053 Forum Appii 3 35 Olla type 3a Ad Medias Ad Medias 3 36 Olla type 3a Ad Medias Ad Medias 3 37 Olla type 3a Ad Medias Ad Medias 3 38 Olla type 3a Site 14040 Ad Medias 3 39 Olla type 3a Ad Medias Ad Medias 3 40 Olla type 3a Site 14053 Forum Appii 3 41 Olla type 3a Site 14003 Ad Medias 3 42 Olla type 3a Site 14034 Ad Medias 4 43 Olla type 3a Ad Medias Ad Medias 4 44 Olla type 3a Site 14038 Ad Medias 4 45 Olla type 3a Site 14034 Ad Medias 4 46 Olla type 3a Site 12305 Pontinia 4 The samples in Fabric 2 were made from a tuff deposit. 3.1.2 Fabric Group 2: Coarse Augite Fabric Group (Figure 4.B) This fabric is characterised by well-sorted, sand-sized augite and sanidine inclusions (< 2 mm), set in a red B. BORGERS ET AL. 4 4 Table 1. 46 samples of Roman cooking vessels from rural sites and off-site contexts in the Pontine region. Table 1. 46 samples of Roman cooking vessels from rural sites and off-site contexts in the Pontine region. Table 2. Four different fabrics were identified among the 46 Roman cooking vessels. Table 2. Four different fabrics were identified among the 46 Roman cooking vessels. Vessel shape Samples Petrographic Fabric Nr. of Samples Olla type 2 1, 2, 3, 4, 5, 6 Coarse Sanidine and Augite Fabric 6 Olla type 3a 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 Coarse Sanidine and Augite Fabric 13 Olla type 2 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 Coarse Augite Fabric 10 Olla type 3a 30, 31 Coarse Augite Fabric 2 Olla type 3a 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 Coarse Calcite Fabric 10 Olla type 3a 42, 43, 44, 45, 46 Coarse Sanidine Fabric 5 b l ith fi idi d bi tit (0 2 ) Th Table 1. 46 samples of Roman cooking vessels from rural sites and off-site contexts in the Pontine region. Sample Vessel shape Context Survey Petro. 3.1.2 Fabric Group 2: Coarse Augite Fabric Group (Figure 4.B) Coarse inclusions and voids show a preferred alignment, suggesting that the vessels were wheel thrown. Some vessels were well fired in an oxidising atmosphere, while most show evidence for different fir- ing horizons. All but two vessels were fired at a high temperature, and this fabric comprises both olla type 2 and olla type 3a cooking jars. 3.1.3 Fabric Group 3: Coarse Calcite Fabric Group (Figure 4.C) This fabric is defined by the presence of moderately to well-sorted, angular to sub-angular inclusions of calcite (< 3.5 mm) and sanidine (< 2-1 mm), set in a red base- clay with fine sanidine and iron (< 0.2 mm). The coarse fraction is about 18%. The fresh nature of the calcite inclusions suggests that they were added to the clay matrix, rather than being the product of weathering, and they appear to derive from limestone rock. The principal minerals include sanidine, plus less common iron. Given that the sanidine inclusions are well-sorted, and sub-angular to sub-rounded, the raw materials used to produce these ceramics appear to have come from a volcanic clay. The presence of micrite in voids, and the absence of calcareous textural features in the clay matrix, suggests that these fine-grained min- erals are secondary, rather than the result of the mixing of two clay sources. The samples in this fabric were made from a volca- nic clay deposit. It appears that crushed calcite has been used as temper, and added in a moderately sorted con- dition to this base clay. Inclusions and voids are ran- domly orientated in the core of the section, and aligned near the rim, suggesting that the vessels were handmade and wheel finished. Most, if not all, samples were fired at a high temperature, and calcite inclusions have been fired out, leaving characteristic voids behind. There also appears to be considerable variety in firing atmosphere, given that some samples are oxidised, others are reduced, but the majority of the samples are characterised by different firing horizons. This evi- dence suggests that the firing process was considerably base-clay with fine sanidine and biotite (0.2 mm). The principal mineral inclusions include augite and sani- dine, plus less common biotite and chert. Occasionally, zeolite can be identified as euhedral microphenocrysts of leucite in an opaque to brownish groundmass (< 3 mm), whereas leucite and olivine occur rarely (< 3 mm). The coarse fraction is about 10%. The rounded nature of the augite and sanidine inclusions suggests that they were the product of weathering. The presence of these volcanic mineral inclusions, as well as the com- position of rock fragments, suggests that the clay used to produce these ceramics was a volcanic tuff. STAR: SCIENCE & TECHNOLOGY OF ARCHAEOLOGICAL RESEARCH 5 Figure 4. 3.1.4 Fabric Group 4: Coarse Sanidine Fabric Group (Figure 4.D) This coarse to fine-grained fabric is characterised by the presence of generally moderately well-sorted, sub- angular to sub-rounded inclusions of sanidine (gener- ally around 2 mm, rarely < 3 mm), in a red base-clay with fine sanidine, biotite and iron (< 0.2 mm). The coarse inclusions of sanidine show cracks and are pre- dominant, whereas iron and augite occur rarely. The coarse fraction varies between 10% and 15%. Some tex- tural variation has been included in the Coarse Sani- dine Fabric, with some samples characterised by a higher proportion of poorly-sorted, coarse sanidine inclusions (< 3 mm), and others with comparatively more fine-grained and well-sorted inclusions of sani- dine (< 2 mm). This evidence might be taken to suggest that a different size fraction of temper material was used to manufacture the vessels. 3.1.3 Fabric Group 3: Coarse Calcite Fabric Group (Figure 4.C) Micrographs of ceramic thin sections in XP: Coarse Sanidine and Augite Fabric (A), Coarse Augite Fabric (B), Coarse Calcite Fabric (C), Coarse Sanidine Fabric (D). Width of individual images = 5.8 mm. Figure 4. Micrographs of ceramic thin sections in XP: Coarse Sanidine and Augite Fabric (A), Coarse Augite Fabric (B), Coarse Calcite Fabric (C), Coarse Sanidine Fabric (D). Width of individual images = 5.8 mm. uncontrolled. All the samples of Fabric 3 belong to olla type 3a vessels. Sanidine and Augite Fabric), but with the difference that the clay matrix in this fabric comprises a larger fine-grained fraction, and coarse augite inclusions occur only rarely. 3.2 Comparison with other ceramic fabrics Fabric 1 is the largest group in this study. This fabric is closely related to the ‘Rome and Tiber Valley Fabric’, which is characterised by volcanic temper, dominated by sand-sized inclusions of sanidine, and variable amounts of augite (clinopyroxene), plus less common biotite and plagioclase, and rarely leucite (Bertoldi, 2011: 116; Thierrin-Michael, 2003: 55-59). The ‘Rome and Tiber Valley Fabric’ has been identified on various sites in and around Rome and Northern Lazio, and was used to produce a wide range of cooking vessels, including different types of ollae (Bertoldi, 2011: 113- 121; Thierrin-Michael, 2003: 55-59; Schuring 1987; Schuring 1986). This fabric is likely to comprise several local fabrics from production centres around Rome and further upstream the Tiber River, such as Vasa- nello (Peña, 1992), given that it includes considerable compositional variation (Thierrin-Michael, 2003: 55-59). Also, Fabric 1 in our study is characterised by a mixture of different volcanic mineral inclusions, and it is likely that it can be subdivided into several smaller groups. However, a larger dataset of ceramic thin sections would have to be examined before this can be accomplished. The raw materials used to produce the ceramics in Fabric 4 appear to have come from a volcanic clay. Vol- canic temper, in the form of sanidine inclusions, has been added in a moderately sorted condition. All the vessels were wheel thrown, and fired at a high tempera- ture. However, differences can be noted in the firing atmosphere: some vessels were well fired in either an oxidising or a reducing atmosphere, whereas others show evidence for different firing horizons. All the samples in this fabric comprise olla type 3a cooking vessels. This fabric is similar to Fabric 1 (the Coarse Fabric 2 is closely related to the ‘Mineralogical Group 5’ Fabric, which is defined by the presence 6 B. BORGERS ET AL. B. BORGERS ET AL. 6 Table 3. Geological samples analysed in this study. 3.3 Comparison with raw material samples Fabric 3 in this study does not appear to be related to any published Central Italian ceramic fabric. Yet still, the surviving coarse inclusions indicate that they derive from limestone rocks, which characterise the Lepine Mountains to the southeast of Rome (Figure 5). In this mountainous area, pottery is known to having been pro- duced at the site of Segni during the Mid-Republican period (Olcese, 2011: 149-150, 203-207; Olcese, 2003: 14). Two misfired cooking vessels from the site have been analysed chemically (Olcese, 2003: 160 table 6), one of which is low in calcite (1, 42%), whereas the other is calcareous (14,27%) (Olcese, 2003: 167). How- ever, these samples were not examined in thin section petrography, making it difficult, therefore, to confirm whether Fabric 3 in this study was produced at Segni. Alternatively, Fabric 3 might have been produced in the Pontine region, and there is some evidence to sup- port this hypothesis: in four samples of Fabric 3, it can be seen that coarse calcite inclusions have burnt out (section 3.1.3). Given that temperatures reached during cooking are not sufficient to result in decomposition of mineral inclusions, the calcite must have burnt out during the initial firing process. All four samples derive from the site at Ad Medias (Table 1), suggesting that this site was the place of production (Tol & Borgers, 2016). Pottery is known to having been produced at Ad Medias during the Late Republican period (Tol, et al., 2014). The ceramics include tiles and amphorae, which occur in two petrographic fabrics, respectively the ‘Colluvial Fabric’ and the ‘Clay Mixing Fabric’ (Borgers, et al., in press). However, neither fabric is related to Fabric 3 in this study. In order to identify the possible raw materials used to produce the Roman cooking vessels examined in this study, geological samples and loose sandy deposits, which have been collected by the GIA in the Pontine region, were examined and compared with the ident- ified ceramic fabrics (Table 3) (see Attema, et al., 2001/2: 378; Nijboer, 1998: 121-129 for geological sampling carried out at Satricum; see Borgers, et al., in press, for clay prospection carried out in the context of Roman pottery production in the region). The Pontine region lies on the Tyrrhenian coast, c. 60 km south of Rome. 3.2 Comparison with other ceramic fabrics Sample Site Type Description Before firing After firing 1 Forum Appii Clay Fluvio-colluvial clay Deep brown Deep red 2 Forum Appii Clay Fluvio-colluvial clay Deep brown Deep red 3 North of Forum Appii Clay Fluvio-colluvial clay Deep brown Deep red 4 North of Forum Appii Clay Fluvio-colluvial clay Deep brown Deep red 5 Forum Appii Sand Clinopyroxene, leucite, olivine - - 6 Satricum Clay Mid-Pleistocene tuffs Light-coloured Pale pink to orange 7 Sezze/Bassiano Clay Weathered volcanic tuff Light-coloured Orange 8 Sezze/Bassiano Clay Weathered volcanic tuff Light-coloured Orange 9 Site 11232 Sand Clinopyroxene - - 10 Site 12317 Sand Sanidine, clinopyroxene, biotite, iron-manganese concretions - - Table 3. Geological samples analysed in this study. Campania has been proposed (Thierrin-Michael, 2003: 56). One reason why these samples may not have been produced in Northern Campania is that, according to the results of the chemical analysis, they are grouped with the samples of the large ‘Rome and Tiber Valley Fabric’ (Olcese, 2003: 49-50, 87). of coarse weathered volcanic rock inclusions, sani- dine, plagioclase, clinopyroxene, and rare biotite, leu- cite and olivine, in a red base-clay (Capelli, 2016: 196- 198). The ‘Mineralogical Group 5’ Fabric has been identified in olla type 3a vessels from Ostia (Olcese & Coletti, 2016), for which a provenance from either Southern Lazio or Campania has been proposed (Capelli, 2016: 198). 3.3 Comparison with raw material samples It consists of a plain, which is bounded by the Alban Hills in the north and the Lepine Mountains to the east. From a geological point view, the region can be described as a graben, which is filled with deposits of several marine terraces, and Holocene fluvio-colluvial deposits. In the north- west, these sediments merge with the lower slopes of the Volcano Laziale, whereas limestone rocks border the graben to the north and east (Figure 5) (Sevink et al., 1984). Relevant deposits for the purpose of this study are a) poorly-sorted Holocene fluvio-colluvial clay deposits, which contain abundant volcanic material (sanidine, clinopyroxene, biotite and leucite), b) Mid-Pleistocene volcanic tuff deposits with abundant volcanic material (leucite, clinopyroxene, olivine, biotite), and their weathered deposits, and c) limestone-based colluvial deposits with carbonate rock fragments and admixture of volcanic material (clinopyroxene, sanidine) (Sevink, et al., 1984). All these sediments are high in kandite/ kaolinite clay minerals, and fire to a red, or a pale pink to orange colour (Table 3) (Sevink, et al., 1984). Fabric 4 appears to be similar to the well-sorted, fine-grained sanidine-rich fabric that has been ident- ified at the Tempio della Concordia in Rome (Thier- rin-Michael, 2003: 56). For the samples from the Tempio della Concordia, a provenance from either the Rome and Tiber Valley region, or Northern Volcanic tuff deposits, containing a mixture of coarse inclusions of clinopyroxene, biotite, olivine, leu- cite, and chert, may have been used as a base-clay for Fabric 2 (‘Coarse Augite Fabric’). This fabric comprises STAR: SCIENCE & TECHNOLOGY OF ARCHAEOLOGICAL RESEARCH 7 STAR: SCIENCE & TECHNOLOGY OF ARCHAEOLOGICAL RESEARCH Figure 5. Geological map of the Pontine region (after Sevink, et al., 1984), with indication of Roman pottery production sites and geological samples. Figure 5. Geological map of the Pontine region (after Sevink, et al., 1984), with indication of Roman pottery production sites and geological samples. (Borgers, et al., in press). However, besides the pres- ence of coarse inclusions of sanidine, these sediments also comprise considerable quantities of clinopyroxene. Indeed, this mineral occurs everywhere in the Pontine Plain and the coastal area (Sevink, et al., 1984) (Figure 5; Table 3: geological sample 9). In addition, clay deposits containing alkaline volcanic sands (sanidine, plagioclase) appear to occur in restricted areas of Northern Lazio and Northern Campania (D’Ambrosio, et al., 2015; Thierrin-Michael, 2003: 56; Peña, 1992: 116 footnote 10; Giannetti & Luhr, 1983). 4.1 Technology of cooking vessels The petrographic analysis of the Roman cooking vessels that were presented in this study show impor- tant similarities and differences at the compositional, technological or typological levels. All the studied cookware samples were produced with a red base-clay of volcanic origin. The use of red-firing clay for the manufacture of these utilitarian vessels appears to have been a tradition that potters shared and passed on for centuries (Olcese, 2003: 60- 61; Thierrin-Michael, 2003: 55-59). Fabric 2 is a case in point. This fabric appears to be closely related to the ‘Coarse gritty/leucite-lava and leucite/tuff’ Fabric from Satricum (Fabric 12 in Attema, et al., 2001/2: 375, 378; fig. 5). This fabric was identified in waster products from a kiln site, dated to around the late 6th century BC, and used to produce storage jars, bowls, ollae and tiles (Nijboer, 1998: 121-129). Both Fabric 2 in this study and Fabric 12 in the Late Archaic kiln site from Satricum are characterised by the pres- ence of sand-sized inclusions of predominant augite, plus less common biotite, chert, and rare leucite, zeolite and olivine, suggesting that similar raw materials were used to manufacture ollae between the Late Archaic and Roman periods. However, there appear to exist technological differences between the two fabrics. More specifically, the Late Archaic vessels in Fabric 12 are characterised by the presence of poorly-sorted coarse inclusions, and they appear to have been mod- elled on a turntable (Nijboer, 1998: 121-129). By com- parison, the Roman vessels in Fabric 2 are defined by the presence of well-sorted, smaller coarse inclusions, set in a homogeneous clay matrix. This evidence might be taken to suggest that the clay deposit was refined - i.e. coarse inclusions were removed - for the manufacture of Roman cooking vessels. Also, the evi- dence in thin section suggests that the Roman vessels were wheel thrown. However, it is difficult to support this hypothesis by macroscopic observations, because the sherds tend to be abraded, and comprise the rims of the vessels most of the time. Some samples in Fabric 1, Fabric 2 and Fabric 4 are well fired in oxidising atmosphere, but most samples are characterised by different firing horizons. Histori- cal sources suggest that ollae may have had various functions (Hilgers, 1969: 39-40). However, samples from the Pontine region are typically scorched by fire, suggesting that they were primarily used as cook- ing pots. 4.1 Technology of cooking vessels Both Fabric 1 and Fabric 2 comprise olla type 2 and olla type 3a cooking vessels, indicating that the work- shops that produced them were active between the 4th and the 1st centuries BC. By contrast, Fabric 3 and Fabric 4 comprise exclusively ollae type 3a of vessels, suggesting that the activity of the workshops that produced them was mainly between the 2nd and the 1st centuries BC. 3.3 Comparison with raw material samples If Fabric 3 is a local fabric from this site, then the raw materials used to produce the cooking vessels should have been transported, given that Ad Medias is situated on marine sediments of the Borgo Ermada complex (Figure 5). One argument that sup- ports this hypothesis is that the raw materials used to produce tiles and amphorae at the site during the Late Republican period were also transported (Borgers, et al., in press). Fabric 4, comprising the bulk of the samples analysed, are defined by the presence of predominant sanidine inclusions, and less common augite, Fabric 2 by the pres- ence of augite inclusions, whereas Fabric 3 is character- ised by coarse calcite. Utilitarian performance is one design premise alongside cultural or social factors relat- ing tospecific times orplaces,and availability.Given that these types of inclusions appear to have been confined to the Tiber Valley region/Northern Campania, Southern Lazio, and the Lepine Mountains respectively, they might be relevant in the light of the latter premise, and taken to reflect the specific natural landscape. production centre is Ad Medias, which is located in the Pontine Plain. If Fabric 3 is a local fabric from this site, then the raw materials used to produce the cooking vessels should have been transported, given that Ad Medias is situated on marine sediments of the Borgo Ermada complex (Figure 5). One argument that sup- ports this hypothesis is that the raw materials used to produce tiles and amphorae at the site during the Late Republican period were also transported (Borgers, et al., in press). 4. Discussion The orientation of the coarse inclusions and voids, as seen in thin section analysis, help interpreting the form- ing technique of the vessels. Following on from the evi- dence seen in the ceramic thin sections, it seems that the samples of Fabric 1, Fabric 2, and Fabric 4 were wheel made, whereas those of Fabric 3 appear to have been handmade and wheel finished. Ideally, these obser- vations are combined with macroscopic analysis of the sherds analysed. However, this proved to be difficult, because the sherds that were analysed in this study derive from surveys, and tend to be weathered. 3.3 Comparison with raw material samples Therefore, it seems unlikely that Fabric 1 and Fabric 4 were pro- duced in the Pontine region. both olla type 2 and olla type 3a vessels, and occurs throughout the Mid- and Late Republican periods in the Pontine region. Geological prospection indicated that volcanic tuffs outcrop near the site at Satricum (Figure 5; Table 3: geological sample 6), where they have been used to manufacture pottery and tiles during the Late Archaic period (Attema et al., 2001/2: 378). Weathered volcanic clay deposits, rich in fine frac- tion inclusions of sanidine and biotite, are related to the fine, red base-clay that has been used to produce the vessels in Fabric 1 (‘Coarse Sanidine and Augite Fabric’), and Fabric 4 (‘Coarse Sanidine Fabric’). Geo- logical prospection indicates that such fine-grained red clay deposits occur around Sezze/Bassiano (Figure 5; Table 3: geological samples 7-8). However, it should be noted that the clay deposits are characterised by the presence of iron-rich clay pellets, which have not been identified in the two ceramic fabrics. In addition, from the evidence seen in thin section, both ceramic fabric groups appear to have been tempered with coarse inclusions of sanidine, and less common augite. Sandy fluvio-colluvial sediments are abundant in ditches in the Pontine plain (Figure 5; Table 3: geologi- cal samples 5 and 10), and they appear to have been used as tempering material to manufacture Roman cer- amic building material and storage jars in the region Limestone-based colluvial deposits may have been used as a base-clay for Fabric 3 (‘Coarse Calcite Fab- ric’), as they typically comprise carbonate rock frag- ments, iron, as well as volcanic minerals (clinopyroxene, sanidine) (Sevink, et al., 1984; Spaarga- ren, 1979). These deposits occur on the slopes of the Lepine Mountains (Figure 5). It is difficult to allocate a centre of production for Fabric 3 with certainty, nevertheless two centres of production are potential candidates: the first is Segni, because of its location in the Lepine Mountains. If Fabric 3 was produced here, indeed, then did pottery production at the site continue until the Late Republican period. The second potential 8 B. BORGERS ET AL. 8 8 production centre is Ad Medias, which is located in the Pontine Plain. 4.2 Correlation of fabrics and surveys: distribution patterns A careful examination of the fabrics and their distri- bution allow us to gain insight in the economic net- works wherein the Pontine region was integrated, and changes that occurred therein between the 4th and the 1st centuries BC. Of the sixteen samples of olla type 2 cooking vessels, dated to the Mid-Republican period, ten belong to Fab- ric 2. Hence, the majority of the cooking vessels were regional products, which were manufactured in the northwestern area of the Pontine region, possibly at or near Satricum. They circulated widely within the region, given that they were identified on various sites in the Pontine Plain, as well as around Norba (Figure 6; Table 2). The six remaining samples belong The samples also differ in terms of the nature and size of the coarse inclusions. More specifically, Fabric 1 and STAR: SCIENCE & TECHNOLOGY OF ARCHAEOLOGICAL RESEARCH 9 9 Figure 6. Map showing the distribution of Fabric 1 and Fabric 2 of olla type 2 cookware samples in the Pontine region, dated between the 4th and the 1st centuries BC. Figure 6. Map showing the distribution of Fabric 1 and Fabric 2 of olla type 2 cookware samples in the Pontine region, dated between the 4th and the 1st centuries BC. to Fabric 1, which appears to have been produced in the wider Rome and Tiber Valley region. Whilst pre- vious studies have demonstrated that pottery from the Rome and Tiber Valley region were distributed to Northern Italy (Peña, 1992: 117; Olcese, 1990), and Rome (Thierrin-Michael, 2003), the results of our study suggest that the Pontine region was also inte- grated in the extensive distribution network of this fab- ric (Figure 6; Table 2). to Fabric 1, which appears to have been produced in the wider Rome and Tiber Valley region. Whilst pre- vious studies have demonstrated that pottery from the Rome and Tiber Valley region were distributed to Northern Italy (Peña, 1992: 117; Olcese, 1990), and Rome (Thierrin-Michael, 2003), the results of our study suggest that the Pontine region was also inte- grated in the extensive distribution network of this fab- ric (Figure 6; Table 2). distribution systems, with fewer cooking vessels from the northwestern part of the Pontine region, and comparatively more cooking vessels from other parts of the region. 4.2 Correlation of fabrics and surveys: distribution patterns Further research is needed to explore the distri- bution of Roman cooking pots in the Pontine region in more depth, but the developments described above are consistent with the archaeological background of the Pontine region. Satricum, which is located in the northwestern part of the Pontine region (Figure 5), seems to have been of regional importance with local pottery production during the Archaic and the Mid- Republican periods (Nijboer, 1998). However, other sites and centres of production in the region gained importance in the Late Republican period. For instance, pottery production has been attested on coastal sites, including the Roman villa of Le Grot- tacce (de Haas, et al., 2008), and on sites that were located on the Via Appia, such as Ad Medias and Forum Appii (Borgers, et al., in press; Tol & Borgers, 2016; Tol, et al., 2014; fig. 1). Potters (or middlemen) would have sold their products at or close to the pro- duction site, or they might have participated in peri- odic markets, which would have taken place at most regional centres or sanctuaries. In this context, it might be interesting to note that Forum Appii is thought to have been such a centre where various A more complex picture emerges for the Late Republican period. Of the thirty samples of olla type 3a cooking vessels, thirteen belong to Fabric 1, whereas ten samples belong to Fabric 3, and five to Fabric 4. Fabric 2 is also present, albeit with two samples only (Figure 7; Table 2). Hence, the supra- regional and regional products (Fabric 1 and Fabric 2 respectively) that were distributed in the region during the Mid-Republican period continued to cir- culate during the Late Republican period, but with the difference that the region had access to other pro- ducts of supra-regional and regional origin as well (Fabric 4 and Fabric 3 respectively). In addition, when the changing ratio in the number of supra- regional and regional fabrics is considered, it might tentatively be suggested that the trade of supra- regional cooking vessels increased during this period, and a shift occurred in regional production and 10 10 B. BORGERS ET AL. 10 Figure 7. Map showing the distribution of Fabric 1, Fabric 2, Fabric 3 and Fabric 4 of olla type 3a cookware samples in the Pontine region, dated between the 2nd and the 1st centuries BC. Figure 7. 4.2 Correlation of fabrics and surveys: distribution patterns Map showing the distribution of Fabric 1, Fabric 2, Fabric 3 and Fabric 4 of olla type 3a cookware samples in the Pontine region, dated between the 2nd and the 1st centuries BC. products were distributed (Tol & Borgers, 2016; Tol, et al., 2014). to the Late Archaic period, indicating that technologi- cal knowledge dates back at least to that time. The other two fabrics, Fabric 3 and Fabric 4, had a regional and supra-regional provenance respectively, and they circulated in the region during the 2nd and the 1st cen- turies BC. 5. Conclusions This paper has presented a pilot study of a limited number of two types of Roman cooking vessels from the Pontine region, Central Italy, dated between the 4th and the 1st centuries BC. This study had three main aims. The first aim was to identify and character- ise the ceramic fabrics present among the cooking vessels studied, using petrographic analysis. The second aim was to identify their provenance on the basis of published ceramic fabrics and geological samples, whilst the third aim was to reconstruct distri- bution patterns of this utilitarian ware within the Pon- tine region, and to identify possible changes therein. As for the second and the third aim, the production and distribution patterns that are tentatively inferred from the Roman cooking vessels in this study show both continuity and change between the 4th and the 1st centuries BC. The workshops that produced cook- ing pots of Fabric 1 and Fabric 2 seem to have been located in the Tiber Valley region and the northwestern part of the Pontine region respectively. These cooking vessels were distributed to different parts of the Pontine Plain and around Norba, the Lepine foothills, indicat- ing that the region of study was integrated in supra- regional and regional distribution networks during the 4th and the 3rd centuries BC. The Pontine region continued to participate in these networks during the 2nd and the 1st centuries BC, but other supra-regional and regional products also circulated in the region at this time. With respect to the first aim, four different fabrics were identified among the Roman cooking vessels. Fab- ric 1 and Fabric 2 had a supra-regional and regional provenance respectively, and were being produced and used between the 4th and the 1st centuries BC. Similar types of raw materials appear to have been used to manufacture both olla type 2 and olla type 3a vessels, suggesting that potters passed on technological knowledge through time. In addition, Fabric 2 appears to be related to a regional fabric from Satricum, dated More research would be needed in order to better understand the observed developments, and how they relate to broader economic and demographic develop- ments, such as the integration of regional markets in STAR: SCIENCE & TECHNOLOGY OF ARCHAEOLOGICAL RESEARCH 11 11 11 Rome’s Suburbium, and population growth (Morley, 1996). 5. Conclusions Future research would need to have a more robust quantitative basis, and use an integrated analyti- cal approach. More specifically, a large group of samples would need to be selected from all parts of the Pontine region, in order to gain insight in the var- ious distribution networks wherein the region partici- pated. Also, focused chemical investigation is needed to locate the production centres of Roman cooking vessels. Hence, large-scale scientific research on these utilitarian vessels would illuminate key aspects of their production organisation and trade networks. De Haas, T., P. A. J. Attema, and H. Pape, 2008. “Amphorae from the coastal zone between Anzio and Torre Astura (Pontine Region, Central Italy): the GIA excavations at Le Grottacce, a local amphora collection and material from surveys in the Nettuno area.” Palaeohistoria, 49/ 50: 517–616. Giannetti, B., and J. F. Luhr. 1983. “The white trachytic tuff of Roccamonfina Volcano (Roman Region, Italy).” Contrib. Mineral. Petrol. 83:235–252. Hilgers, W. 1969. “Lateinische Gefässnamen. Bezeichnungen, Funktion und Form römischer Gefäße nach den antiken Schriftquellen.” Beihefte der Bonner Jahrbücher, 31. LaboratoryforConservationandMaterialStudiesGroningenI- nstituteofArchaeology. [online] [Accessed 15 March 2017]. Available at: < www.lcm.rug.nl>. Leitch, V. 2011. Harbour to Desert, Emporium to Sanctuary: Trade in North African Cookwares. Proceedings of the XVII Congresso Internazionale di Archeologia Classica: 11–23. G. Tol http://orcid.org/0000-0003-0743-5173 Olcese, G. 2003. Ceramiche comuni a Roma e in area romana: produzione, circolazione e tecnologia (tarda età repubblicana – prima età imperiale). Mantova: Documenti di Archeologia, 28. Acknowledgements The research presented in this paper is part of the project ‘Fora, stationes, and sanctuaries: the role of minor centres in the economy of Roman Central Italy’, which has been funded by the Netherlands Organisation for Scientific Research (grant number 360-61-030). The authors would like to thank the Groningen Institute for Archaeology, Uni- versity of Groningen, for their additional support, and Prof. Jan Sevink (UvA), for sharing his knowledge on the geology of the Pontine region. Thanks are also owed to STAR’s anon- ymous reviewers, who read and commented on an earlier draft of this paper. Any mistakes that remain are entirely the authors’ responsibility. MinorCentresProjectGroningenInstituteofArchaeology. [online] [Accessed 15 March 2017]. Available at: < http:// minorcenters.gia-mediterranean.nl/petrographic-data/>. Morley, N. 1996. Metropolis and hinterland: The City of Rome and the Italian Economy, 200 BC-AD 200. Cambridge: Cambridge University Press. Nijboer, A. 1998. From household production to work- shops: Archaeological evidence for economic transform- ations, pre-monetary exchange and urbanisation in central Italy from 800 to 400 BC. PhD thesis, University of Groningen. 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M. 1986. “The Roman, early Medieval and Medieval coarse kitchen wares of the San Sisto Vecchio in Rome: continuity and break in tradition.” BABesh, 61: 158–207. Tol, G. 2012. A fragmented history: A methodological and artefactual approach to the study of ancient settlement in the territories of Satricum and Antium. Eelde/ Groningen: Barkhuis Publishing. Schuring, J. M. 1987. “Supplementary note to the Roman, early Medieval and Medieval coarse kitchen wares of the San Sisto Vecchio in Rome: the distribution of the fab- rics.” BABesh, 62: 109–129. Tol, G., and B. Borgers. 2016. “An Integrated Approach to the study of local production and exchange in the lower Pontine Plain.” Journal of Roman Archaeology, 29: 349–370. Sevink, J., J. A. Remmelzwaal, and O. C. Spaargaren. 1984. The Soils of Southern Lazio and Adjacent Campania. Fysisch Geografisch en Bodemkundig Laboratorium. University of Amsterdam, 38. Tol, G., T. De Haas, K. Armstrong, and P. A. J. Attema. 2014. “Minor Centres in the Pontine Plain: the cases of Forum Appii and Ad Medias.” Papers of the British School at Rome, 82: 109–134. Spaargaren, O. C. 1979. “Weathering and soil formation in a limestone area near Pastena (Fr., Italy).” Fysisch
https://openalex.org/W1992630997	https://sajlis.journals.ac.za/pub/article/download/1263/1194	English	null	How LIS professionals can use alerting services, Ina Fourie	South African journal of libraries and information science/South African journal of library and information science	2,013	cc-by-sa	593	97 97 How LIS professionals can use alerting services Ina Fourie Chandos: Oxford, 2006. 178p ISBN I 84334 128X (pbk.) Price: unknown How LIS professionals can use alerting services Ina Fourie Chandos: Oxford, 2006. 178p ISBN I 84334 128X (pbk.) Price: unknown This book focuses on the usefulnessof alerting services for LIS professionals. It is a valuable and useful resource as it draws attention to the new and existing current awareness services which can be easily retrieved for updating ones information data banks.The availability of these services does not always mean that they are exploited for advancement of knowledge either by individuals or groups. This book provides a variety of definitions of current awarenessservices by severalauthors. The book is divided into seven chapters, a detailed bibliogr~phy and an alphabetical index at the end. In the introductory chapter, the author explains about the need for alerting services and reasons why professionals must use these services. Shedescribes how the challengesaffect the professionalsin their practice and how they can monitor such challenges through alerting services. For example, she says that there is a need for information professionals to understand their own information behaviour in order for them to make use of the alerting services to improve their knowledge generation. In Chapter Two, the author explainsthe concepts of alerting servicesand the early development of current awareness services (CAS), and the rationale for offering these services. Chapter Three explains the relationship between the environment and the LISprofessionals in practice, aswell as LISeducators. The variety of alerting servicesavailablefor the benefit of LISprofessionalsare dealt with in the next chapter. The web site addressesprovided can be used by novices for monitoring events, keeping track of trends and new research reports. Chapter Fivedescribes in relative detail the information-seeking behaviour of LISprofessionalsand how this relates to the use of seeking and alerting services. The questions that can be asked are: (I) How do they try to meet their information needs (e.g. bridging the gap)?(2) What knowledge do they need and what do they know that will enable them to use alerting services successfully?(3) What barriers influence their use of alerting services? The range of topics covered by the different chapters is quite diverse. This makes it a valuable resource for researchers and novice researcherswho want an overview of current and alerting services. Reviewed by: Dr DaisyJacobs,Senior Lecturer, Department of Library and Information Science,University of Zululand, South Africa. Tel.: (035) 902 6832 Email: djacobs@pan.uzulu.ac.za 97 The book not only gives a wealth of information on current awarenessservices both manual and web-based, but also provides detailed accounts of electronic newsletters, discussion lists, article alerting services, weblogs, as well as automated tracking tools. Much hasbeen detailed about the positive aspects of alerting services without neglecting the details of the negative aspectsof alerting services and how to deal with them. As seen from the background information provided about each alerting service, one is made to think of the benefits more than the disadvantagesof usingthem. It is hoped that all LIS professionals would be able to relate to the issuesdiscussed concerning alerting services and make greater use of them. g The book will be useful to LIS professionals but to academics and all those interested in keeping abreast with their research. It is very readable, providing valuable information that can be used to benchmark a library, aswell as ideas on how to introduce the alerting servicesto students and staff. Reviewed by: Dr DaisyJacobs,Senior Lecturer, Department of Library and Information Science,University of Zululand, South Africa. Tel.: (035) 902 6832 Email: djacobs@pan.uzulu.ac.za SA Jnl Libs & Info Sci 2008, 74(I)
https://openalex.org/W2972169767	http://cds.cern.ch/record/2687051/files/scoap3-fulltext.pdf	English	null	Search for non-resonant Higgs boson pair production in the bbℓνℓν final state with the ATLAS detector in pp collisions at <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si1.svg"><mml:msqrt><mml:mrow><mml:mi>s</mml:mi></mml:mrow></mml:msqrt><mml:mo linebreak="goodbreak" linebreakstyle="after">=</mml:mo><mml:mn>13</mml:mn></mml:math> TeV	Physics letters. B	2,020	cc-by	36,006	1. Introduction In 2012, the ATLAS and CMS Collaborations reported the obser- vation of a new particle in the search for the Standard Model (SM) Higgs boson (H) [1,2]. So far, measurements of the spin and cou- plings of the new particle are consistent with those predicted by the Brout–Englert–Higgs (BEH) mechanism of the SM [3–12]. The SM predicts non-resonant production of Higgs boson pairs (H H) in proton–proton (pp) collisions, referred to as non-resonant H H production, with the dominant production modes at the LHC pro- ceeding via the gluon–gluon fusion (ggF) process. The ggF process has two leading order contributions: the ﬁrst corresponds to the so-called ‘triangle diagram’, which includes the Higgs boson self- coupling, and the second is the so-called ‘box diagram’, which in- cludes a heavy-quark loop with two fermion–fermion–Higgs ( f f H) vertices. These two amplitudes interfere destructively, resulting in a low cross-section of only 31.05 ± 1.90 fb for the ggF H H pro- duction mode, computed at next-to-next-to-leading order (NNLO) and including ﬁnite top-quark mass effects [13–20]. Feynman dia- grams illustrating these two contributions are shown in Fig. 1. The measurement of non-resonant H H production at the LHC stands as an important test of the BEH mechanism. In many beyond-the-SM (BSM) theories, H H production can be enhanced by modifying the Higgs boson self-coupling, λH H H , or the top-quark Yukawa cou- pling, yt, and/or by introducing new contact interactions between Fig. 1. Feynman diagrams for leading order ggF production of Higgs boson pairs: the ‘triangle diagram’ sensitive to the Higgs boson self-coupling on the left and the ‘box diagram’ on the right. two top-quarks or gluons and two Higgs bosons or introducing production mechanisms via intermediate BSM particles [21–23]. The ATLAS and CMS Collaborations have performed searches for non-resonant H H production in a variety of ﬁnal states at 13 TeV [24–33]. No signiﬁcant excess of events beyond SM ex- pectations is observed in these searches, with the ATLAS and CMS data-analyses setting observed (expected) limits on non-resonant H H production to be no larger than 6.9 (10.0) and 22.2 (12.8) times the predicted rate in the SM, respectively [34,35]. a r t i c l e i n f o Article history: Received 19 August 2019 Received in revised form 4 December 2019 Accepted 5 December 2019 Available online 13 December 2019 Editor: M. Doser A search for non-resonant Higgs boson pair production, as predicted by the Standard Model, is presented, where one of the Higgs bosons decays via the H →bb channel and the other via one of the H → W W ∗/Z Z∗/ττ channels. The analysis selection requires events to have at least two b-tagged jets and exactly two leptons (electrons or muons) with opposite electric charge in the ﬁnal state. Candidate events consistent with Higgs boson pair production are selected using a multi-class neural network discriminant. The analysis uses 139 fb−1 of pp collision data recorded at a centre-of-mass energy of 13 TeV by the ATLAS detector at the Large Hadron Collider. An observed (expected) upper limit of 1.2 (0.9+0.4 −0.3) pb is set on the non-resonant Higgs boson pair production cross-section at 95% conﬁdence level, which is equivalent to 40 (29+14 −9 ) times the value predicted in the Standard Model. 9 © 2019 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funded by SCOAP3. Fig. 1. Feynman diagrams for leading order ggF production of Higgs boson pairs: the ‘triangle diagram’ sensitive to the Higgs boson self-coupling on the left and the ‘box diagram’ on the right. Search for non-resonant Higgs boson pair production in the bbℓνℓν ﬁnal state with the ATLAS detector in pp collisions at √ s = 13 TeV .The ATLAS Collaboration ⋆ ⋆E-mail address: atlas .publications @cern .ch. ed by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funded by Physics Letters B 801 (2020) 135145 Physics Letters B 801 (2020) 135145 Contents lists available at ScienceDirect ⋆E-mail address: atlas .publications @cern .ch. ses searching for non-resonant H H production via similar decay channels were performed previously in the single-lepton ﬁnal state https://doi.org/10.1016/j.physletb.2019.135145 0370-2693/© 2019 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funded by SCOAP3. 4. Event selection and object deﬁnitions Selected events are required to have at least one pp interaction vertex reconstructed from at least two ID tracks with pT > 0.4 GeV. The primary vertex for each event is deﬁned as the vertex with the highest (pT)2 of associated ID tracks [102]. Events that contain at least one jet arising from non-collision sources or detector noise are rejected by a set of quality criteria [103]. p g gg Monte Carlo (MC) simulation [41] is used to model the signal processes and in the estimation of SM background processes. A GEANT4 [42] simulation of the ATLAS detector was used for the background processes. The signal MC samples were processed with a fast simulation that relies on a parameterisation of the calorime- ter response [41] and on GEANT4 for the tracking detectors. Simu- lated events are reconstructed using the same algorithms as used for data and include the effects of multiple pp interactions in the same or neighbouring bunch crossings, collectively referred to as pile-up. The simulation of pile-up collisions was performed with Pythia 8.186 [43] using the ATLAS A3 set of tuned parameters [44] and the NNPDF2.3LO parton distribution function (PDF) set [45]. Simulated events were reweighted to match the distribution of pile-up interactions in data. The average amount of pile-up in the data collected during 2015–2018 was 33.7. Loose and signal criteria are deﬁned in order to select recon- structed lepton and jet candidates, where the latter is a subset of the former. Compared to the loose objects, the signal objects are required to satisfy tighter identiﬁcation or quality criteria that are designed to suppress background contributions. Reconstructed loose (signal) electrons are required to satisfy the ‘Loose’ (‘Tight’) likelihood identiﬁcation criteria [104]. Loose electrons are required to have pT > 10 GeV and to be within \|η\| < 2.47. In addition, sig- nal electrons are required to be outside the range 1.37 < \|η\| < 1.52, which corresponds to the transition regions between the bar- rel and endcaps of the electromagnetic calorimeters. In order to reduce background contributions from jets misidentiﬁed as elec- trons, signal electrons are required to be isolated according to the ‘Gradient’ selection criteria [104]. Reconstructed loose and signal muon candidates are required to have pT > 10 GeV, to be within \|η\| < 2.4, and to satisfy the ‘Medium’ identiﬁcation criteria [105]. by ATLAS in searches for H H →bbW W ∗[28] and in the dilepton channel by CMS in searches for H H →bbW W ∗/bbZ Z∗[31]. by ATLAS in searches for H H →bbW W ∗[28] and in the dilepton channel by CMS in searches for H H →bbW W ∗/bbZ Z∗[31]. Lagrangian in the inﬁnite top-quark mass approximation. The gen- erated signal events were reweighted with form factors that take into account the ﬁnite mass of the top-quark [46,47]. SM back- ground processes were simulated using different MC event gener- ators. The MC matrix element (ME) event generators and PDF sets, the parton showering (PS) and the underlying event (UE) mod- elling, UE tuned parameters (tune), and the accuracy of the the- oretical cross-sections used to normalise the simulated processes are summarised in Table 1. Each SM background process is nor- malised to the best available respective theoretical cross-section. The mass of the Higgs boson was set to 125 GeV for all signal and background processes. The H H branching fractions (BF) predicted by the SM [13] are used for all Higgs boson decays. MadSpin [48] was used to model top-quark spin correlations and EvtGen [49] was used to model properties of b- and c-hadron decays for pro- cesses using Pythia and for the signal processes. 3. Dataset and simulated events The data used for this search were collected in pp collisions at the LHC with a centre-of-mass energy of 13 TeV. Only those data collected during stable LHC beam conditions and with all ATLAS detector subsystems fully operational are used, and correspond to an integrated luminosity of 139 fb−1. The selection of candidate events with oppositely charged leptons is based on a combination of single-lepton and dilepton triggers.2 The use of a given trig- ger depends on the ﬂavour and the transverse momenta (pT) of the two (pT-ordered) leptons in the event, and on the data-taking period. Single-lepton triggers with pT thresholds between 22 and 28 GeV are given priority over dilepton triggers. The criteria of the dilepton triggers are checked only if no single-lepton trigger crite- ria are met and have pT thresholds as low as 19 (10) GeV for the leading (subleading) lepton. At least one reconstructed lepton (or lepton pair) has to match a corresponding trigger object, in which case their oﬄine pT must be higher than the trigger threshold by at least 2 GeV, in order to be on the eﬃciency plateau of the cor- responding trigger. 2. ATLAS detector The ATLAS detector [37–39] is a general-purpose particle de- tector with forward–backward symmetric cylindrical geometry.1 It includes an inner tracking detector (ID), immersed in an axial mag- netic ﬁeld, which provides precision tracking of charged particles over the range of \|η\| < 2.5. Calorimeter systems with either liq- uid argon or scintillator tiles as the active medium provide energy measurements over the range of \|η\| < 4.9. The muon spectrome- ter (MS) is positioned outside the calorimeters and includes three air-core toroidal magnets. The MS is composed of several types of muon detectors which provide trigger and high-precision tracking capabilities for \|η\| < 2.4 and \|η\| < 2.7, respectively. A hardware- based trigger followed by a software-based trigger reduce the recorded event rate to an average of 1 kHz [40]. SM top-quark pair production (t¯t) and the production of sin- gle top-quarks in association with W bosons (W t) contribute with signiﬁcant background contamination in the bbℓνℓν ﬁnal state. At next-to-leading-order (NLO) accuracy, there exists non-trivial in- terference between these two processes that may be enhanced in phase-space regions wherein there are high fractions of W t events [50]. Two schemes are typically used to remove the overlap be- tween these two processes: the so-called diagram removal (DR) and diagram subtraction (DS) schemes [51]; the former is used in the present analysis to remove the overlapping events and the latter is used to evaluate the systematic uncertainty in correspond- ing background event yields. Because of these effects, the sum of the simulated t¯t and W t processes is considered as a single background process and referred to as the ‘Top’ process in what follows. 1 ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point (IP) in the centre of the detector and the z-axis along the beam pipe. The x-axis points from the IP to the centre of the LHC ring, and the y-axis points upwards. Cylindrical coordinates (r, φ) are used in the transverse plane, φ being the azimuthal angle around the z-axis. The pseudorapidity is deﬁned in terms of the polar angle θ as η = − ln tan(θ/2). The angular distance is measured in units of R ≡ ( η)2 + ( φ)2. 2 Distinct sets of single-lepton triggers are used for electrons and muons. Dilepton triggers require either two electrons, two muons, or one electron and one muon. 1. Introduction This Letter describes a search for non-resonant H H produc- tion in the bbℓνℓν ﬁnal state, where ℓrefers to a lepton (either an electron or a muon), using 13 TeV pp collision data collected with the ATLAS detector during 2015–2018 and corresponding to a total integrated luminosity of 139 fb−1. The analysis uses machine-learning techniques based on feedforward neural network architectures [36] to construct an event-level classiﬁer trained to distinguish between the H H signal and SM backgrounds. Analy- ses searching for non-resonant H H production via similar decay channels were performed previously in the single-lepton ﬁnal state The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 2 5.1. Deep learning approach to target H H The discriminant uses the outputs of a DNN classiﬁer that is built using the Keras library with Tensorﬂow as a backend [114, 115] and uses the lwtnn library [116] to interface with the analy- sis software infrastructure of the ATLAS experiment. The sample of events used for training is composed of equal numbers of events from the signal and each of the dominant background processes: Top (as deﬁned in Section 3), Z/γ ∗→ℓℓ(Z-ℓℓ), and Z/γ ∗→ττ (Z-ττ ) production. The signal sample used in the training of the classiﬁer contains only the H H →bbW W ∗component due to its larger BF relative to the H H →bbττ and H H →bbZ Z∗compo- nents. However, the sum of all three signal components is evalu- ated as the signal when performing the statistical analysis. Addi- tionally, all processes that make up the training sample (H H → bbW W ∗, Top, Z-ℓℓ, and Z-ττ ) have the same weight during the training of the classiﬁer. The training sample is composed of simu- lated candidate events with mℓℓ> 20 GeV and having one or more b-tagged jets, where events with exactly one b-tagged jet are in- cluded to increase the number of events available for training. For the training events with exactly one b-tagged jet, each observable that requires at least two b-tagged jets is set to its mean value as computed with the full set of training events that contain at least two b-tagged jets. Observables that require two b-tagged jets are deﬁned using the leading two b-tagged jets. The classiﬁer contains two fully connected hidden layers each with 250 nodes. Rectiﬁed linear unit (ReLU) activations are used for each layer [117]. In or- der to improve the robustness of the training and to reduce effects due to overtraining, there is a dropout layer that randomly drops 50% of the nodes between the two fully connected layers during training [118]. The classiﬁer produces four outputs that are passed through a softmax activation, constraining their sum to one [36]. The missing transverse momentum pmiss T , the magnitude of which is denoted by Emiss T , is constructed from the negative vec- torial sum of the transverse momenta of calibrated loose objects in the event. 3 A calorimeter-tagged muon has only a reconstructed track in the ID matched to energy deposits in the calorimeter compatible with a minimum ionising particle, but no corresponding track segment in the MS. Table 1 and to maximise rejection of the expected SM backgrounds, the analysis uses a multivariate approach to select signal events. Jets are reconstructed from topological clusters of energy de- posits in the calorimeters [106] using the anti-kt algorithm [107, 108] with a radius parameter of R = 0.4 and calibrated as de- scribed in Ref. [109]. Candidate loose jets are required to have pT > 20 GeV. Signal jets are required to have \|η\| < 2.8 and must satisfy pile-up suppression requirements based on the output of a multivariate classiﬁer [110], which identiﬁes jets consistent with a primary vertex in the region \|η\| < 2.4 and pT < 120 GeV. The MV2c10 multivariate algorithm [111] is used to identify jets containing b-hadrons (b-tagged jets). An MV2c10 working point with a b-tagging eﬃciency of 70%, estimated from simulated t¯t events [112], is used. The b-tagged jets must have pT > 20 GeV and \|η\| < 2.5. The momentum of b-tagged jets is adjusted us- ing the muon-in-jet correction, as described in Ref. [6], by ac- counting for momentum losses due to muons originating from in- ﬂight semileptonic b-hadron decays occurring within the b-tagged jet. 5. Analysis strategy The analysis relies on the use of a multivariate discriminant de- signed to select candidate events consistent with non-resonant H H production. Section 5.1 describes the architecture and the training of the deep neural network (DNN) classiﬁer from which the dis- criminant is constructed. Section 5.2 describes the signal region selection criteria. Section 5.3 describes the ﬁnal background esti- mation procedure. 4. Event selection and object deﬁnitions Additionally, signal muons are required to be isolated according to the ‘FixedCutLoose’ selection criteria [105]. Signal electron (muon) candidates are required to originate from the primary vertex by demanding that the signiﬁcance of the transverse impact param- eter, deﬁned as the absolute value of the track transverse impact parameter, d0, measured relative to the primary vertex, divided by its uncertainty, σd0, satisfy \|d0\|/σd0 < 5 (3). The difference z0 between the value of the z coordinate of the point on the track at which d0 is deﬁned and the longitudinal position of the primary The signal processes with ggF-initiated non-resonant H H pro- duction in the bbℓνℓν ﬁnal state were generated with an effective The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 3 Table 1 Table 1 List of the ME generators and PS/UE modelling algorithms used in the simulation. Alternative generators and PS/UE models, used to estimate systematic uncertainties, are shown in parentheses. The PDF sets, tunes, and the perturbative QCD highest-order accuracy (leading-order, LO; next-to-leading-order, NLO; next-to-next-to-leading-order, NNLO; next-to-next-to-leading-logarithm, NNLL) used for the normalisation of the samples are also included. The top-quark mass is set to 172.5 GeV. Table 1 List of the ME generators and PS/UE modelling algorithms used in the simulation. Alternative generators and PS/UE models, used to estimate systematic uncertainties, are shown in parentheses. The PDF sets, tunes, and the perturbative QCD highest-order accuracy (leading-order, LO; next-to-leading-order, NLO; next-to-next-to-leading-order, NNLO; next-to-next-to-leading-logarithm, NNLL) used for the normalisation of the samples are also included. The top-quark mass is set to 172.5 GeV. Process ME generator (alternative) ME PDF PS/UE model (alternative) UE tune Prediction order for total cross-section t¯t [52,53] Powheg-Box v2 [54,55] NNPDF3.0NLO [56] Pythia 8.230 [57] A14 [58] NNLO + NNLL [59–65] (MadGraph 5_aMC@NLO) (Herwig 7.0.4) (H7-MMHT14) Single-top s-channel, W t [52,66,67] Powheg-Box NNPDF3.0NLO Pythia 8.230 A14 NLO + NNLL [68,69] (MadGraph 5_aMC@NLO) (Herwig 7.0.4) (H7-MMHT14) Single-top t-channel [52,66] Powheg-Box, MadSpin [48] NNPDF3.04fNLO Pythia 8.230 A14 NLO + NNLL [70] (MadGraph 5_aMC@NLO) (Herwig 7.0.4) (H7-MMHT14) W , Z/γ ∗+ jets [71] Sherpa 2.2.1 [72,73] NNPDF3.0NNLO Sherpa 2.2.1 Sherpa default NLO(LO) ≤2(4) partons [74–78] (Z/γ ∗+ jets) (MadGraph 5_aMC@NLO) (Pythia 8.230) (A14) Diboson (W W , W Z, Z Z) [79] Sherpa 2.2.2 NNPDF3.0NNLO Sherpa 2.2.2 Sherpa default NLO(LO) ≤1(3) partons [75–78] t¯tW , t¯t Z [80] MadGraph 5_aMC@NLO [81] NNPDF3.0NLO Pythia 8.210 A14 NLO [82,83] t¯tH [80] MadGraph 5_aMC@NLO NNPDF3.0NLO Pythia 8.210 A14 NLO [84,85] W H, Z H [86] Pythia 8.186 [43] NNPDF2.3LO [45] Pythia 8.186 A14 NNLO QCD + NLO EW [87–93] ggF H [94] Powheg-Box v2 NNLOPS [95] CT10 [96] Pythia 8.212 AZNLO [97] NNNLO QCD + NLO EW [98] SM H H →bbℓνℓν [99] MadGraph 5_aMC@NLO 2.6.2 CT10 Herwig 7.0.4 [100] H7-MMHT14 [101] NNLO [14–20] vertex is required to satisfy \| z0 × sinθ\| < 0.5 mm, where θ is the polar angle of the track with respect to the z-axis. and to maximise rejection of the expected SM backgrounds, the analysis uses a multivariate approach to select signal events. and to maximise rejection of the expected SM backgrounds, the analysis uses a multivariate approach to select signal events. 5.2. Signal selection criteria To deﬁne signal selection criteria, the analysis relies on the invariant mass of the two leptons, mℓℓ, and the invariant mass of the two leading (pT-ordered) b-tagged jets, mbb. Due to spin- correlation effects present in the H →W W ∗→ℓνℓν decay within the dominant H H →bbW W ∗signal process, the signal events exhibit values of mℓℓthat are typically below 60 GeV. By se- lecting low values of mℓℓ, the signal purity can therefore be en- hanced while rejecting a large component of the SM Z boson and Top backgrounds. Additionally, mbb has a peak at the mass of the Higgs boson for the signal process and therefore provides an effective means to deﬁne selections in which the H H contri- bution is enhanced. The signal selection criteria therefore require mℓℓ∈(20, 60) GeV and mbb ∈(110, 140) GeV. The mℓℓ> 20 GeV requirement is enforced in order to remove contamination from low-mass resonances and Z/γ ∗processes. The signal selection cri- teria are further broken down into same-ﬂavour (SF), i.e. ee or μμ, or different-ﬂavour (DF), i.e. eμ, regions. Separating by dilep- ton ﬂavour enhances the separation power between the signal and Z/γ ∗background; the former has roughly equal probabilities for the SF and DF ﬁnal states and the latter leads predominantly to SF ﬁnal states. H H pH H/ pTop pZ ℓℓ pZ ττ The H H →bbℓνℓν signal events are characterised by two distinct ‘Higgs hemispheres’. One hemisphere contains the two b-tagged jets from the H →bb decay and it is typically opposite in the transverse plane to the second hemisphere that contains the two leptons and Emiss T from the H →W W ∗/Z Z∗/ττ decay. The ﬁnal-state objects in the SM backgrounds, the Top process in par- ticular, are distributed more uniformly within the event and they typically do not exhibit the same opposite hemispheres topology as the H H signal. These Higgs hemispheres thus provide a topo- logical criterion that distinguishes the signal from the background and motivates the choice of input observables that are provided to the classiﬁer. Thirty-ﬁve such variables are provided as inputs to the classiﬁer, ranging from momentum components of the visi- ble ﬁnal-state objects to observables using event-wide information, and are constructed using only calibrated ﬁnal state objects that have well-understood uncertainties (Section 6). A complete list is provided in Table 2. Table 2 found to be compatible to its performance when presented with those of the training sample. The resulting four outputs, each constrained to values between 0 and 1, are referred to as pi (i ∈{H H, Top, Z-ℓℓ, Z-ττ}). Values of pi nearer to 1 indicate that the event likely belongs to class i and val- ues nearer to 0 indicate otherwise. The main discriminant in the analysis, dH H , is constructed from the four pi and is deﬁned as dH H = ln pH H/ pTop + pZ-ℓℓ+ pZ-ττ . Table 2 (pT , η, φ) pT , η, and φ of the leptons, leading two jets (not necessarily b-tagged), and leading two b-tagged jets Dilepton ﬂavour Whether the event is composed of two electrons, two muons, or one of each (encoded as 3 booleans) Rℓℓ, \| φℓℓ\| R and magnitude of the φ between the two leptons mℓℓ, pℓℓ T Invariant mass and the transverse momentum of the dilepton system Emiss T , Emiss T -φ Magnitude of the missing transverse momentum vector and its φ component \| φ(pmiss T , pℓℓ T )\| Magnitude of the φ between the pmiss T and the transverse momentum of the dilepton system \|pmiss T + pℓℓ T \| Magnitude of the vector sum of the pmiss T and the transverse momentum of the dilepton system Jet multiplicities Numbers of b-tagged and non-b-tagged jets \| φbb\| Magnitude of the φ between the leading two b-tagged jets mbb T2 mT2 [120] using the leading two b-tagged jets as the visible inputs and pmiss T as invisible input HT2 Scalar sum of the magnitudes of the momenta of the H →ℓνℓν and H →bb systems, HT2 = \|pmiss T + pℓ,0 T + pℓ,1 T \| + \|pb,0 T + pb,1 T \| HR T2 Ratio of HT2 and scalar sum of the transverse momenta of the H decay products, HR T2 = HT2/(Emiss T + \|pℓ,0 T \| + \|pℓ,1 T \| + \|pb,0 T \| + \|pb,1 T \|), where pℓ(b) ,0{1} T are the transverse momenta of the leading {subleading} lepton (b-tagged jet) pT , η, and φ of the leptons, leading two jets (not necessarily b-tagged), and leading two b-tagged jets Whether the event is composed of two electrons, two muons, or one of each (encoded as 3 booleans) R and magnitude of the φ between the two leptons mT2 [120] using the leading two b-tagged jets as the visible inputs and pmiss T as invisible input S l f h i d f h f h H ℓℓ d H bb HT2 = \|pmiss T + pℓ,0 T + pℓ,1 T \| + \|pb,0 T + pb,1 T \| Ratio of HT2 and scalar sum of the transverse momenta of the H decay products, HR T2 = HT2/(Emiss T + \|pℓ,0 T \| + \|pℓ,1 T \| + \|pb,0 T \| + \|pb,1 T \|), HR T2 = HT2/(Emiss T + \|pℓ,0 T \| + \|pℓ,1 T \| + \|pb,0 T \| + \|pb,1 T \|), HR T2 = HT2/(Emiss T + \|pℓ,0 T \| + \|pℓ,1 T \| + \|pb,0 T \| + \|pb,1 T \|), where pℓ(b) ,0{1} T are the transverse momenta of the leading {subleading} lepton (b-tagged jet) found to be compatible to its performance when presented with those of the training sample. 5.2. Signal selection criteria The event-wide input observables are sensitive to the presence of Higgs hemispheres in the signal and are largely angular in nature or take advantage of the fact that the ﬁnal state objects from each of the Higgs bosons in the signal tend to be near to each other. The observables HR T2 and mbb T2 are non-standard high-level observables that are not straightforward functions of the momenta of the ﬁnal-state objects. By construction, HR T2 can take values between zero and one; it peaks near one for signal and is more broadly distributed for background. The mbb T2 observable is deﬁned similarly to the Mbb T2 observable in Ref. [119] but does not include the ﬁnal-state leptons. As discussed in Ref. [119], for the Top backgrounds mbb T2 generally has values below the mass of the top-quark due to kinematic constraints while for the Z/γ ∗pro- cesses, which have little-to-no Emiss T , mbb T2 is typically below 45 GeV. The use of dropout regularisation during the training of the classiﬁer allows it to more effectively use the information con- tained in the full set of inputs presented in Table 2 by reducing its susceptibility to overtraining effects that may otherwise appear as a result of using such an extended input feature space in the case where no such regularisation is performed. To verify this, the performance of the classiﬁer was checked using an independent sample of events not used in the training of the classiﬁer and was In addition to the mℓℓand mbb requirements, the same-ﬂavour and different-ﬂavour signal regions, SR-SF and SR-DF, respectively, are deﬁned by requiring high values of dH H and are presented in Table 3. The chosen threshold values of dH H > 5.45 (5.55) for SR-SF (SR-DF) are found to maximise the expected sensitivity to the non-resonant H H process. The predicted H H →bbℓνℓν signal yields in SR-SF and SR-DF are shown in Table 3, and are composed of 90% H H →bbW W ∗, 9% H H →bbττ , and 1% H H →bbZ Z∗. The predominance of the H H →bbW W ∗process over the other two is a result of both its larger overall BF and of the classiﬁer having been trained only on this component of the signal. 5.1. Deep learning approach to target H H An additional term is included to account for the en- ergy of ID tracks that are matched to the primary vertex in the event but not to any of the selected loose objects [113]. To avoid double-counting, loose objects are subject to the over- lap removal procedure deﬁned as follows. If a reconstructed elec- tron and muon share a track in the ID, the electron is removed. However, if the muon sharing the track with the electron is calorimeter-tagged,3 then the muon is removed instead of the elec- tron. If a jet and an electron are reconstructed within R = 0.2 of each other, then the jet is removed. If a jet and a muon are within R = 0.2 of each other, and the jet has less than three tracks or carries less than 50% of the muon pT, then the jet is removed; oth- erwise, the muon is removed. Electrons or muons separated from the remaining jets by R < 0.4 are removed. The analysis selects candidate events with exactly two oppo- sitely charged signal leptons, electrons or muons, and at least two signal b-tagged jets. To enhance sensitivity to the signal process The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 Table 2 Description of the variables used as inputs to the DNN classiﬁer. Table 3 Analysis region and background estimation summary. Shown are the deﬁnitions of the control, validation, and signal regions used in the analysis as well as the predicted and observed event yields in each of these regions. The predicted yields are shown after background-only ﬁts to data in the control regions. The Top and Z/γ ∗+ HF post-ﬁt normalisation factors, obtained from background-only ﬁts in the corresponding control regions, are shown at the bottom of the table. Also shown is the predicted H H →bbℓνℓν signal yield in each of the regions, multiplied by a factor of 20. Of the H H yield in the signal regions, 90% comes from the H H →bbW W ∗process, 9% from the H H →bbττ process, and 1% from the H H →bbZ Z∗process. The uncertainties in each yield and in the normalisation correction factors account for the statistical and systematic uncertainties described in Section 6, with those on the normalisation corrections due only to experimental sources. Region deﬁnitions Observable CR-Top VR-1 CR-Z+HF VR-2 SR-SF SR-DF Dilepton ﬂavour DF SF DF or SF SF SF DF mℓℓ[GeV] (20, 60) (20, 60) (81.2, 101.2) (71.2, 81.2) or (101.2, 115) (20, 60) (20, 60) mbb [GeV] /∈(100, 140) > 140 (100, 140) (100, 140) (110, 140) (110, 140) dH H > 4.5 > 4.5 > 0 > 0 > 5.45 > 5.55 Event yields Data 108 171 852 157 16 9 Total Bkg. 108 ± 10 162 ± 10 852 ± 29 147 ± 11 14.9 ± 2.1 4.9 ± 1.2 Top 92 ± 11 77 ± 10 55 ± 7 71 ± 10 4.8 ± 1.4 3.8 ± 1.1 Z/γ ∗+ HF 3.2 ± 0.5 70 ± 4 686 ± 33 60 ± 4 7.8 ± 1.4 0.21 ± 0.05 Other 13.1 ± 3.4 14.2 ± 1.9 110 ± 13 15.8 ± 1.2 2.3 ± 0.5 0.9 ± 0.4 H H (×20) 2.70 ± 0.25 1.03 ± 0.22 1.97 ± 0.11 1.22 ± 0.05 5.0 ± 0.6 4.8 ± 0.8 Post-ﬁt normalisation μTop = 0.79 ± 0.10 μZ/γ ∗+ HF = 1.36 ± 0.07 originating from heavy-ﬂavour hadrons (bb, bc, or cc), subse- quently referred to as Z/γ ∗+ HF. Subdominant SM processes con- tribute via t¯t production in association with an electroweak vec- tor boson, single Higgs boson production (predominantly via the t¯tH mode), Z/γ ∗production in association with light-ﬂavour jets, and electroweak diboson processes. Table 3 There is additionally a minor contribution of background events from non-prompt leptons pro- duced in semileptonic decays of heavy-ﬂavour hadrons and from misidentiﬁed electron candidates arising from photon conversions and jets. This background is estimated using events with a same- charge lepton pair, following procedures described in Ref. [121], after subtracting the prompt-lepton contribution. The rest of the SM background processes detailed in Table 1 are estimated pri- marily using simulation. terference effects as described in Section 3. For this reason, CR-Top and the validation regions are deﬁned so that they have predicted t¯t and W t compositions similar to that of the signal regions. This ensures that the normalisation correction determined in the ﬁt for the Top background results in an accurate estimate of the com- bined t¯t and W t process in the signal regions, accounting for potential interference effects present in data but not necessar- ily modelled in MC simulation. Table 3 compares the observed and predicted event yields, where the background event yields obtained after background-only ﬁts in the corresponding control regions are also shown. The post-ﬁt normalisation correction fac- tors for the Top and Z/γ ∗+ HF background processes, respectively μTop = 0.79 ± 0.10 and μZ/γ ∗+ HF = 1.36 ± 0.07, are also shown in Table 3. The uncertainties in μTop and μZ/γ ∗+ HF take into account the statistical and systematic uncertainties due to the experimental sources, as described in Section 6. Dedicated control regions are deﬁned to derive data-driven nor- malisation corrections for the dominant background processes: CR- Top for Top and CR-Z+HF for Z/γ ∗+ HF. These normalisation cor- rections have a uniform prior and are checked in two validation regions, VR-1 and VR-2, enriched with events from the Top and Z/γ ∗+ HF processes. The control and validation regions are de- ﬁned in Table 3 and are kinematically close to the signal regions. CR-Top (CR-Z+HF) and VR-1 (VR-2) are deﬁned by inverting the mbb (mℓℓ) requirements relative to those of the signal regions but retain a selection of the high dH H region similar to the signal regions. The dH H selections were relaxed to increase statistical power, indepen- dent checks showed that this did not have a signiﬁcant impact on the post-ﬁt normalisation corrections in Table 3. Table 3 Distributions of dH H in the control regions after performing background-only ﬁts to data in the control regions and applying the Top and Z/γ ∗+ HF normalisation corrections are shown in Fig. 2. In the control and validation regions, good agreement be- tween the data and SM prediction provided by the post-ﬁt MC simulation is observed for the observables relevant to the analy- sis. 5.3. Background estimation As mentioned in Section 5.1, the dominant backgrounds ex- pected to contaminate the signal regions are the Top and Z/γ ∗ processes, speciﬁcally Z/γ ∗production in association with jets 5 The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 6. Systematic uncertainties The un- certainties in the background estimates in SR-DF are dominated by the uncertainty due to the parton shower affecting the Top background process (12%), the uncertainty in the Top normalisa- tion correction μTop (10%), the uncertainty due to the comparison between the generators used for the Top process (7.5%), and the uncertainty due to the modelling of ISR and FSR in the Top pro- cess (5%). Systematic uncertainties in the signal acceptance due to vary- ing μr and μf, as well as PDF-induced uncertainties, are evaluated using the same procedure as for the Top background process. The resulting scale (PDF) uncertainties are < 3% (< 1%) in both sig- nal regions. The uncertainty due to the parton shower modelling is computed by comparing Herwig7 with Pythia8, and is found to be 8% (9%) in SR-SF (SR-DF). The uncertainty in the H H pro- duction cross-section, evaluated to be 5%, is included as an uncer- tainty in σ SM(gg →H H) when computing the upper limits on the cross-section ratio in Table 5. This value is the quadrature sum of the scale, PDF+αs, and top mass contributions as reported by the LHCXSWG [20]. The uncertainties due to experimental sources arise primarily from the mismeasurement of reconstructed object momenta and from the mismodelling of reconstruction eﬃciencies. These un- certainties include uncertainties from the mismodelling of the jet energy scale (JES) [109] and jet energy resolution (JER) [126]. Addi- tional uncertainties for b-tagged jets arise from the mismodelling of the b-tagging eﬃciency [111] and from the mismodelling of the rates at which charm- and light-ﬂavoured jets are selected as b-tagged jets [127,128]. Lepton-related uncertainties arise from the mismodelling of the electron [104] (muon [105]) reconstructed energy (momentum) measurements, as well as in the mismod- elling of their reconstruction and identiﬁcation eﬃciencies [104, 105]. The Emiss T scale and resolution [113] uncertainties, as well as uncertainties from the mismodelling of pile-up, trigger eﬃciency and luminosity, are also taken into account. The uncertainty in the combined 2015–2018 integrated luminosity is 1.7% [129], ob- tained using the LUCID-2 detector [130] for the primary luminosity measurements. The combined effect of the experimental sources of systematic uncertainty in the predicted background yields is sum- marised in Table 4 and is dominated by the JER, with all other contributions found to be negligible. 6. Systematic uncertainties The ratio of the data to the sum of the backgrounds is shown in the lower panel. The hatched bands indicate the combined statistical and systematic uncertainty. the corrected predictions in the signal regions using the follow- ing procedures. The uncertainties in the estimated Top background event yields due to parton shower modelling are assessed as the difference between the predictions of Powheg-Box showered with Pythia or Herwig, and those due to the choice of event generator are assessed by comparing the predictions of Powheg-Box or Mad- Graph 5_aMC@NLO [122], both showered with Pythia. The uncer- tainties due to missing higher-order corrections are estimated by changing the renormalisation and factorisation scales (μr and μf, respectively) up and down by a factor of two (8-points variation). The uncertainties due to the modelling of initial- and ﬁnal-state ra- diation (ISR and FSR, respectively) in the generators used to simu- late the Top background processes are evaluated using the method described in Ref. [122]. The Top background composition is var- ied within the uncertainties in the theoretical predictions for the t¯t and single-top-quark W t cross-sections [65,68,123]. The uncer- tainty arising from the interference between the NLO predictions for t¯t and W t processes is estimated by taking the difference be- tween the predicted Top background yields obtained with the DR and DS schemes used for the NLO W t calculation [122]. The un- certainties due to PDF variations are computed as the envelope of the central values of the nominal NNPDF3.0 PDF set and the CT14, MMHT14, and PDF4LHC15_30PDF PDF sets [124]. All uncertainties except those in the scale variations, cross-section, and interference are considered as fully correlated between the t¯t and W t pro- cesses. The Z/γ ∗+ HF modelling uncertainties are estimated using the nominal Sherpa 2.2.1 samples by considering different merging (CKKW-L) [125] and resummation scales. The uncertainties due to PDF variations and changes in μr and μf are calculated using the same procedures as for the Top backgrounds. An additional uncer- tainty in the Z/γ ∗process is computed by taking the difference between the nominal Sherpa 2.2.1 samples with samples generated using MadGraph 5_aMC@NLO+Pythia8. The dominant uncertain- ties in the total background estimates in SR-SF are the Z/γ ∗+ HF modelling uncertainties (8%), primarily that due to comparison of Sherpa 2.2.1 and MadGraph 5_aMC@NLO, and the parton shower uncertainty affecting the Top background process (5%). 6. Systematic uncertainties The analysis evaluates several sources of systematic uncertainty for the signal and background processes, which are classiﬁed as either experimental (detector or luminosity related) or theoretical modelling uncertainties. Statistical uncertainties of the simulated event samples are also taken into account. The main uncertainty components are summarised in Table 4. MC modelling uncertain- ties in the Top and Z/γ ∗+ HF background estimates are dominant, followed by statistical and detector uncertainties. VR-1 keeps only those events with mbb > 140 GeV, excluding the region mbb < 100 GeV which is included in CR-Top, due to signiﬁcant contamination of Z/γ ∗+ HF events. The correlations be- tween the mℓℓand mbb observables and dH H after the preselection are observed to be small and do not prevent the use of the former two in the construction of the analysis regions deﬁned in Table 3, as dH H is found to rely mainly on the information provided by the additional input observables listed in Table 2. This absence of strong correlation ensures that the measurements made in the tails of dH H in the control regions can be extrapolated to those in the signal regions. The normalisation corrections of the Top and Z/γ ∗+ HF back- ground processes are determined primarily by the data events in the control regions when performing the statistical analysis. These corrections take into account the statistical and systematic uncer- tainties due to the experimental sources, as described later in this section. In addition, the systematic uncertainties in the theoret- ical modelling of these processes are applied as uncertainties in The Top background in the signal regions is expected to be composed of approximately equal contributions from the t¯t and single-top-quark W t process and therefore susceptible to the in- The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 6 Fig. 2. Distributions of dH H in CR-Top (left) and CR-Z+HF (right). Distributions are shown after the ﬁt to data under the background-only hypothesis in the shown control regions. The ratio of the data to the sum of the backgrounds is shown in the lower panel. The hatched bands indicate the combined statistical and systematic uncertainty. Fig. 2. Distributions of dH H in CR-Top (left) and CR-Z+HF (right). Distributions are shown after the ﬁt to data under the background-only hypothesis in the shown control regions. Table 4 Table 4 Breakdown of the main uncertainty components in the background estimates in the two signal regions for the Top, Z/γ ∗+ HF, and all other (“Other”) backgrounds. The uncertainty components associated with the total background estimate in the signal regions (the sum of Top, Z/γ ∗+ HF, and Other) is listed under “Total Bkg.”. As in the upper half of Table 3, all uncertainties are shown “post-ﬁt”. The percentages show the size of the uncertainty relative to the expected background in each column and uncertainties can be correlated, not necessarily adding in quadrature to the total uncertainty in each column or across each row. Uncertainties in the post-ﬁt normalisation factors, μTop and μZ/γ ∗+ HF, are only applicable for the Top and Z/γ ∗+ HF processes. Uncertainty [%] SR-SF SR-DF Top Z/γ ∗+ HF Other Total Bkg. Top Z/γ ∗+ HF Other Total Bkg. Total uncertainty 28 18 20 14 30 26 41 25 Theoretical 21 15 17 11 20 15 40 17 Experimental 12 < 5 8 < 5 15 17 8 12 MC statistics 8 8 6 8 13 13 7 11 μTop, μZ/γ ∗+ HF 13 5 n/a 5 13 5 n/a 10 Fig. 3. Distributions of mbb (left), mℓℓ(middle), and the discriminant dH H (right). The distributions are shown after the ﬁt to data in the control regions under the background- only hypothesis. Each distribution includes both the SF and DF events and imposes signal selection requirements on all quantities except the one being plotted, but the requirement on dH H has been relaxed to dH H > 5 for the distributions of mbb and mℓℓ. The H H →bbℓνℓν signal (“H H”) is overlaid and has its cross-section scaled by a factor of 20 relative to the SM prediction for visualisation purposes. The ratio of the data to the sum of the backgrounds is shown in the lower panel of each ﬁgure. The hatched bands indicate the combined statistical and systematic uncertainty. Fig. 3. Distributions of mbb (left), mℓℓ(middle), and the discriminant dH H (right). The distributions are shown after the ﬁt to data in the control regions under the background- only hypothesis. Table 5 The p-value corresponding to the background-only hypothesis, giv- ing the probability that the data in the signal regions be at least as incompatible with the background-only hypothesis as that ob- served in SR-SF and SR-DF, is p0 = 0.15 and corresponds to 1.05σ signiﬁcance. Distributions of mbb, mℓℓ, and dH H after performing background-only ﬁts to data in the control regions and apply- ing the Top and Z/γ ∗+ HF normalisation corrections are shown in Fig. 3. The signal selection criteria are imposed on all observ- ables shown in Fig. 3 apart from the one being plotted, except that the dH H requirement for the mbb and mℓℓdistributions is relaxed to dH H > 5. No signiﬁcant excess of events over the expected SM background is observed and upper limits are set on non-resonant Higgs boson pair production at 95% conﬁdence level (CL) using the CLs method [132]. Table 5 presents these upper limits and com- parisons with the SM prediction. The observed (expected) limit at Table 4 Each distribution includes both the SF and DF events and imposes signal selection requirements on all quantities except the one being plotted, but the requirement on dH H has been relaxed to dH H > 5 for the distributions of mbb and mℓℓ. The H H →bbℓνℓν signal (“H H”) is overlaid and has its cross-section scaled by a factor of 20 relative to the SM prediction for visualisation purposes. The ratio of the data to the sum of the backgrounds is shown in the lower panel of each ﬁgure. The hatched bands indicate the combined statistical and systematic uncertainty. 7. Results In order to extract information about the H H →bbℓνℓν signal cross-section, a counting experiment is performed with a proﬁle- likelihood ﬁt [131] simultaneously across the CR-Top, CR-Z+HF, SR-SF, and SR-DF regions using the predicted and observed event counts in each region as inputs. The Top and Z/γ ∗+ HF normal- 7 Table 5 Table 5 Observed and expected upper limits on the ggF-initiated non-resonant H H production cross-section at 95% CL and their ratios to the SM prediction (σ SM(gg →H H) = 31.05 ± 1.90 fb [13–20]). The ±1σ and ±2σ variations about the expected limit are also shown. Uncertainties in the SM cross-section are taken into account when computing the upper limits on the cross-section ratio. −2σ −1σ Expected +1σ +2σ Observed σ (gg →H H) [pb] 0.5 0.6 0.9 1.3 1.9 1.2 σ (gg →H H)/σ SM (gg →H H) 14 20 29 43 62 40 95% CL is 1.2 (0.9) pb, corresponding to 40 (29) times the SM pre- diction. 95% CL is 1.2 (0.9) pb, corresponding to 40 (29) times the SM pre- diction. isation corrections are also extracted from this ﬁt and are found to differ negligibly from those presented in Table 3. All sources of systematic and statistical uncertainty in the signal and back- ground models are implemented as deviations from the nominal model, scaled by nuisance parameters that are proﬁled in the ﬁt. The p-value corresponding to the background-only hypothesis, giv- ing the probability that the data in the signal regions be at least as incompatible with the background-only hypothesis as that ob- served in SR-SF and SR-DF, is p0 = 0.15 and corresponds to 1.05σ signiﬁcance. Distributions of mbb, mℓℓ, and dH H after performing background-only ﬁts to data in the control regions and apply- ing the Top and Z/γ ∗+ HF normalisation corrections are shown in Fig. 3. The signal selection criteria are imposed on all observ- ables shown in Fig. 3 apart from the one being plotted, except that the dH H requirement for the mbb and mℓℓdistributions is relaxed to dH H > 5. No signiﬁcant excess of events over the expected SM background is observed and upper limits are set on non-resonant Higgs boson pair production at 95% conﬁdence level (CL) using the CLs method [132]. Table 5 presents these upper limits and com- parisons with the SM prediction. The observed (expected) limit at isation corrections are also extracted from this ﬁt and are found to differ negligibly from those presented in Table 3. 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Deciphering the nature of the Higgs sector, arXiv:1610 .07922 [hep -ph], 2016. p y We acknowledge the support of ANPCyT, Argentina; YerPhI, Ar- menia; ARC, Australia; BMWFW and FWF, Austria; ANAS, Azer- baijan; SSTC, Belarus; CNPq and FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR and VSC CR, Czech Republic; DNRF and DNSRC, Denmark; IN2P3-CNRS, CEA-DRF/IRFU, France; SRNSFG, Georgia; BMBF, HGF, and MPG, Germany; GSRT, Greece; RGC, Hong Kong SAR, China; ISF and Benoziyo Center, Is- rael; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; NWO, Netherlands; RCN, Norway; MNiSW and NCN, Poland; FCT, Portu- gal; MNE/IFA, Romania; MES of Russia and NRC KI, Russian Fed- eration; JINR; MESTD, Serbia; MSSR, Slovakia; ARRS and MIZŠ, Slovenia; DST/NRF, South Africa; MINECO, Spain; SRC and Wallen- berg Foundation, Sweden; SERI, SNSF and Cantons of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK, Turkey; STFC, United Kingdom; DOE and NSF, United States of America. In addition, in- dividual groups and members have received support from BCKDF, Canarie, CRC and Compute Canada, Canada; COST, ERC, ERDF, Hori- zon 2020, and Marie Skłodowska-Curie Actions, European Union; Investissements d’Avenir Labex and Idex, ANR, France; DFG and AvH Foundation, Germany; Herakleitos, Thales and Aristeia pro- grammes co-ﬁnanced by EU-ESF and the Greek NSRF, Greece; BSF- NSF and GIF, Israel; CERCA Programme Generalitat de Catalunya, Spain; The Royal Society and Leverhulme Trust, United Kingdom. [14] S. Dawson, S. Dittmaier, M. Spira, Neutral Higgs-boson pair production at hadron colliders: QCD corrections, Phys. Rev. D 58 (1998) 115012, arXiv: hep -ph /9805244 [hep -ph]. [15] S. Borowka, et al., Higgs boson pair production in gluon fusion at next-to- leading order with full top-quark mass dependence, Phys. Rev. Lett. 117 (1) (2016) 012001, arXiv:1604 .06447 [hep -ph]. [16] J. 8. Conclusions A search for non-resonant Higgs boson pair production, as pre- dicted by the SM, is presented in the ﬁnal state with at least two b-tagged jets and exactly two leptons with opposite electric charge, where one of the Higgs bosons decays to bb and the other de- cays to either W W ∗, Z Z∗, or ττ . The analysis uses pp collision data recorded at √ s = 13 TeV by the ATLAS detector at the LHC, corresponding to an integrated luminosity of 139 fb−1. The data are in agreement with the predictions for the SM background pro- cesses. An observed (expected) 95% CL upper limit is set on the cross-section for the production of Higgs boson pairs, correspond- ing to 40 (29) times the SM prediction. These limits are com- parable to the previous leading searches for non-resonant Higgs boson pair production performed by the ATLAS and CMS experi- ments. 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Barnea 16 S.L. Barnes 60c, B.M. Barnett 144, R.M. Barnett 18, Z. Barnovska-Blenessy 60a, A. Baroncelli 60a, G. Barone 29 A.J. Barr 135, L. Barranco Navarro 45a,45b, F. Barreiro 98, J. Barreiro Guimarães da Costa 15a, S. Barsov 138, R. Bartoldus 153, G. Bartolini 101, A.E. Barton 89, P. Bartos 28a, A. Basalaev 46, A. Bassalat 132,aq, M.J. Basso 167, R.L. Bates 57, S. Batlamous 35e, J.R. The ATLAS Collaboration Batley 32, B. Batool 151, M. Battaglia 146, M. Bauce 72a,72 F. Bauer 145, K.T. Bauer 171, H.S. Bawa 31,m, J.B. Beacham 49, T. Beau 136, P.H. Beauchemin 170, F. Becherer P. Bechtle 24, H.C. Beck 53, H.P. Beck 20,s, K. Becker 52, M. Becker 99, C. Becot 46, A. Beddall 12d, A.J. Beddall 12a, V.A. Bednyakov 79, M. Bedognetti 119, C.P. Bee 155, T.A. Beermann 76, M. Begalli 80b, M. Begel 29, A. Behera 155, J.K. Behr 46, F. Beisiegel 24, A.S. Bell 94, G. Bella 161, L. Bellagamba 23b, A. Bellerive 34, P. Bellos 9, K. Beloborodov 121b,121a, K. Belotskiy 111, N.L. Belyaev 111, D. Benchekroun 35a, N. Benekos 10, Y. Benhammou 161, D.P. Benjamin 6, M. Benoit 54, J.R. Bensinger 26, S. Bentvelsen 119, L. Beresford 135, M. Beretta 51, D. Berge 46, E. Bergeaas Kuutmann 172, N. Berger 5, B. Bergmann 142, L.J. Bergsten 26, J. Beringer 18, S. Berlendis 7, N.R. Bernard 102, G. Bernardi 136, C. Bernius 153, F.U. Bernlochner 24, T. Berry 93, P. Berta 99, C. Bertella 15a, I.A. Bertram 89, O. Bessidskaia Bylund 182, N. Besson 145, A. Bethani 100, S. Bethke 114, A. Betti 24, A.J. Bevan 92, J. Beyer 114, D.S. Bhattacharya 177, P. Bhattarai 26, R. Bi 139, R.M. Bianchi 139, O. Biebel 113, D. Biedermann 19, R. Bielski 36, K. Bierwagen 99, N.V. Biesuz 71a,71b, M. Biglietti 74a, T.R.V. Billoud 109, M. Bindi 53, A. Bingul 12d, C. Bini 72a,72b, S. Biondi 23b,23a, M. Birman 180, T. Bisanz 53, J.P. Biswal 161, D. Biswas 181,j, A. Bitadze 100, C. Bittrich 48, K. Bjørke 134, K.M. Black 25, T. Blazek 28a, I. Bloch 46, C. Blocker 26, A. Blue 57, U. Blumenschein 92, G.J. Bobbink 119, V.S. Bobrovnikov 121b,121a, S.S. Bocchetta 96, A. Bocci 49, D. Boerner 46, D. Bogavac 14, A.G. Bogdanchikov 121b,121a, C. Bohm 45a, V. Boisvert 93, P. Bokan 53,172, T. Bold 83a, A.S. Boldyrev 112, A.E. Bolz 61b, M. Bomben 136, M. Bona 92, J.S. Bonilla 131, M. Boonekamp 145, C.D. Booth 93, H.M. Borecka-Bielska 90, A. Borisov 122, G. Borissov 89, J. Bortfeldt 36, D. Bortoletto 135, D. Boscherini 23b, M. Bosman 14, J.D. Bossio Sola 103, K. Bouaouda 35a, J. Boudreau 139, E.V. Bouhova-Thacker 89, D. Boumediene 38, S.K. Boutle 57, A. Boveia 126, J. Boyd 36, D. Boye 33b,ar, I.R. Boyko 79, A.J. Bozson 93, J. Bracinik 21, N. The ATLAS Collaboration Chekanov 6, S.V. Chekulaev 168a, G.A. Chelkov 79,aw, M.A. Chelstowska 36, B. Chen 78, C. Chen 60a, C.H. Chen 78, H. Chen 29, J. Chen 60a, J. Chen 39, S. Chen 137, S.J. Chen 15c, X. Chen 15b,av, Y. Chen 82, Y-H. Chen 46, H.C. Cheng 63a, H.J. Cheng 15a,15d, A. Cheplakov 79, E. Cheremushkina 122, R. Cherkaoui El Moursli 35e, E. Cheu 7, K. Cheung 64, T.J.A. Chevalérias 145, L. Chevalier 145, V. Chiarella 51, G. Chiarelli 71a, G. Chiodini 67a, A.S. Chisholm 21, A. Chitan 27b, I. Chiu 163, Y.H. Chiu 176, M.V. Chizhov 79, K. Choi 65, A.R. Chomont 72a,72b, S. Chouridou 162, Y.S. Chow 119, M.C. Chu 63a, X. Chu 15a, J. Chudoba 141, A.J. Chuinard 103, J.J. Chwastowski 84, L. Chytka 130, D. Cieri 114, K.M. Ciesla 84, D. Cinca 47, V. Cindro 91, I.A. Cioar˘a 27b, A. Ciocio 18, F. Cirotto 69a,69b, Z.H. Citron 180,k, M. Citterio 68a, D.A. Ciubotaru 27b, B.M. Ciungu 167, A. Clark 54, M.R. Clark 39, P.J. Clark 50, C. Clement 45a,45b, Y. Coadou 101, M. Cobal 66a,66c, A. Coccaro 55b, J. Cochran 78, H. Cohen 161, A.E.C. Coimbra 36, L. Colasurdo 118, B. Cole 39, A.P. Colijn 119, J. Collot 58, P. Conde Muiño 140a,f , E. Coniavitis 52, S.H. Connell 33b, I.A. Connelly 57, S. Constantinescu 27b, F. Conventi 69a,ay, A.M. Cooper-Sarkar 135, F. Cormier 175, K.J.R. Cormier 167, L.D. Corpe 94, M. Corradi 72a,72b, E.E. Corrigan 96, F. Corriveau 103,ae, A. Cortes-Gonzalez 36, M.J. Costa 174, F. Costanza 5, D. Costanzo 149, G. Cowan 93, J.W. Cowley 32, J. Crane 100, K. Cranmer 124, S.J. Crawley 57, R.A. Creager 137, S. Crépé-Renaudin 58, F. Crescioli 136, M. Cristinziani 24, V. Croft 119, G. Crosetti 41b,41a, A. Cueto 5, T. Cuhadar Donszelmann 149, A.R. Cukierman 153, W.R. Cunningham 57, S. Czekierda 84, P. Czodrowski 36, R. Di Nardo 102, K.F. Di Petrillo 59, R. Di Sipio 167, D. Di Valentino 34, C. Diaconu 101, F.A. Dias 40, T. Dias Do Vale 140a, M.A. Diaz 147a, J. Dickinson 18, E.B. Diehl 105, J. Dietrich 19, S. Díez Cornell 46, A. Dimitrievska 18, W. Ding 15b, J. Dingfelder 24, F. Dittus 36, F. Djama 101, T. Djobava 159b, J.I. Djuvsland 17, M.A.B. Do Vale 80c, M. Dobre 27b, D. Dodsworth 26, C. Doglioni 96, J. Dolejsi 143, Z. Dolezal 143, M. Donadelli 80d, B. Dong 60c, J. Donini 38, A. The ATLAS Collaboration Cabrera Urbán 174, D. Caforio 56, H. Cai 173, V.M.M. Cairo 153, O. Cakir 4a, N. Calace 36, P. Calaﬁura 18, A. Calandri 101, G. Calderini 136, P. Calfayan 65, G. Callea 57, L.P. Caloba 80b, S. Calvente Lopez 98, D. Calvet 38, S. Calvet 38, T.P. Calvet 155, M. Calvetti 71a,71b, 136 36 98 73 73b 134 12 The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 I. Carli 143, T. Carli 36, G. Carlino 69a, B.T. Carlson 139, L. Carminati 68a,68b, R.M.D. Carney 45a,45b, S. Caron 118, E. Carquin 147c, S. Carrá 46, J.W.S. Carter 167, M.P. Casado 14,e, A.F. Casha 167, D.W. Casper 171, R. Castelijn 119, F.L. Castillo 174, V. Castillo Gimenez 174, N.F. Castro 140a,140e, A. Catinaccio 36, J.R. Catmore 134, A. Cattai 36, J. Caudron 24, V. Cavaliere 29, E. Cavallaro 14, M. Cavalli-Sforza 14, V. Cavasinni 71a,71b, E. Celebi 12b, F. Ceradini 74a,74b, L. Cerda Alberich 174, K. Cerny 130, A.S. Cerqueira 80a, A. Cerri 156, L. Cerrito 73a,73b, F. Cerutti 18, A. Cervelli 23b,23a, S.A. Cetin 12b, Z. Chadi 35a, D. Chakraborty 120, S.K. Chan 59, W.S. Chan 119, W.Y. Chan 90, J.D. Chapman 32, B. Chargeishvili 159b, D.G. Charlton 21, T.P. Charman 92, C.C. Chau 34, S. Che 126, S. Chekanov 6, S.V. Chekulaev 168a, G.A. Chelkov 79,aw, M.A. Chelstowska 36, B. Chen 78, C. Chen 60a, C.H. Chen 78, H. Chen 29, J. Chen 60a, J. Chen 39, S. Chen 137, S.J. Chen 15c, X. Chen 15b,av, Y. Chen 82, Y-H. Chen 46, H.C. Cheng 63a, H.J. Cheng 15a,15d, A. Cheplakov 79, E. Cheremushkina 122, R. Cherkaoui El Moursli 35e, E. Cheu 7, K. Cheung 64, T.J.A. Chevalérias 145, L. Chevalier 145, V. Chiarella 51, G. Chiarelli 71a, G. Chiodini 67a, A.S. Chisholm 21, A. Chitan 27b, I. Chiu 163, Y.H. Chiu 176, M.V. Chizhov 79, K. Choi 65, A.R. Chomont 72a,72b, S. Chouridou 162, Y.S. Chow 119, M.C. Chu 63a, X. Chu 15a, J. Chudoba 141, A.J. Chuinard 103, J.J. Chwastowski 84, L. Chytka 130, D. Cieri 114, K.M. Ciesla 84, D. Cinca 47, V. Cindro 91, I.A. Cioar˘a 27b, A. Ciocio 18, F. Cirotto 69a,69b, Z.H. Citron 180,k, M. Citterio 68a, D.A. Ciubotaru 27b, B.M. Ciungu 167, A. Clark 54, M.R. Clark 39, P.J. Clark 50, C. Clement 45a,45b, Y. Coadou 101, M. Cobal 66a,66c, A. Coccaro 55b, J. Cochran 78, H. Cohen 161, A.E.C. The ATLAS Collaboration Coimbra 36, L. Colasurdo 118, B. Cole 39, A.P. Colijn 119, J. Collot 58, P. Conde Muiño 140a,f , E. Coniavitis 52, S.H. Connell 33b, I.A. Connelly 57, S. Constantinescu 27b, F. Conventi 69a,ay, A.M. Cooper-Sarkar 135, F. Cormier 175, K.J.R. Cormier 167, L.D. Corpe 94, M. Corradi 72a,72b, E.E. Corrigan 96, F. Corriveau 103,ae, A. Cortes-Gonzalez 36, M.J. Costa 174, F. Costanza 5, D. Costanzo 149, G. Cowan 93, J.W. Cowley 32, J. Crane 100, K. Cranmer 124, S.J. Crawley 57, R.A. Creager 137, S. Crépé-Renaudin 58, F. Crescioli 136, M. Cristinziani 24, V. Croft 119, G. Crosetti 41b,41a, A. Cueto 5, T. Cuhadar Donszelmann 149, A.R. Cukierman 153, W.R. Cunningham 57, S. Czekierda 84, P. Czodrowski 36, M.J. Da Cunha Sargedas De Sousa 60b, J.V. Da Fonseca Pinto 80b, C. Da Via 100, W. Dabrowski 83a, T. Dado 28a, S. Dahbi 35e, T. Dai 105, C. Dallapiccola 102, M. Dam 40, G. D’amen 29, V. D’Amico 74a,74b, J. Damp 99, J.R. Dandoy 137, M.F. Daneri 30, N.P. Dang 181,j, N.S. Dann 100, M. Danninger 175, V. Dao 36, G. Darbo 55b, O. Dartsi 5, A. Dattagupta 131, T. Daubney 46, S. D’Auria 68a,68b, W. Davey 24, C. David 46, T. Davidek 143, D.R. Davis 49, I. Dawson 149, K. De 8, R. De Asmundis 69a, M. De Beurs 119, S. De Castro 23b,23a, S. De Cecco 72a,72b, N. De Groot 118, P. de Jong 119, H. De la Torre 106, A. De Maria 15c, D. De Pedis 72a, A. De Salvo 72a, U. De Sanctis 73a,73b, M. De Santis 73a,73b, A. De Santo 156, K. De Vasconcelos Corga 101, J.B. De Vivie De Regie 132, C. Debenedetti 146, D.V. Dedovich 79, A.M. Deiana 42, M. Del Gaudio 41b,41a, J. Del Peso 98, Y. Delabat Diaz 46, D. Delgove 132, F. Deliot 145,r, C.M. Delitzsch 7, M. Della Pietra 69a,69b, D. Della Volpe 54, A. Dell’Acqua 36, L. Dell’Asta 73a,73b, M. Delmastro 5, C. Delporte 132, P.A. Delsart 58, D.A. DeMarco 167, S. Demers 183, M. Demichev 79, G. Demontigny 109, S.P. Denisov 122, D. Denysiuk 119, L. D’Eramo 136, D. Derendarz 84, J.E. Derkaoui 35d, F. Derue 136, P. Dervan 90, K. Desch 24, C. Deterre 46, K. Dette 167, C. Deutsch 24, M.R. Devesa 30, P.O. Deviveiros 36, A. Dewhurst 144, F.A. Di Bello 54, A. Di Ciaccio 73a,73b, L. Di Ciaccio 5, W.K. The ATLAS Collaboration Di Clemente 137, C. Di Donato 69a,69b, A. Di Girolamo 36, G. Di Gregorio 71a,71b, B. Di Micco 74a,74b, R. Di Nardo 102, K.F. Di Petrillo 59, R. Di Sipio 167, D. Di Valentino 34, C. Diaconu 101, F.A. Dias 40, T. Dias Do Vale 140a, M.A. Diaz 147a, J. Dickinson 18, E.B. Diehl 105, J. Dietrich 19, S. Díez Cornell 46, A. Dimitrievska 18, W. Ding 15b, J. Dingfelder 24, F. Dittus 36, F. Djama 101, T. Djobava 159b, J.I. Djuvsland 17, M.A.B. Do Vale 80c, M. Dobre 27b, D. Dodsworth 26, C. Doglioni 96, J. Dolejsi 143, Z. Dolezal 143, M. Donadelli 80d, B. Dong 60c, J. Donini 38, A. D’onofrio 92, M. D’Onofrio 90, J. Dopke 144, A. Doria 69a, M.T. Dova 88, A.T. Doyle 57, E. Drechsler 152, E. Dreyer 152, T. Dreyer 53, A.S. Drobac 170, D. Du 60b, Y. Duan 60b, F. Dubinin 110, M. Dubovsky 28a, A. Dubreuil 54, E. Duchovni 180, G. Duckeck 113, A. Ducourthial 136, O.A. Ducu 109, D. Duda 114, A. Dudarev 36, A.C. Dudder 99, E.M. Duﬃeld 18, L. Duﬂot 132, M. Dührssen 36, C. Dülsen 182, M. Dumancic 180, A.E. Dumitriu 27b, A.K. Duncan 57, M. Dunford 61a, A. Duperrin 101, H. Duran Yildiz 4a, M. Düren 56, A. Durglishvili 159b, D. Duschinger 48, B. Dutta 46, D. Duvnjak 1, G.I. Dyckes 137, M. Dyndal 36, S. Dysch 100, B.S. Dziedzic 84, K.M. Ecker 114, R.C. Edgar 105, 49 36 17 18 172 35 35 The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 12 I. Carli 143, T. Carli 36, G. Carlino 69a, B.T. Carlson 139, L. Carminati 68a,68b, R.M.D. Carney 45a,45b, S. Caron 118, E. Carquin 147c, S. Carrá 46, J.W.S. Carter 167, M.P. Casado 14,e, A.F. Casha 167, D.W. Casper 171, R. Castelijn 119, F.L. Castillo 174, V. Castillo Gimenez 174, N.F. Castro 140a,140e, A. Catinaccio 36, J.R. Catmore 134, A. Cattai 36, J. Caudron 24, V. Cavaliere 29, E. Cavallaro 14, M. Cavalli-Sforza 14, V. Cavasinni 71a,71b, E. Celebi 12b, F. Ceradini 74a,74b, L. Cerda Alberich 174, K. Cerny 130, A.S. Cerqueira 80a, A. Cerri 156, L. Cerrito 73a,73b, F. Cerutti 18, A. Cervelli 23b,23a, S.A. Cetin 12b, Z. Chadi 35a, D. Chakraborty 120, S.K. Chan 59, W.S. Chan 119, W.Y. Chan 90, J.D. Chapman 32, B. Chargeishvili 159b, D.G. Charlton 21, T.P. Charman 92, C.C. Chau 34, S. Che 126, S. The ATLAS Collaboration D’onofrio 92, M. D’Onofrio 90, J. Dopke 144, A. Doria 69a, M.T. Dova 88, A.T. Doyle 57, E. Drechsler 152, E. Dreyer 152, T. Dreyer 53, A.S. Drobac 170, D. Du 60b, Y. Duan 60b, F. Dubinin 110, M. Dubovsky 28a, A. Dubreuil 54, E. Duchovni 180, G. Duckeck 113, A. Ducourthial 136, O.A. Ducu 109, D. Duda 114, A. Dudarev 36, A.C. Dudder 99, E.M. Duﬃeld 18, L. Duﬂot 132, M. Dührssen 36, C. Dülsen 182, M. Dumancic 180, A.E. Dumitriu 27b, A.K. Duncan 57, M. Dunford 61a, A. Duperrin 101, H. Duran Yildiz 4a, M. Düren 56, A. Durglishvili 159b, D. Duschinger 48, B. Dutta 46, D. Duvnjak 1, G.I. Dyckes 137, M. Dyndal 36, S. Dysch 100, B.S. Dziedzic 84, K.M. Ecker 114, R.C. Edgar 105, M.G. Eggleston 49, T. Eifert 36, G. Eigen 17, K. Einsweiler 18, T. Ekelof 172, H. El Jarrari 35e, M. El Kacimi 35c, R. El Kosseiﬁ101, V. Ellajosyula 172, M. Ellert 172, F. Ellinghaus 182, A.A. Elliot 92, N. Ellis 36, The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 13 The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 1 J. Elmsheuser 29, M. Elsing 36, D. Emeliyanov 144, A. Emerman 39, Y. Enari 163, M.B. Epland 49, J. Erdmann 47, A. Ereditato 20, M. Errenst 36, M. Escalier 132, C. Escobar 174, O. Estrada Pastor 174, E. Etzion 161, H. Evans 65, A. Ezhilov 138, F. Fabbri 57, L. Fabbri 23b,23a, V. Fabiani 118, G. Facini 94, R.M. Faisca Rodrigues Pereira 140a, R.M. Fakhrutdinov 122, S. Falciano 72a, P.J. Falke 5, S. Falke 5, J. Faltova 143, Y. Fang 15a, Y. Fang 15a, G. Fanourakis 44, M. Fanti 68a,68b, M. Faraj 66a,66c,u, A. Farbin 8, A. Farilla 74a, E.M. Farina 70a,70b, T. Farooque 106, S. Farrell 18, S.M. Farrington 50, P. Farthouat 36, F. Fassi 35e P. Fassnacht 36, D. Fassouliotis 9, M. Faucci Giannelli 50, W.J. Fawcett 32, L. Fayard 132, O.L. Fedin 138,p, W. Fedorko 175, M. Feickert 42, L. Feligioni 101, A. Fell 149, C. Feng 60b, E.J. Feng 36, M. Feng 49, M.J. Fenton 57, A.B. Fenyuk 122, J. Ferrando 46, A. Ferrante 173, A. Ferrari 172, P. Ferrari 119, R. Ferrari 70a, D.E. Ferreira de Lima 61b, A. Ferrer 174, D. Ferrere 54, C. Ferretti 105, F. Fiedler 99, A. Filipˇciˇc 91, F. Filthaut 118, K.D. Finelli 25, M.C.N. Fiolhais 140a,140c,a, L. The ATLAS Collaboration Fiorini 174, F. Fischer 113, W.C. Fisher 106, I. Fleck 151, P. Fleischmann 105, R.R.M. Fletcher 137, T. Flick 182, B.M. Flierl 113, L. Flores 137, L.R. Flores Castillo 63a, F.M. Follega 75a,75b, N. Fomin 17, J.H. Foo 167, G.T. Forcolin 75a,75b, A. Formica 145, F.A. Förster 14, A.C. Forti 100, A.G. Foster 21, M.G. Foti 135, D. Fournier 132, H. Fox 89, P. Francavilla 71a,71b, S. Francescato 72a,72b, M. Franchini 23b,23a, S. Franchino 61a, D. Francis 36, L. Franconi 20, M. Franklin 59, A.N. Fray 92, P.M. Freeman 21, B. Freund 109, W.S. Freund 80b, E.M. Freundlich 47, D.C. Frizzell 128, D. Froidevaux 36, J.A. Frost 135, C. Fukunaga 164, E. Fullana Torregrosa 174, E. Fumagalli 55b,55a, T. Fusayasu 115, J. Fuster 174, A. Gabrielli 23b,23a, A. Gabrielli 18, G.P. Gach 83a, S. Gadatsch 54, P. Gadow 114, G. Gagliardi 55b,55a, L.G. Gagnon 109, C. Galea 27b, B. Galhardo 140a, G.E. Gallardo 135, E.J. Gallas 135, B.J. Gallop 144, G. Galster 40, R. Gamboa Goni 92, K.K. Gan 126, S. Ganguly 180, J. Gao 60a, Y. Gao 50, Y.S. Gao 31,m, C. García 174, J.E. García Navarro 174, J.A. García Pascual 15a, C. Garcia-Argos 52, M. Garcia-Sciveres 18, R.W. Gardner 37, N. Garelli 153, S. Gargiulo 52, V. Garonne 134, A. Gaudiello 55b,55a, G. Gaudio 70a, I.L. Gavrilenko 110, A. Gavrilyuk 123, C. Gay 175, G. Gaycken 46, E.N. Gazis 10, A.A. Geanta 27b, C.M. Gee 146, C.N.P. Gee 144, J. Geisen 53, M. Geisen 99, M.P. Geisler 61a, C. Gemme 55b, M.H. Genest 58, C. Geng 105, S. Gentile 72a,72b, S. George 93, T. Geralis 44, L.O. Gerlach 53, P. Gessinger-Befurt 99, G. Gessner 47, S. Ghasemi 151, M. Ghasemi Bostanabad 176, A. Ghosh 132, A. Ghosh 77, B. Giacobbe 23b, S. Giagu 72a,72b, N. Giangiacomi 23b,23a, P. Giannetti 71a, A. Giannini 69a,69b, G. Giannini 14, S.M. Gibson 93, M. Gignac 146, D. Gillberg 34, G. Gilles 182, D.M. Gingrich 3,ax, M.P. Giordani 66a,66c, F.M. Giorgi 23b, P.F. Giraud 145, G. Giugliarelli 66a,66c, D. Giugni 68a, F. Giuli 73a,73b, S. Gkaitatzis 162, I. Gkialas 9,h, E.L. Gkougkousis 14, P. Gkountoumis 10, L.K. Gladilin 112, C. Glasman 98, J. Glatzer 14, P.C.F. Glaysher 46, A. Glazov 46, G.R. Gledhill 131, M. Goblirsch-Kolb 26, D. Godin 109, S. Goldfarb 104, T. Golling 54, D. Golubkov 122, A. Gomes 140a,140b, R. Goncalves Gama 53, R. Gonçalo 140a,140b, G. Gonella 52, L. The ATLAS Collaboration Gonella 21, A. Gongadze 79, F. Gonnella 21, J.L. Gonski 59, S. González de la Hoz 174, S. Gonzalez-Sevilla 54, G.R. Gonzalvo Rodriguez 174, L. Goossens 36, P.A. Gorbounov 123, H.A. Gordon 29, B. Gorini 36, E. Gorini 67a,67b, A. Gorišek 91, A.T. Goshaw 49, M.I. Gostkin 79, C.A. Gottardo 118, M. Gouighri 35b, D. Goujdami 35c, A.G. Goussiou 148, N. Govender 33b, C. Goy 5, E. Gozani 160, I. Grabowska-Bold 83a, E.C. Graham 90, J. Gramling 171, E. Gramstad 134, S. Grancagnolo 19, M. Grandi 156, V. Gratchev 138, P.M. Gravila 27f, F.G. Gravili 67a,67b, C. Gray 57, H.M. Gray 18, C. Grefe 24, K. Gregersen 96, I.M. Gregor 46, P. Grenier 153, K. Grevtsov 46, C. Grieco 14, N.A. Grieser 128, J. Griﬃths 8, A.A. Grillo 146, K. Grimm 31,l, S. Grinstein 14,z, J.-F. Grivaz 132, S. Groh 99, E. Gross 180, J. Grosse-Knetter 53, Z.J. Grout 94, C. Grud 105, A. Grummer 117, L. Guan 105, W. Guan 181, J. Guenther 36, A. Guerguichon 132, J.G.R. Guerrero Rojas 174, F. Guescini 114, D. Guest 171, R. Gugel 52, T. Guillemin 5, S. Guindon 36, U. Gul 57, J. Guo 60c, W. Guo 105, Y. Guo 60a,t, Z. Guo 101, R. Gupta 46, S. Gurbuz 12c, G. Gustavino 128, M. Guth 52, P. Gutierrez 128, C. Gutschow 94, C. Guyot 145, C. Gwenlan 135, C.B. Gwilliam 90, A. Haas 124, C. Haber 18, H.K. Hadavand 8, N. Haddad 35e, A. Hadef 60a, S. Hageböck 36, M. Haleem 177, J. Haley 129, G. Halladjian 106 G.D. Hallewell 101, K. Hamacher 182, P. Hamal 130, K. Hamano 176, H. Hamdaoui 35e, G.N. Hamity 149, K. Han 60a,ak, L. Han 60a, S. Han 15a,15d, Y.F. Han 167, K. Hanagaki 81,x, M. Hance 146, D.M. Handl 113, B. Haney 137, R. Hankache 136, E. Hansen 96, J.B. Hansen 40, J.D. Hansen 40, M.C. Hansen 24, P.H. Hansen 40, E C H 100 K H 169 T H b 182 S H k h 107 PF H i 178 N M H 113 J. Elmsheuser 29, M. Elsing 36, D. Emeliyanov 144, A. Emerman 39, Y. Enari 163, M.B. Epland 49, J. Erdmann 47, A. Ereditato 20, M. Errenst 36, M. Escalier 132, C. Escobar 174, O. Estrada Pastor 174, E. Etzion 161, H. Evans 65, A. Ezhilov 138, F. Fabbri 57, L. Fabbri 23b,23a, V. Fabiani 118, G. Facini 94, R.M. The ATLAS Collaboration Faisca Rodrigues Pereira 140a, R.M. Fakhrutdinov 122, S. Falciano 72a, P.J. Falke 5, S. Falke 5, J. Faltova 143, Y. Fang 15a, Y. Fang 15a, G. Fanourakis 44, M. Fanti 68a,68b, M. Faraj 66a,66c,u, A. Farbin 8, A F ill 74a E M F i 70a 70b T F 106 S F ll 18 S M F i 50 P F h 36 F F i 35e A. Farilla 74a, E.M. Farina 70a,70b, T. Farooque 106, S. Farrell 18, S.M. Farrington 50, P. Farthouat 36, F. Fassi 35e, P. Fassnacht 36, D. Fassouliotis 9, M. Faucci Giannelli 50, W.J. Fawcett 32, L. Fayard 132, O.L. Fedin 138,p, 175 42 101 149 60b 36 49 , , g , , g , J g , g , M.J. Fenton 57, A.B. Fenyuk 122, J. Ferrando 46, A. Ferrante 173, A. Ferrari 172, P. Ferrari 119, R. Ferrari 70a, i d i 61b A 174 54 i 105 i dl 99 A ili ˇiˇ 91 g , j , , , y , , Grabowska-Bold 83a, E.C. Graham 90, J. Gramling 171, E. Gramstad 134, S. Grancagnolo 19, M. Grandi 156, V. Gratchev 138, P.M. Gravila 27f, F.G. Gravili 67a,67b, C. Gray 57, H.M. Gray 18, C. Grefe 24, K. Gregersen 96, M. Gregor 46, P. Grenier 153, K. Grevtsov 46, C. Grieco 14, N.A. Grieser 128, J. Griﬃths 8, A.A. Grillo 146, K. Grimm 31,l, S. Grinstein 14,z, J.-F. Grivaz 132, S. Groh 99, E. Gross 180, J. Grosse-Knetter 53, Z.J. Grout 94, . Grud 105, A. Grummer 117, L. Guan 105, W. Guan 181, J. Guenther 36, A. Guerguichon 132, The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 14 14 The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 K.K. Heidegger 52, W.D. Heidorn 78, J. Heilman 34, S. Heim 46, T. Heim 18, B. Heinemann 46,as, J.J. Heinrich 131, L. Heinrich 36, C. Heinz 56, J. Hejbal 141, L. Helary 61b, A. Held 175, S. Hellesund 134, C.M. Helling 146, S. Hellman 45a,45b, C. Helsens 36, R.C.W. Henderson 89, Y. Heng 181, S. Henkelmann 175, A.M. Henriques Correia 36, G.H. Herbert 19, H. Herde 26, V. Herget 177, Y. Hernández Jiménez 33c, H. Herr 99, M.G. Herrmann 113, T. Herrmann 48, G. Herten 52, R. Hertenberger 113, L. Hervas 36, T.C. Herwig 137, G.G. Hesketh 94, N.P. Hessey 168a, A. Higashida 163, S. Higashino 81, E. The ATLAS Collaboration Higón-Rodriguez 174 K. Hildebrand 37, E. Hill 176, J.C. Hill 32, K.K. Hill 29, K.H. Hiller 46, S.J. Hillier 21, M. Hils 48, I. Hinchliffe 18, F. Hinterkeuser 24, M. Hirose 133, S. Hirose 52, D. Hirschbuehl 182, B. Hiti 91, O. Hladik 141, D.R. Hlaluku 33c X. Hoad 50, J. Hobbs 155, N. Hod 180, M.C. Hodgkinson 149, A. Hoecker 36, F. Hoenig 113, D. Hohn 52, D. Hohov 132, T.R. Holmes 37, M. Holzbock 113, L.B.A.H Hommels 32, S. Honda 169, T.M. Hong 139, J.C. Honig 52, A. Hönle 114, B.H. Hooberman 173, W.H. Hopkins 6, Y. Horii 116, P. Horn 48, L.A. Horyn 37, S. Hou 158, A. Hoummada 35a, J. Howarth 100, J. Hoya 88, M. Hrabovsky 130, J. Hrdinka 76, I. Hristova 19, J. Hrivnac 132, A. Hrynevich 108, T. Hryn’ova 5, P.J. Hsu 64, S.-C. Hsu 148, Q. Hu 29, S. Hu 60c, Y.F. Hu 15a, D.P. Huang 94, Y. Huang 60a, Y. Huang 15a, Z. Hubacek 142, F. Hubaut 101, M. Huebner 24, F. Huegging 24, T.B. Huffman 135, M. Huhtinen 36, R.F.H. Hunter 34, P. Huo 155, A.M. Hupe 34, N. Huseynov 79,ag, J. Huston 106, J. Huth 59, R. Hyneman 105, S. Hyrych 28a, G. Iacobucci 54, G. Iakovidis 29, I. Ibragimov 151, L. Iconomidou-Fayard 132, Z. Idrissi 35e, P. Iengo 36, R. Ignazzi 40, O. Igonkina 119,ab,∗, R. Iguchi 163, T. Iizawa 54, Y. Ikegami 81, M. Ikeno 81, D. Iliadis 162, N. Ilic 118,167,ae, F. Iltzsche 48, G. Introzzi 70a,70b, M. Iodice 74a, K. Iordanidou 168a, V. Ippolito 72a,72b, M.F. Isacson 172, M. Ishino 163, W. Islam 129, C. Issever 135, S. Istin 160, F. Ito 169, J.M. Iturbe Ponce 63a, R. Iuppa 75a,75b, A. Ivina 180, H. Iwasaki 81, J.M. Izen 43, V. Izzo 69a, P. Jacka 141, P. Jackson 1, R.M. Jacobs 24, B.P. Jaeger 152, V. Jain 2, G. Jäkel 182, K.B. Jakobi 99, K. Jakobs 52, S. Jakobsen 76, T. Jakoubek 141, J. Jamieson 57, K.W. Janas 83a, R. Jansky 54, J. Janssen 24, M. Janus 53, P.A. Janus 83a, G. Jarlskog 96, N. Javadov 79,ag, T. Jav ˚urek 36, M. Javurkova 52, F. Jeanneau 145, L. Jeanty 131, J. Jejelava 159a,ah, A. Jelinskas 178, P. Jenni 52,b, J. Jeong 46, N. Jeong 46, S. Jézéquel 5, H. Ji 181, J. Jia 155, H. Jiang 78, Y. Jiang 60a, Z. Jiang 153,q, S. The ATLAS Collaboration Jiggins 52, F.A. Jimenez Morales 38, J. Jimenez Pena 114, S. Jin 15c, A. Jinaru 27b, O. Jinnouchi 165, H. Jivan 33c, P. Johansson 149, K.A. Johns 7, C.A. Johnson 65, K. Jon-And 45a,45b, R.W.L. Jones 89, S.D. Jones 156, S. Jones 7, T.J. Jones 90, J. Jongmanns 61a, P.M. Jorge 140a, J. Jovicevic 36, X. Ju 18, J.J. Junggeburth 114, A. Juste Rozas 14,z, A. Kaczmarska 84, M. Kado 72a,72b, H. Kagan 126, M. Kagan 153, C. Kahra 99, T. Kaji 179, E. Kajomovitz 160, C.W. Kalderon 96, A. Kaluza 99, A. Kamenshchikov 122, M. Kaneda 163, L. Kanjir 91, Y. Kano 163, V.A. Kantserov 111, J. Kanzaki 81, L.S. Kaplan 181, D. Kar 33c, K. Karava 135, M.J. Kareem 168b, S.N. Karpov 79, Z.M. Karpova 79, V. Kartvelishvili 89, A.N. Karyukhin 122, L. Kashif 181, R.D. Kass 126, A. Kastanas 45a,45b, C. Kato 60d,60c, J. Katzy 46, K. Kawade 150, K. Kawagoe 87, T. Kawaguchi 116, T. Kawamoto 163, G. Kawamura 53, E.F. Kay 176 V.F. Kazanin 121b,121a, R. Keeler 176, R. Kehoe 42, J.S. Keller 34, E. Kellermann 96, D. Kelsey 156, J.J. Kempster 21, J. Kendrick 21, O. Kepka 141, S. Kersten 182, B.P. Kerševan 91, S. Ketabchi Haghighat 167, M. Khader 173, F. Khalil-Zada 13, M. Khandoga 145, A. Khanov 129, A.G. Kharlamov 121b,121a, T. Kharlamova 121b,121a, E.E. Khoda 175, A. Khodinov 166, T.J. Khoo 54, E. Khramov 79, J. Khubua 159b, S. Kido 82, M. Kiehn 54, C.R. Kilby 93, Y.K. Kim 37, N. Kimura 94, O.M. Kind 19, B.T. King 90,∗, D. Kirchmeier 48, J. Kirk 144, A.E. Kiryunin 114, T. Kishimoto 163, D.P. Kisliuk 167, V. Kitali 46, O. Kivernyk 5, T. Klapdor-Kleingrothaus 52, M. Klassen 61a, M.H. Klein 105, M. Klein 90, U. Klein 90, K. Kleinknecht 99, P. Klimek 120, A. Klimentov 29, T. Klingl 24, T. Klioutchnikova 36, F.F. Klitzner 113, P. Kluit 119, S. Kluth 114, E. Kneringer 76, E.B.F.G. Knoops 101, A. Knue 52, D. Kobayashi 87, T. Kobayashi 163, M. Kobel 48, M. Kocian 153, P. Kodys 143, P.T. Koenig 24, T. Koffas 34, N.M. Köhler 36, T. Koi 153, M. Kolb 61b, I. Koletsou 5, T. Komarek 130, T. Kondo 81, N. Kondrashova 60c, K. Köneke 52, A.C. König 118, T. Kono 125, R. Konoplich 124,an, V. Konstantinides 94, N. Konstantinidis 94, B. Konya 96, R. Kopeliansky 65, S. Koperny 83a, K. Korcyl 84, K. The ATLAS Collaboration Kordas 162, G. Koren 161, A. Korn 94, I. Korolkov 14, E.V. Korolkova 149, N. Korotkova 112, O. Kortner 114, S. Kortner 114, T. Kosek 143, V.V. Kostyukhin 166, A. Kotsokechagia 132, A. Kotwal 49, A. Koulouris 10, A. Kourkoumeli-Charalampidi 70a,70b, C. Kourkoumelis 9, E. Kourlitis 149, V. Kouskoura 29, A.B. Kowalewska 84, R. Kowalewski 176, C. Kozakai 163, W. Kozanecki 145, A.S. Kozhin 122, V.A. Kramarenko 112, G. Kramberger 91, D. Krasnopevtsev 60a, M.W. Krasny 136, A. Krasznahorkay 36, D. Krauss 114, J.A. Kremer 83a, J. Kretzschmar 90, P. Krieger 167, F. Krieter 113, A. Krishnan 61b, K. Krizka 18, K. Kroeninger 47, H. Kroha 114, J. Kroll 141, J. Kroll 137, K.S. Krowpman 106, J. Krstic 16, U. Kruchonak 79, K.K. Heidegger 52, W.D. Heidorn 78, J. Heilman 34, S. Heim 46, T. Heim 18, B. Heinemann 46,as, J.J. Heinrich 131, L. Heinrich 36, C. Heinz 56, J. Hejbal 141, L. Helary 61b, A. Held 175, S. Hellesund 134, C.M. Helling 146, S. Hellman 45a,45b, C. Helsens 36, R.C.W. Henderson 89, Y. Heng 181, S. Henkelmann 175, A.M. Henriques Correia 36, G.H. Herbert 19, H. Herde 26, V. Herget 177, Y. Hernández Jiménez 33c, H. Herr 99, M.G. Herrmann 113, T. Herrmann 48, G. Herten 52, R. Hertenberger 113, L. Hervas 36, T.C. Herwig 137, G.G. Hesketh 94, N.P. Hessey 168a, A. Higashida 163, S. Higashino 81, E. Higón-Rodriguez 17 K. Hildebrand 37, E. Hill 176, J.C. Hill 32, K.K. Hill 29, K.H. Hiller 46, S.J. Hillier 21, M. Hils 48, I. Hinchliffe 18, F. Hinterkeuser 24, M. Hirose 133, S. Hirose 52, D. Hirschbuehl 182, B. Hiti 91, O. Hladik 141, D.R. Hlaluku 33c X. Hoad 50, J. Hobbs 155, N. Hod 180, M.C. Hodgkinson 149, A. Hoecker 36, F. Hoenig 113, D. Hohn 52, D. Hohov 132, T.R. Holmes 37, M. Holzbock 113, L.B.A.H Hommels 32, S. Honda 169, T.M. Hong 139, J.C. Honig 52, A. Hönle 114, B.H. Hooberman 173, W.H. Hopkins 6, Y. Horii 116, P. Horn 48, L.A. Horyn 37, S. Hou 158, A. Hoummada 35a, J. Howarth 100, J. Hoya 88, M. Hrabovsky 130, J. Hrdinka 76, I. Hristova 19, J. Hrivnac 132, A. Hrynevich 108, T. Hryn’ova 5, P.J. Hsu 64, S.-C. Hsu 148, Q. Hu 29, S. Hu 60c, Y.F. Hu 15a, D.P. Huang 94, Y. Huang 60a, Y. Huang 15a, Z. Hubacek 142, F. Hubaut 101, M. The ATLAS Collaboration Huebner 24, F. Huegging 24, T.B. Huffman 135, M. Huhtinen 36, R.F.H. Hunter 34, P. Huo 155, A.M. Hupe 34, N. Huseynov 79,ag, J. Huston 106, J. Huth 59, R. Hyneman 105, S. Hyrych 28a, G. Iacobucci 54, G. Iakovidis 29, I. Ibragimov 151, L. Iconomidou-Fayard 132, Z. Idrissi 35e, P. Iengo 36, R. Ignazzi 40, O. Igonkina 119,ab,∗, R. Iguchi 163, T. Iizawa 54, Y. Ikegami 81, M. Ikeno 81, D. Iliadis 162, N. Ilic 118,167,ae, F. Iltzsche 48, G. Introzzi 70a,70b, M. Iodice 74a, K. Iordanidou 168a, V. Ippolito 72a,72b, M.F. Isacson 172, M. Ishino 163, W. Islam 129, C. Issever 135, S. Istin 160, F. Ito 169, J.M. Iturbe Ponce 63a, R. Iuppa 75a,75b, A. Ivina 180, H. Iwasaki 81, J.M. Izen 43, V. Izzo 69a, P. Jacka 141, P. Jackson 1, R.M. Jacobs 24, B.P. Jaeger 152, V. Jain 2, G. Jäkel 182, K.B. Jakobi 99, K. Jakobs 52, S. Jakobsen 76, T. Jakoubek 141, J. Jamieson 57, K.W. Janas 83a, R. Jansky 54, J. Janssen 24, M. Janus 53, P.A. Janus 83a, G. Jarlskog 96, N. Javadov 79,ag, T. Jav ˚urek 36, M. Javurkova 52, F. Jeanneau 145, L. Jeanty 131, J. Jejelava 159a,ah, A. Jelinskas 178, P. Jenni 52,b, J. Jeong 46, N. Jeong 46, S. Jézéquel 5, H. Ji 181, J. Jia 155, H. Jiang 78, Y. Jiang 60a, Z. Jiang 153,q, S. Jiggins 52, F.A. Jimenez Morales 38 J. Jimenez Pena 114, S. Jin 15c, A. Jinaru 27b, O. Jinnouchi 165, H. Jivan 33c, P. Johansson 149, K.A. Johns 7, C.A. Johnson 65, K. Jon-And 45a,45b, R.W.L. Jones 89, S.D. Jones 156, S. Jones 7, T.J. Jones 90, J. Jongmanns 61a P.M. Jorge 140a, J. Jovicevic 36, X. Ju 18, J.J. Junggeburth 114, A. Juste Rozas 14,z, A. Kaczmarska 84, M. Kado 72a,72b, H. Kagan 126, M. Kagan 153, C. Kahra 99, T. Kaji 179, E. Kajomovitz 160, C.W. Kalderon 96, A. Kaluza 99, A. Kamenshchikov 122, M. Kaneda 163, L. Kanjir 91, Y. Kano 163, V.A. Kantserov 111, J. Kanzaki 81, L.S. Kaplan 181, D. Kar 33c, K. Karava 135, M.J. Kareem 168b, S.N. Karpov 79, Z.M. Karpova 79, V. Kartvelishvili 89, A.N. Karyukhin 122, L. Kashif 181, R.D. Kass 126, A. Kastanas 45a,45b, C. Kato 60d,60c, J. Katzy 46, K. Kawade 150, K. Kawagoe 87, T. Kawaguchi 116, T. Kawamoto 163, G. Kawamura 53, E.F. Kay 176 V.F. The ATLAS Collaboration Mc Kee 105, T.G. McCarthy 114, W.P. McCormack 18, E.F. McDonald 104, J.A. Mcfayden 36, G. Mchedlidze 159b, M.A. McKay 42, K.D. McLean 176, S.J. McMahon 144, P.C. McNamara 104, C.J. McNicol 178, R.A. McPherson 176,ae, J.E. Mdhluli 33c, Z.A. Meadows 102, S. Meehan 36, T. Megy 52, S. Mehlhase 113, A. Mehta 90, T. Meideck 58, B. Meirose 43, D. Melini 174, B.R. Mellado Garcia 33c, J.D. Mellenthin 53, M. Melo 28a, F. Meloni 46, A. Melzer 24, S.B. Menary 100, The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 15 H. Krüger 24, N. Krumnack 78, M.C. Kruse 49, J.A. Krzysiak 84, T. Kubota 104, O. Kuchinskaia 166, S. Kuday 4b, J.T. Kuechler 46, S. Kuehn 36, A. Kugel 61a, T. Kuhl 46, V. Kukhtin 79, R. Kukla 101, Y. Kulchitsky 107,aj, S. Kuleshov 147c, Y.P. Kulinich 173, M. Kuna 58, T. Kunigo 85, A. Kupco 141, T. Kupfer 47, O. Kuprash 52, H. Kurashige 82, L.L. Kurchaninov 168a, Y.A. Kurochkin 107, A. Kurova 111, M.G. Kurth 15a,15d, E.S. Kuwertz 36 M. Kuze 165, A.K. Kvam 148, J. Kvita 130, T. Kwan 103, A. La Rosa 114, L. La Rotonda 41b,41a, F. La Ruffa 41b,41a C. Lacasta 174, F. Lacava 72a,72b, D.P.J. Lack 100, H. Lacker 19, D. Lacour 136, E. Ladygin 79, R. Lafaye 5, B. Laforge 136, T. Lagouri 33c, S. Lai 53, S. Lammers 65, W. Lampl 7, C. Lampoudis 162, E. Lançon 29, U. Landgraf 52, M.P.J. Landon 92, M.C. Lanfermann 54, V.S. Lang 46, J.C. Lange 53, R.J. Langenberg 36, A.J. Lankford 171, F. Lanni 29, K. Lantzsch 24, A. Lanza 70a, A. Lapertosa 55b,55a, S. Laplace 136, J.F. Laporte 145 T. Lari 68a, F. Lasagni Manghi 23b,23a, M. Lassnig 36, T.S. Lau 63a, A. Laudrain 132, A. Laurier 34, M. Lavorgna 69a,69b, S.D. Lawlor 93, M. Lazzaroni 68a,68b, B. Le 104, E. Le Guirriec 101, M. LeBlanc 7, T. LeCompte 6, F. Ledroit-Guillon 58, A.C.A. Lee 94, C.A. Lee 29, G.R. Lee 17, L. Lee 59, S.C. Lee 158, S.J. Lee 34, S. Lee 78, B. Lefebvre 168a, H.P. Lefebvre 93, M. Lefebvre 176, F. Legger 113, C. Leggett 18, K. Lehmann 152, N. Lehmann 182, G. Lehmann Miotto 36, W.A. Leight 46, A. Leisos 162,y, M.A.L. Leite 80d, C.E. Leitgeb 113, R. Leitner 143, D. Lellouch 180,∗, K.J.C. Leney 42, T. Lenz 24, B. Lenzi 36, R. Leone 7, S. The ATLAS Collaboration Leone 71a, C. Leonidopoulos 50, A. Leopold 136, G. Lerner 156, C. Leroy 109, R. Les 167, C.G. Lester 32, M. Levchenko 138, J. Levêque 5, D. Levin 105, L.J. Levinson 180, D.J. Lewis 21, B. Li 15b, B. Li 105, C-Q. Li 60a, F. Li 60c, H. Li 60a, H. Li 60b, J. Li 60c, K. Li 153, L. Li 60c, M. Li 15a, Q. Li 15a,15d, Q.Y. Li 60a, S. Li 60d,60c, X. Li 46, Y. Li 46, Z. Li 60b, Z. Liang 15a, B. Liberti 73a, A. Liblong 167, K. Lie 63c, C.Y. Lin 32, K. Lin 106, T.H. Lin 99, R.A. Linck 65, J.H. Lindon 21, A.L. Lionti 54, E. Lipeles 137, A. Lipniacka 17, M. Lisovyi 61b, T.M. Liss 173,au, A. Lister 175, A.M. Litke 146, J.D. Little 8, B. Liu 78, B.L Liu 6, H.B. Liu 29, H. Liu 105, J.B. Liu 60a, J.K.K. Liu 135, K. Liu 136, M. Liu 60a, P. Liu 18, Y. Liu 15a,15d, Y.L. Liu 105, Y.W. Liu 60a, M. Livan 70a,70b, A. Lleres 58, J. Llorente Merino 152, S.L. Lloyd 92, C.Y. Lo 63b, F. Lo Sterzo 42, E.M. Lobodzinska 46, P. Loch 7, S. Loffredo 73a,73b, T. Lohse 19, K. Lohwasser 149, M. Lokajicek 141, J.D. Long 173, R.E. Long 89, L. Longo 36, K.A. Looper 126, J.A. Lopez 147c, I. Lopez Paz 100, A. Lopez Solis 149, J. Lorenz 113, N. Lorenzo Martinez 5, M. Losada 22, P.J. Lösel 113, A. Lösle 52, X. Lou 46, X. Lou 15a, A. Lounis 132, J. Love 6, P.A. Love 89, J.J. Lozano Bahilo 174, M. Lu 60a, Y.J. Lu 64, H.J. Lubatti 148, C. Luci 72a,72b, A. Lucotte 58, C. Luedtke 52, F. Luehring 65, I. Luise 136, L. Luminari 72a, B. Lund-Jensen 154, M.S. Lutz 102, D. Lynn 29, R. Lysak 141, E. Lytken 96, F. Lyu 15a, V. Lyubushkin 79, T. Lyubushkina 79, H. Ma 29, L.L. Ma 60b, Y. Ma 60b, G. Maccarrone 51, A. Macchiolo 114, C.M. Macdonald 149, J. Machado Miguens 137, D. Madaffari 174, R. Madar 38, W.F. Mader 48, N. Madysa 48, J. Maeda 82, S. Maeland 17, T. Maeno 29, M. Maerker 48, A.S. Maevskiy 112, V. Magerl 52, N. Magini 78, D.J. Mahon 39, C. Maidantchik 80b, T. Maier 113, A. Maio 140a,140b,140d, K. Maj 83a, O. Majersky 28a, S. Majewski 131, Y. Makida 81, N. Makovec 132, B. The ATLAS Collaboration Malaescu 136, Pa. Malecki 84, V.P. Maleev 138, F. Malek 58, U. Mallik 77, D. Malon 6, C. Malone 32, S. Maltezos 10, S. Malyukov 79, J. Mamuzic 174, G. Mancini 51, I. Mandi´c 91, L. Manhaes de Andrade Filho 80a, I.M. Maniatis 162, J. Manjarres Ramos 48, K.H. Mankinen 96, A. Mann 113, A. Manousos 76, B. Mansoulie 145, I. Manthos 162, S. Manzoni 119, A. Marantis 162, G. Marceca 30, L. Marchese 135, G. Marchiori 136, M. Marcisovsky 141, L. Marcoccia 73a,73b, C. Marcon 96, C.A. Marin Tobon 36, M. Marjanovic 128, Z. Marshall 18, M.U.F Martensson 172, S. Marti-Garcia 174, C.B. Martin 126, T.A. Martin 178, V.J. Martin 50, B. Martin dit Latour 17, L. Martinelli 74a,74b, M. Martinez 14,z V.I. Martinez Outschoorn 102, S. Martin-Haugh 144, V.S. Martoiu 27b, A.C. Martyniuk 94, A. Marzin 36, S.R. Maschek 114, L. Masetti 99, T. Mashimo 163, R. Mashinistov 110, J. Masik 100, A.L. Maslennikov 121b,121a, L. Massa 73a,73b, P. Massarotti 69a,69b, P. Mastrandrea 71a,71b, A. Mastroberardino 41b,41a, T. Masubuchi 163, D. Matakias 10, A. Matic 113, P. Mättig 24, J. Maurer 27b, B. Maˇcek 91, D.A. Maximov 121b,121a, R. Mazini 158, I. Maznas 162, S.M. Mazza 146, S.P. Mc Kee 105, T.G. McCarthy 114, W.P. McCormack 18, E.F. McDonald 104, J.A. Mcfayden 36, G. Mchedlidze 159b, M.A. McKay 42, K.D. McLean 176, S.J. McMahon 144, P.C. McNamara 104, C.J. McNicol 178, R.A. McPherson 176,ae, J.E. Mdhluli 33c, Z.A. Meadows 102, S. Meehan 36, T. Megy 52, S. Mehlhase 113, A. Mehta 90, T. Meideck 58, B. Meirose 43, D. Melini 174, B. Malaescu 136, Pa. Malecki 84, V.P. Maleev 138, F. Malek 58, U. Mallik 77, D. Malon 6, C. Malone 32, S. Maltezos 10, S. Malyukov 79, J. Mamuzic 174, G. Mancini 51, I. Mandi´c 91, L. Manhaes de Andrade Filho 80a, I.M. Maniatis 162, J. Manjarres Ramos 48, K.H. Mankinen 96, A. Mann 113, A. Manousos 76, B. Mansoulie 145, I. Manthos 162, S. Manzoni 119, A. Marantis 162, G. Marceca 30, y C.A. Marin Tobon 36, M. Marjanovic 128, Z. Marshall 18, M.U.F Martensson 172, S. Marti-Garcia 174, 126 178 50 17 74 74b 1 C.A. Marin Tobon , M. Marjanovic , Z. Marshall , M.U.F Martensson , S. Marti-Garcia , C.B. Martin 126, T.A. Martin 178, V.J. Martin 50, B. Martin dit Latour 17, L. Martinelli 74a,74b, M. Martinez 14,z, V.I. The ATLAS Collaboration Kazanin 121b,121a, R. Keeler 176, R. Kehoe 42, J.S. Keller 34, E. Kellermann 96, D. Kelsey 156, J.J. Kempster 21, J. Kendrick 21, O. Kepka 141, S. Kersten 182, B.P. Kerševan 91, S. Ketabchi Haghighat 167, M. Khader 173, F. Khalil-Zada 13, M. Khandoga 145, A. Khanov 129, A.G. Kharlamov 121b,121a, T. Kharlamova 121b,121a, E.E. Khoda 175, A. Khodinov 166, T.J. Khoo 54, E. Khramov 79, J. Khubua 159b, S. Kido 82, M. Kiehn 54, C.R. Kilby 93, Y.K. Kim 37, N. Kimura 94, O.M. Kind 19, B.T. King 90,∗, D. Kirchmeier 48, J. Kirk 144, A.E. Kiryunin 114, T. Kishimoto 163, D.P. Kisliuk 167, V. Kitali 46, O. Kivernyk 5, T. Klapdor-Kleingrothaus 52, M. Klassen 61a, M.H. Klein 105, M. Klein 90, U. Klein 90, K. Kleinknecht 99, P. Klimek 120, A. Klimentov 29, T. Klingl 24, T. Klioutchnikova 36, F.F. Klitzner 113, P. Kluit 119, S. Kluth 114, E. Kneringer 76, E.B.F.G. Knoops 101, A. Knue 52, D. Kobayashi 87, T. Kobayashi 163, M. Kobel 48, M. Kocian 153, P. Kodys 143, P.T. Koenig 24, T. Koffas 34, N.M. Köhler 36, T. Koi 153, M. Kolb 61b, I. Koletsou 5 T. Komarek 130, T. Kondo 81, N. Kondrashova 60c, K. Köneke 52, A.C. König 118, T. Kono 125, R. Konoplich 124,an, V. Konstantinides 94, N. Konstantinidis 94, B. Konya 96, R. Kopeliansky 65, S. Koperny 83a, K. Korcyl 84, K. Kordas 162, G. Koren 161, A. Korn 94, I. Korolkov 14, E.V. Korolkova 149, N. Korotkova 112, O. Kortner 114, S. Kortner 114, T. Kosek 143, V.V. Kostyukhin 166, A. Kotsokechagia 132, A Kotwal 49 A Koulouris 10 A Kourkoumeli-Charalampidi 70a,70b C Kourkoumelis 9 E Kourlitis 149 The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 H. Krüger 24, N. Krumnack 78, M.C. Kruse 49, J.A. Krzysiak 84, T. Kubota 104, O. Kuchinskaia 166, S. Kuday 4b J.T. Kuechler 46, S. Kuehn 36, A. Kugel 61a, T. Kuhl 46, V. Kukhtin 79, R. Kukla 101, Y. Kulchitsky 107,aj, S. Kuleshov 147c, Y.P. Kulinich 173, M. Kuna 58, T. Kunigo 85, A. Kupco 141, T. Kupfer 47, O. Kuprash 52, H. Kurashige 82, L.L. Kurchaninov 168a, Y.A. Kurochkin 107, A. Kurova 111, M.G. Kurth 15a,15d, E.S. Kuwertz 3 M. Kuze 165, A.K. Kvam 148, J. Kvita 130, T. Kwan 103, A. La Rosa 114, L. La Rotonda 41b,41a, F. La Ruffa 41b,41 C. Lacasta 174, F. The ATLAS Collaboration Lacava 72a,72b, D.P.J. Lack 100, H. Lacker 19, D. Lacour 136, E. Ladygin 79, R. Lafaye 5, B. Laforge 136, T. Lagouri 33c, S. Lai 53, S. Lammers 65, W. Lampl 7, C. Lampoudis 162, E. Lançon 29, U. Landgraf 52, M.P.J. Landon 92, M.C. Lanfermann 54, V.S. Lang 46, J.C. Lange 53, R.J. Langenberg 36, A.J. Lankford 171, F. Lanni 29, K. Lantzsch 24, A. Lanza 70a, A. Lapertosa 55b,55a, S. Laplace 136, J.F. Laporte 14 T. Lari 68a, F. Lasagni Manghi 23b,23a, M. Lassnig 36, T.S. Lau 63a, A. Laudrain 132, A. Laurier 34, M. Lavorgna 69a,69b, S.D. Lawlor 93, M. Lazzaroni 68a,68b, B. Le 104, E. Le Guirriec 101, M. LeBlanc 7, T. LeCompte 6, F. Ledroit-Guillon 58, A.C.A. Lee 94, C.A. Lee 29, G.R. Lee 17, L. Lee 59, S.C. Lee 158, S.J. Lee 34, S. Lee 78, B. Lefebvre 168a, H.P. Lefebvre 93, M. Lefebvre 176, F. Legger 113, C. Leggett 18, K. Lehmann 152, N. Lehmann 182, G. Lehmann Miotto 36, W.A. Leight 46, A. Leisos 162,y, M.A.L. Leite 80d, C.E. Leitgeb 113, R. Leitner 143, D. Lellouch 180,∗, K.J.C. Leney 42, T. Lenz 24, B. Lenzi 36, R. Leone 7, S. Leone 71a, C. Leonidopoulos 50, A. Leopold 136, G. Lerner 156, C. Leroy 109, R. Les 167, C.G. Lester 32, M. Levchenko 138 J. Levêque 5, D. Levin 105, L.J. Levinson 180, D.J. Lewis 21, B. Li 15b, B. Li 105, C-Q. Li 60a, F. Li 60c, H. Li 60a, H. Li 60b, J. Li 60c, K. Li 153, L. Li 60c, M. Li 15a, Q. Li 15a,15d, Q.Y. Li 60a, S. Li 60d,60c, X. Li 46, Y. Li 46, Z. Li 60b, Z. Liang 15a, B. Liberti 73a, A. Liblong 167, K. Lie 63c, C.Y. Lin 32, K. Lin 106, T.H. Lin 99, R.A. Linck 65, J.H. Lindon 21, A.L. Lionti 54, E. Lipeles 137, A. Lipniacka 17, M. Lisovyi 61b, T.M. Liss 173,au, A. Lister 175, A.M. Litke 146, J.D. Little 8, B. Liu 78, B.L Liu 6, H.B. Liu 29, H. Liu 105, J.B. Liu 60a, J.K.K. Liu 135, K. Liu 136, M. Liu 60a, P. Liu 18, Y. Liu 15a,15d, Y.L. Liu 105, Y.W. Liu 60a, M. Livan 70a,70b, A. Lleres 58, J. Llorente Merino 152, S.L. Lloyd 92, C.Y. Lo 63b, F. Lo Sterzo 42, E.M. Lobodzinska 46, P. Loch 7, S. Loffredo 73a,73b, T. The ATLAS Collaboration Lohse 19, K. Lohwasser 149, M. Lokajicek 141, J.D. Long 173, R.E. Long 89, L. Longo 36, K.A. Looper 126, J.A. Lopez 147c, I. Lopez Paz 100, A. Lopez Solis 149, J. Lorenz 113, N. Lorenzo Martinez 5, M. Losada 22, P.J. Lösel 113, A. Lösle 52, X. Lou 46, X. Lou 15a, A. Lounis 132, J. Love 6, P.A. Love 89, J.J. Lozano Bahilo 174, M. Lu 60a, Y.J. Lu 64, H.J. Lubatti 148, C. Luci 72a,72b, A. Lucotte 58, C. Luedtke 52, F. Luehring 65, I. Luise 136, L. Luminari 72a, B. Lund-Jensen 154, M.S. Lutz 102, D. Lynn 29, R. Lysak 141, E. Lytken 96, F. Lyu 15a, V. Lyubushkin 79, T. Lyubushkina 79, H. Ma 29, L.L. Ma 60b, Y. Ma 60b, G. Maccarrone 51, A. Macchiolo 114, C.M. Macdonald 149, J. Machado Miguens 137, D. Madaffari 174, R. Madar 38, W.F. Mader 48, N. Madysa 48, J. Maeda 82, S. Maeland 17, T. Maeno 29, M. Maerker 48, A.S. Maevskiy 112, V. Magerl 52, N. Magini 78, D.J. Mahon 39, C. Maidantchik 80b, T. Maier 113, A. Maio 140a,140b,140d, K. Maj 83a, O. Majersky 28a, S. Majewski 131, Y. Makida 81, N. Makovec 132, B. Malaescu 136, Pa. Malecki 84, V.P. Maleev 138, F. Malek 58, U. Mallik 77, D. Malon 6, C. Malone 32, S. Maltezos 10, S. Malyukov 79, J. Mamuzic 174, G. Mancini 51, I. Mandi´c 91, L. Manhaes de Andrade Filho 80a, I.M. Maniatis 162, J. Manjarres Ramos 48, K.H. Mankinen 96, A. Mann 113 A. Manousos 76, B. Mansoulie 145, I. Manthos 162, S. Manzoni 119, A. Marantis 162, G. Marceca 30, L. Marchese 135, G. Marchiori 136, M. Marcisovsky 141, L. Marcoccia 73a,73b, C. Marcon 96, C.A. Marin Tobon 36, M. Marjanovic 128, Z. Marshall 18, M.U.F Martensson 172, S. Marti-Garcia 174, C.B. Martin 126, T.A. Martin 178, V.J. Martin 50, B. Martin dit Latour 17, L. Martinelli 74a,74b, M. Martinez 14 V.I. Martinez Outschoorn 102, S. Martin-Haugh 144, V.S. Martoiu 27b, A.C. Martyniuk 94, A. Marzin 36, S.R. Maschek 114, L. Masetti 99, T. Mashimo 163, R. Mashinistov 110, J. Masik 100, A.L. Maslennikov 121b,121a L. Massa 73a,73b, P. Massarotti 69a,69b, P. Mastrandrea 71a,71b, A. Mastroberardino 41b,41a, T. Masubuchi 163 D. Matakias 10, A. Matic 113, P. Mättig 24, J. Maurer 27b, B. Maˇcek 91, D.A. Maximov 121b,121a, R. Mazini 158 I. Maznas 162, S.M. Mazza 146, S.P. The ATLAS Collaboration O’neill 135, A. Onofre 140a,140e, P.U.E. Onyisi 11, H. Oppen 134, M.J. Oreglia 37, G.E. Orellana 88, D. Orestano 74a,74b, N. Orlando 14, R.S. Orr 167, V. O’Shea 57, R. Ospanov 60a, G. Otero y Garzon 30, H. Otono 87, P.S. Ott 61a, M. Ouchrif 35d, J. Ouellette 29, F. Ould-Saada 134, A. Ouraou 145, Q. Ouyang 15a, M. Owen 57, R.E. Owen 21, V.E. Ozcan 12c, N. Ozturk 8, J. Pacalt 130, H.A. Pacey 32, K. Pachal 49, A. Pacheco Pages 14, C. Padilla Aranda 14, S. Pagan Griso 18, M. Paganini 183, G. Palacino 65, S. Palazzo 50, S. Palestini 36, M. Palka 83b, D. Pallin 38, I. Panagoulias 10, C.E. Pandini 36, J.G. Panduro Vazquez 93, P. Pani 46, G. Panizzo 66a,66c, L. Paolozzi 54, C. Papadatos 109, K. Papageorgiou 9,h, S. Parajuli 43, A. Paramonov 6, D. Paredes Hernandez 63b, S.R. Paredes Saenz 135, B. Parida 166, T.H. Park 167, A.J. Parker 31, M.A. Parker 32, F. Parodi 55b,55a, E.W. Parrish 120, J.A. Parsons 39, U. Parzefall 52, L. Pascual Dominguez 136, V.R. Pascuzzi 167, J.M.P. Pasner 146, F. Pasquali 119, E. Pasqualucci 72a, S. Passaggio 55b, F. Pastore 93, P. Pasuwan 45a,45b, S. Pataraia 99, J.R. Pater 100, A. Pathak 181,j, T. Pauly 36, B. Pearson 114, M. Pedersen 134, L. Pedraza Diaz 118, R. Pedro 140a, T. Peiffer 53, S.V. Peleganchuk 121b,121a, O. Penc 141, H. Peng 60a, B.S. Peralva 80a, M.M. Perego 132, A.P. Pereira Peixoto 140a, D.V. Perepelitsa 29, F. Peri 19, L. Perini 68a,68b, H. Pernegger 36, S. Perrella 69a,69b, K. Peters 46, R.F.Y. Peters 100, B.A. Petersen 36, T.C. Petersen 40, E P i 101 A P idi 1 C P id 162 P P ff 132 M P 135 F P i 74a 74b M P 183 H. Meyer Zu Theenhausen 61a, F. Miano 156, M. Michetti 19, R.P. Middleton 144, L. Mijovi´c 50, G. Mikenberg 180, M. Mikestikova 141, M. Mikuž 91, H. Mildner 149, M. Milesi 104, A. Milic 167, D.A. Millar 92, D.W. Miller 37, A. Milov 180, D.A. Milstead 45a,45b, R.A. Mina 153,q, A.A. Minaenko 122, M. Miñano Moya 174, I.A. Minashvili 159b, A.I. Mincer 124, B. Mindur 83a, M. Mineev 79, Y. Minegishi 163, L.M. Mir 14, A. Mirto 67a,67b, K.P. Mistry 137, T. Mitani 179, J. Mitrevski 113, V.A. Mitsou 174, M. Mittal 60c, O. Miu 167, A. Miucci 20, P.S. The ATLAS Collaboration Miyagawa 149, A. Mizukami 81, J.U. Mjörnmark 96, T. Mkrtchyan 184, M. Mlynarikova 143, T. Moa 45a,45b, K. Mochizuki 109, P. Mogg 52, S. Mohapatra 39, R. Moles-Valls 24, M.C. Mondragon 106, K. Mönig 46, J. Monk 40, E. Monnier 101, A. Montalbano 152, J. Montejo Berlingen 36, M. Montella 94, F. Monticelli 88, S. Monzani 68a, N. Morange 132, D. Moreno 22, M. Moreno Llácer 36, C. Moreno Martinez 14, P. Morettini 55b, M. Morgenstern 119, S. Morgenstern 48, D. Mori 152, M. Morii 59, M. Morinaga 179, V. Morisbak 134, A.K. Morley 36, G. Mornacchi 36, A.P. Morris 94, L. Morvaj 155, P. Moschovakos 36, B. Moser 119, M. Mosidze 159b, T. Moskalets 145, H.J. Moss 149, J. Moss 31,n, E.J.W. Moyse 102, S. Muanza 101, J. Mueller 139, R.S.P. Mueller 113, D. Muenstermann 89, G.A. Mullier 96, J.L. Munoz Martinez 14, F.J. Munoz Sanchez 100, P. Murin 28b, W.J. Murray 178,144, A. Murrone 68a,68b, M. Muškinja 18, C. Mwewa 33a, A.G. Myagkov 122,ao, J. Myers 131, M. Myska 142, B.P. Nachman 18, O. Nackenhorst 47, A. Nag Nag 48, K. Nagai 135, K. Nagano 81, Y. Nagasaka 62, M. Nagel 52, J.L. Nagle 29, E. Nagy 101, A.M. Nairz 36, Y. Nakahama 116, K. Nakamura 81, T. Nakamura 163, I. Nakano 127, H. Nanjo 133, F. Napolitano 61a, R.F. Naranjo Garcia 46, R. Narayan 42, I. Naryshkin 138, T. Naumann 46, G. Navarro 22, P.Y. Nechaeva 110, F. Nechansky 46, T.J. Neep 21, A. Negri 70a,70b, M. Negrini 23b, C. Nellist 53, M.E. Nelson 135, S. Nemecek 141, P. Nemethy 124, M. Nessi 36,d, M.S. Neubauer 173, M. Neumann 182, P.R. Newman 21, Y.S. Ng 19, Y.W.Y. Ng 171, B. Ngair 35e, H.D.N. Nguyen 101, T. Nguyen Manh 109, E. Nibigira 38, R.B. Nickerson 135, R. Nicolaidou 145, D.S. Nielsen 40, J. Nielsen 146, N. Nikiforou 11, V. Nikolaenko 122,ao, I. Nikolic-Audit 136, K. Nikolopoulos 21, P. Nilsson 29, H.R. Nindhito 54, Y. Ninomiya 81, A. Nisati 72a, N. Nishu 60c, R. Nisius 114, I. Nitsche 47, T. Nitta 179, T. Nobe 163, Y. Noguchi 85, I. Nomidis 136, M.A. Nomura 29, M. Nordberg 36, N. Norjoharuddeen 135, T. Novak 91, O. Novgorodova 48, R. Novotny 142, L. Nozka 130, K. Ntekas 171, E. Nurse 94, F.G. Oakham 34,ax, H. Oberlack 114, J. Ocariz 136, A. Ochi 82, I. Ochoa 39, J.P. Ochoa-Ricoux 147a, K. The ATLAS Collaboration Martinez Outschoorn 102, S. Martin-Haugh 144, V.S. Martoiu 27b, A.C. Martyniuk 94, A. Marzin 36, S.R. Maschek 114, L. Masetti 99, T. Mashimo 163, R. Mashinistov 110, J. Masik 100, A.L. Maslennikov 121b,121a, L. Massa 73a,73b, P. Massarotti 69a,69b, P. Mastrandrea 71a,71b, A. Mastroberardino 41b,41a, T. Masubuchi 163, D. Matakias 10, A. Matic 113, P. Mättig 24, J. Maurer 27b, B. Maˇcek 91, D.A. Maximov 121b,121a, R. Mazini 158, I. Maznas 162, S.M. Mazza 146, S.P. Mc Kee 105, T.G. McCarthy 114, W.P. McCormack 18, E.F. McDonald 104, J.A. Mcfayden 36, G. Mchedlidze 159b, M.A. McKay 42, K.D. McLean 176, S.J. McMahon 144, P.C. McNamara 104, C.J. McNicol 178, R.A. McPherson 176,ae, J.E. Mdhluli 33c, Z.A. Meadows 102, S. Meehan 36, T. Megy 52, S. Mehlhase 113, A. Mehta 90, T. Meideck 58, B. Meirose 43, D. Melini 174, B.R. Mellado Garcia 33c, J.D. Mellenthin 53, M. Melo 28a, F. Meloni 46, A. Melzer 24, S.B. Menary 100, E.D. Mendes Gouveia 140a,140e, L. Meng 36, X.T. Meng 105, S. Menke 114, E. Meoni 41b,41a, S. Mergelmeyer 19, S.A.M. Merkt 139, C. Merlassino 20, P. Mermod 54, L. Merola 69a,69b, C. Meroni 68a, O. Meshkov 112,110, J.K.R. Meshreki 151, A. Messina 72a,72b, J. Metcalfe 6, A.S. Mete 171, C. Meyer 65, J. Meyer 160, J-P. Meyer 145, The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 16 H. Meyer Zu Theenhausen 61a, F. Miano 156, M. Michetti 19, R.P. Middleton 144, L. Mijovi´c 50, G. Mikenberg 180, M. Mikestikova 141, M. Mikuž 91, H. Mildner 149, M. Milesi 104, A. Milic 167, D.A. Millar 92, D.W. Miller 37, A. Milov 180, D.A. Milstead 45a,45b, R.A. Mina 153,q, A.A. Minaenko 122, M. Miñano Moya 174, I.A. Minashvili 159b, A.I. Mincer 124, B. Mindur 83a, M. Mineev 79, Y. Minegishi 163, L.M. Mir 14, A. Mirto 67a,67b, K.P. Mistry 137, T. Mitani 179, J. Mitrevski 113, V.A. Mitsou 174, M. Mittal 60c, O. Miu 167, A. Miucci 20, P.S. Miyagawa 149, A. Mizukami 81, J.U. Mjörnmark 96, T. Mkrtchyan 184, M. Mlynarikova 143, T. Moa 45a,45b, K. Mochizuki 109, P. Mogg 52, S. Mohapatra 39, R. Moles-Valls 24, M.C. Mondragon 106, K. Mönig 46, J. Monk 40, E. Monnier 101, A. Montalbano 152, J. Montejo Berlingen 36, M. Montella 94, F. Monticelli 88, S. Monzani 68a, N. Morange 132, D. Moreno 22, M. Moreno Llácer 36, C. Moreno Martinez 14, P. The ATLAS Collaboration Morettini 55b, M. Morgenstern 119, S. Morgenstern 48, D. Mori 152, M. Morii 59, M. Morinaga 179, V. Morisbak 134, A.K. Morley 36, G. Mornacchi 36, A.P. Morris 94, L. Morvaj 155, P. Moschovakos 36, B. Moser 119, M. Mosidze 159b, T. Moskalets 145, H.J. Moss 149, J. Moss 31,n, E.J.W. Moyse 102, S. Muanza 101, J. Mueller 139, R.S.P. Mueller 113, D. Muenstermann 89, G.A. Mullier 96, J.L. Munoz Martinez 14, F.J. Munoz Sanchez 100, P. Murin 28b, W.J. Murray 178,144, A. Murrone 68a,68b, M. Muškinja 18, C. Mwewa 33a, A.G. Myagkov 122,ao, J. Myers 131, M. Myska 142, B.P. Nachman 18, O. Nackenhorst 47, A. Nag Nag 48, K. Nagai 135, K. Nagano 81, Y. Nagasaka 62, M. Nagel 52, J.L. Nagle 29, E. Nagy 101, A.M. Nairz 36, Y. Nakahama 116, K. Nakamura 81, T. Nakamura 163, I. Nakano 127, H. Nanjo 133, F. Napolitano 61a, R.F. Naranjo Garcia 46, R. Narayan 42, I. Naryshkin 138, T. Naumann 46, G. Navarro 22, P.Y. Nechaeva 110, F. Nechansky 46, T.J. Neep 21, A. Negri 70a,70b, M. Negrini 23b, C. Nellist 53, M.E. Nelson 135, S. Nemecek 141, P. Nemethy 124, M. Nessi 36,d, M.S. Neubauer 173, M. Neumann 182, P.R. Newman 21, Y.S. Ng 19, Y.W.Y. Ng 171, B. Ngair 35e, H.D.N. Nguyen 101, T. Nguyen Manh 109, E. Nibigira 38, R.B. Nickerson 135, R. Nicolaidou 145, D.S. Nielsen 40, J. Nielsen 146, N. Nikiforou 11, V. Nikolaenko 122,ao, I. Nikolic-Audit 136, K. Nikolopoulos 21, P. Nilsson 29, H.R. Nindhito 54, Y. Ninomiya 81, A. Nisati 72a, N. Nishu 60c, R. Nisius 114, I. Nitsche 47, T. Nitta 179, T. Nobe 163, Y. Noguchi 85, I. Nomidis 136, M.A. Nomura 29, M. Nordberg 36, N. Norjoharuddeen 135, T. Novak 91, O. Novgorodova 48, R. Novotny 142, L. Nozka 130, K. Ntekas 171, E. Nurse 94, F.G. Oakham 34,ax, H. Oberlack 114, J. Ocariz 136, A. Ochi 82, I. Ochoa 39, J.P. Ochoa-Ricoux 147a, K. O’Connor 26, S. Oda 87, S. Odaka 81, S. Oerdek 53, A. Ogrodnik 83a, A. Oh 100, S.H. Oh 49, C.C. Ohm 154, H. Oide 165, M.L. Ojeda 167, H. Okawa 169, Y. Okazaki 85, Y. Okumura 163, T. Okuyama 81, A. Olariu 27b, L.F. Oleiro Seabra 140a, S.A. Olivares Pino 147a, D. Oliveira Damazio 29, J.L. Oliver 1, M.J.R. Olsson 171, A. Olszewski 84, J. Olszowska 84, D.C. O’Neil 152, A.P. The ATLAS Collaboration O’Connor 26, S. Oda 87, S. Odaka 81, S. Oerdek 53, A. Ogrodnik 83a, A. Oh 100, S.H. Oh 49, C.C. Ohm 154, H. Oide 165, M.L. Ojeda 167, H. Okawa 169, Y. Okazaki 85, Y. Okumura 163, T. Okuyama 81, A. Olariu 27b, L.F. Oleiro Seabra 140a, S.A. Olivares Pino 147a, D. Oliveira Damazio 29, J.L. Oliver 1, M.J.R. Olsson 171, A. Olszewski 84, J. Olszowska 84, D.C. O’Neil 152, A.P. O’neill 135, A. Onofre 140a,140e, P.U.E. Onyisi 11, H. Oppen 134, M.J. Oreglia 37, G.E. Orellana 88, D. Orestano 74a,74b, N. Orlando 14, R.S. Orr 167, V. O’Shea 57, R. Ospanov 60a, G. Otero y Garzon 30, H. Otono 87, P.S. Ott 61a, M. Ouchrif 35d, J. Ouellette 29, F. Ould-Saada 134, A. Ouraou 145, Q. Ouyang 15a, M. Owen 57, R.E. Owen 21, V.E. Ozcan 12c, N. Ozturk 8, J. Pacalt 130, H.A. Pacey 32, K. Pachal 49, A. Pacheco Pages 14, C. Padilla Aranda 14, S. Pagan Griso 18, M. Paganini 183, G. Palacino 65, S. Palazzo 50, S. Palestini 36, M. Palka 83b, D. Pallin 38, I. Panagoulias 10, C.E. Pandini 36, J.G. Panduro Vazquez 93, P. Pani 46, G. Panizzo 66a,66c, L. Paolozzi 54, C. Papadatos 109, K. Papageorgiou 9,h, S. Parajuli 43, A. Paramonov 6, D. Paredes Hernandez 63b, S.R. Paredes Saenz 135, B. Parida 166, T.H. Park 167, A.J. Parker 31, M.A. Parker 32, F. Parodi 55b,55a, E.W. Parrish 120, J.A. Parsons 39, U. Parzefall 52, L. Pascual Dominguez 136, V.R. Pascuzzi 167, J.M.P. Pasner 146, F. Pasquali 119, E. Pasqualucci 72a, S. Passaggio 55b, F. Pastore 93, P. Pasuwan 45a,45b, S. Pataraia 99, J.R. Pater 100, A. Pathak 181,j, T. Pauly 36, B. Pearson 114, M. Pedersen 134, L. Pedraza Diaz 118, R. Pedro 140a, T. Peiffer 53, S.V. Peleganchuk 121b,121a, O. Penc 141, H. Peng 60a, B.S. Peralva 80a, M.M. Perego 132, A.P. Pereira Peixoto 140a, D.V. Perepelitsa 29, F. Peri 19, L. Perini 68a,68b, H. Pernegger 36, S. Perrella 69a,69b, K. Peters 46, R.F.Y. Peters 100, B.A. Petersen 36, T.C. Petersen 40, E. Petit 101, A. Petridis 1, C. Petridou 162, P. Petroff 132, M. Petrov 135, F. Petrucci 74a,74b, M. Pettee 183, N.E. Pettersson 102, K. Petukhova 143, A. Peyaud 145, R. Pezoa 147c, L. Pezzotti 70a,70b, T. Pham 104, F.H. Phillips 106, P.W. Phillips 144, M.W. Phipps 173, G. Piacquadio 155, E. Pianori 18, A. The ATLAS Collaboration Picazio 102, The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 17 The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 D.M. Portillo Quintero 58, S. Pospisil 142, K. Potamianos 46, I.N. Potrap 79, C.J. Potter 32, H. Potti 11, T. Poulsen 96, J. Poveda 36, T.D. Powell 149, G. Pownall 46, M.E. Pozo Astigarraga 36, P. Pralavorio 101, S. Prell 78, D. Price 100, M. Primavera 67a, S. Prince 103, M.L. Proﬃtt 148, N. Proklova 111, K. Prokoﬁev 63c, F. Prokoshin 79, S. Protopopescu 29, J. Proudfoot 6, M. Przybycien 83a, D. Pudzha 138, A. Puri 173, P. Puzo 132 J. Qian 105, Y. Qin 100, A. Quadt 53, M. Queitsch-Maitland 46, A. Qureshi 1, M. Racko 28a, P. Rados 104, F. Ragusa 68a,68b, G. Rahal 97, J.A. Raine 54, S. Rajagopalan 29, A. Ramirez Morales 92, K. Ran 15a,15d, T. Rashid 132, S. Raspopov 5, D.M. Rauch 46, F. Rauscher 113, S. Rave 99, B. Ravina 149, I. Ravinovich 180, J.H. Rawling 100, M. Raymond 36, A.L. Read 134, N.P. Readioff 58, M. Reale 67a,67b, D.M. Rebuzzi 70a,70b, A. Redelbach 177, G. Redlinger 29, K. Reeves 43, L. Rehnisch 19, J. Reichert 137, D. Reikher 161, A. Reiss 99, A. Rej 151, C. Rembser 36, M. Renda 27b, M. Rescigno 72a, S. Resconi 68a, E.D. Resseguie 137, S. Rettie 175, E. Reynolds 21, O.L. Rezanova 121b,121a, P. Reznicek 143, E. Ricci 75a,75b, R. Richter 114, S. Richter 46, E. Richter-Was 83b, O. Ricken 24, M. Ridel 136, P. Rieck 114, C.J. Riegel 182, O. Rifki 46, M. Rijssenbeek 155, A. Rimoldi 70a,70b, M. Rimoldi 46, L. Rinaldi 23b, G. Ripellino 154, I. Riu 14, J.C. Rivera Vergara 176, F. Rizatdinova 129, E. Rizvi 92, C. Rizzi 36, R.T. Roberts 100, S.H. Robertson 103,ae, M. Robin 46, D. Robinson 3 J.E.M. Robinson 46, C.M. Robles Gajardo 147c, A. Robson 57, A. Rocchi 73a,73b, E. Rocco 99, C. Roda 71a,71b, S. Rodriguez Bosca 174, A. Rodriguez Perez 14, D. Rodriguez Rodriguez 174, A.M. Rodríguez Vera 168b, S. Roe 36, O. Røhne 134, R. Röhrig 114, R.A. Rojas 147c, C.P.A. Roland 65, J. Roloff 59, A. Romaniouk 111, M. Romano 23b,23a, N. Rompotis 90, M. Ronzani 124, L. Roos 136, S. Rosati 72a, K. Rosbach 52, G. Rosin 102, B.J. Rosser 137, E. Rossi 46, E. Rossi 74a,74b, E. Rossi 69a,69b, L.P. Rossi 55b, L. Rossini 68a,68b, R. The ATLAS Collaboration Rosten 14, M. Rotaru 27b, J. Rothberg 148, D. Rousseau 132, G. Rovelli 70a,70b, A. Roy 11, D. Roy 33c, A. Rozanov 101, Y. Rozen 160, X. Ruan 33c, F. Rubbo 153, F. Rühr 52, A. Ruiz-Martinez 174, A. Rummler 36, Z. Rurikova 52, N.A. Rusakovich 79, H.L. Russell 103, L. Rustige 38,47, J.P. Rutherfoord 7, E.M. Rüttinger 149, M. Rybar 39, G. Rybkin 132, E.B. Rye 134, A. Ryzhov 122, P. Sabatini 53, G. Sabato 119, S. Sacerdoti 132, H.F-W. Sadrozinski 146, R. Sadykov 79, F. Safai Tehrani 72a, B. Safarzadeh Samani 156, P. Saha 120, S. Saha 10 M. Sahinsoy 61a, A. Sahu 182, M. Saimpert 46, M. Saito 163, T. Saito 163, H. Sakamoto 163, A. Sakharov 124,an D. Salamani 54, G. Salamanna 74a,74b, J.E. Salazar Loyola 147c, P.H. Sales De Bruin 172, A. Salnikov 153, J. Salt 174, D. Salvatore 41b,41a, F. Salvatore 156, A. Salvucci 63a,63b,63c, A. Salzburger 36, J. Samarati 36, D. Sammel 52, D. Sampsonidis 162, D. Sampsonidou 162, J. Sánchez 174, A. Sanchez Pineda 66a,66c, H. Sandaker 134, C.O. Sander 46, I.G. Sanderswood 89, M. Sandhoff 182, C. Sandoval 22, D.P.C. Sankey 144, M. Sannino 55b,55a, Y. Sano 116, A. Sansoni 51, C. Santoni 38, H. Santos 140a,140b, S.N. Santpur 18, A. Santra 174, A. Sapronov 79, J.G. Saraiva 140a,140d, O. Sasaki 81, K. Sato 169, F. Sauerburger 52, E. Sauvan 5, P. Savard 167,ax, N. Savic 114, R. Sawada 163, C. Sawyer 144, L. Sawyer 95,al, C. Sbarra 23b, A. Sbrizzi 23a, T. Scanlon 94, J. Schaarschmidt 148, P. Schacht 114, B.M. Schachtner 113, D. Schaefer 37, L. Schaefer 137, J. Schaeffer 99, S. Schaepe 36, U. Schäfer 99, A.C. Schaffer 132, D. Schaile 113, R.D. Schamberger 155, N. Scharmberg 100, V.A. Schegelsky 138, D. Scheirich 143, F. Schenck 19, M. Schernau 171, C. Schiavi 55b,55a, S. Schier 146, L.K. Schildgen 24, Z.M. Schillaci 26, E.J. Schioppa 36, M. Schioppa 41b,41a, K.E. Schleicher 52, S. Schlenker 36, K.R. Schmidt-Sommerfeld 114, K. Schmieden 36, C. Schmitt 99, S. Schmitt 46, S. Schmitz 99, J.C. Schmoeckel 46, U. Schnoor 52, L. Schoeffel 145, A. Schoening 61b, P.G. Scholer 52, E. Schopf 135, M. Schott 99, J.F.P. Schouwenberg 118, J. Schovancova 36, S. Schramm 54, F. Schroeder 182, A. Schulte 99, H-C. Schultz-Coulon 61a, M. Schumacher 52, B.A. Schumm 146, Ph. Schune 145, A. Schwartzman 153, T.A. Schwarz 105, Ph. Schwemling 145, R. The ATLAS Collaboration Vermeulen 119, 152 147 54 149 149 The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 18 18 The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 S. Simion 132, E. Simioni 99, R. Simoniello 99, S. Simsek 12b, P. Sinervo 167, V. Sinetckii 112,110, N.B. Sinev 131, M. Sioli 23b,23a, I. Siral 105, S.Yu. Sivoklokov 112, J. Sjölin 45a,45b, E. Skorda 96, P. Skubic 128, M. Slawinska 84, K. Sliwa 170, R. Slovak 143, V. Smakhtin 180, B.H. Smart 144, J. Smiesko 28a, N. Smirnov 111, S.Yu. Smirnov 111, Y. Smirnov 111, L.N. Smirnova 112,v, O. Smirnova 96, J.W. Smith 53, M. Smizanska 89, K. Smolek 142, A. Smykiewicz 84, A.A. Snesarev 110, H.L. Snoek 119, I.M. Snyder 131, S. Snyder 29, R. Sobie 176,ae, A. Soffer 161, A. Søgaard 50, F. Sohns 53, C.A. Solans Sanchez 36, E.Yu. Soldatov 111, U. Soldevila 174, A.A. Solodkov 122, A. Soloshenko 79, O.V. Solovyanov 122, V. Solovyev 138, P. Sommer 149, H. Son 170, W. Song 144, W.Y. Song 168b, A. Sopczak 142, F. Sopkova 28b, C.L. Sotiropoulou 71a,71b, S. Sottocornola 70a,70b, R. Soualah 66a,66c,g, A.M. Soukharev 121b,121a, D. South 46, S. Spagnolo 67a,67b, M. Spalla 114, M. Spangenberg 178, F. Spanò 93, D. Sperlich 52, T.M. Spieker 61a, R. Spighi 23b, G. Spigo 36, M. Spina 156, D.P. Spiteri 57, M. Spousta 143, A. Stabile 68a,68b, B.L. Stamas 120, R. Stamen 61a, M. Stamenkovic 119, E. Stanecka 84, B. Stanislaus 135, M.M. Stanitzki 46, M. Stankaityte 135, B. Stapf 119, E.A. Starchenko 122, G.H. Stark 146, J. Stark 58, S.H. Stark 40, P. Staroba 141, P. Starovoitov 61a, S. Stärz 103, R. Staszewski 84, G. Stavropoulos 44, M. Stegler 46, P. Steinberg 29, A.L. Steinhebel 131, B. Stelzer 152, H.J. Stelzer 139, O. Stelzer-Chilton 168a, H. Stenzel 56, T.J. Stevenson 156, G.A. Stewart 36, M.C. Stockton 36, G. Stoicea 27b, M. Stolarski 140a, S. Stonjek 114, A. Straessner 48, J. Strandberg 154, S. Strandberg 45a,45b, M. Strauss 128, P. Strizenec 28b, R. Ströhmer 177, D.M. Strom 131, R. Stroynowski 42, A. Strubig 50, S.A. Stucci 29, B. Stugu 17, J. Stupak 128, N.A. Styles 46, D. Su 153, S. Suchek 61a, V.V. Sulin 110, M.J. Sullivan 90, D.M.S. Sultan 54, S. Sultansoy 4c, T. Sumida 85, S. Sun 105, X. Sun 3, K. Suruliz 156, C.J.E. Suster 157, M.R. Sutton 156, S. Suzuki 81, M. Svatos 141, M. The ATLAS Collaboration Swiatlowski 37, S.P. Swift 2, T. Swirski 177, A. Sydorenko 99, I. Sykora 28a, M. Sykora 143, T. Sykora 143, D. Ta 99, K. Tackmann 46,aa, J. Taenzer 161, A. Taffard 171, R. Taﬁrout 168a, H. Takai 29, R. Takashima 86, K. Takeda 82, T. Takeshita 150, E.P. Takeva 50, Y. Takubo 81, M. Talby 101, A.A. Talyshev 121b,121a, N.M. Tamir 161, J. Tanaka 163, M. Tanaka 165, R. Tanaka 132, S. Tapia Araya 173, S. Tapprogge 99, A. Tarek Abouelfadl Mohamed 136, S. Tarem 160, K. Tariq 60b, G. Tarna 27b,c, G.F. Tartarelli 68a, P. Tas 143, M. Tasevsky 141, T. Tashiro 85, E. Tassi 41b,41a, A. Tavares Delgado 140a,140b, Y. Tayalati 35e, A.J. Taylor 50, G.N. Taylor 104, W. Taylor 168b, A.S. Tee 89, R. Teixeira De Lima 153, P. Teixeira-Dias 93, H. Ten Kate 36, J.J. Teoh 119, S. Terada 81, K. Terashi 163, J. Terron 98, S. Terzo 14, M. Testa 51, R.J. Teuscher 167,ae, S.J. Thais 183, T. Theveneaux-Pelzer 46, F. Thiele 40, D.W. Thomas 93, J.O. Thomas 42, J.P. Thomas 21, A.S. Thompson 57, P.D. Thompson 21, L.A. Thomsen 183, E. Thomson 137, E.J. Thorpe 92, R.E. Ticse Torres 53, V.O. Tikhomirov 110,ap, Yu.A. Tikhonov 121b,121a, S. Timoshenko 111, P. Tipton 183, S. Tisserant 101, K. Todome 23b,23a, S. Todorova-Nova 5, S. Todt 48, J. Tojo 87, S. Tokár 28a, K. Tokushuku 81, E. Tolley 126, K.G. Tomiwa 33c, M. Tomoto 116, L. Tompkins 153,q, B. Tong 59, P. Tornambe 102, E. Torrence 131, H. Torres 48, E. Torró Pastor 148, C. Tosciri 135, J. Toth 101,ad, D.R. Tovey 149, A. Traeet 17, C.J. Treado 124, T. Trefzger 177, F. Tresoldi 156, A. Tricoli 29, I.M. Trigger 168a, S. Trincaz-Duvoid 136, W. Trischuk 167, B. Trocmé 58, A. Trofymov 145, C. Troncon 68a, M. Trovatelli 176, F. Trovato 156, L. Truong 33b, M. Trzebinski 84, A. Trzupek 84, F. Tsai 46, J.C-L. Tseng 135, P.V. Tsiareshka 107,aj A. Tsirigotis 162, V. Tsiskaridze 155, E.G. Tskhadadze 159a, M. Tsopoulou 162, I.I. Tsukerman 123, V. Tsulaia 18 S. Tsuno 81, D. Tsybychev 155, Y. Tu 63b, A. Tudorache 27b, V. Tudorache 27b, T.T. Tulbure 27a, A.N. Tuna 59, S. Turchikhin 79, D. Turgeman 180, I. Turk Cakir 4b,w, R.J. Turner 21, R.T. Turra 68a, P.M. Tuts 39, S. Tzamarias 162, E. Tzovara 99, G. Ucchielli 47, K. Uchida 163, I. Ueda 81, M. Ughetto 45a,45b, F. The ATLAS Collaboration Schwienhorst 106, A. Sciandra 146, G. Sciolla 26, M. Scodeggio 46, M. Scornajenghi 41b,41a, F. Scuri 71a, F. Scutti 104, L.M. Scyboz 114, C.D. Sebastiani 72a,72b, P. Seema 19, S.C. Seidel 117, A. Seiden 146, B.D. Seidlitz 29, T. Seiss 37, J.M. Seixas 80b, G. Sekhniaidze 69a, K. Sekhon 105, S.J. Sekula 42, N. Semprini-Cesari 23b,23a, S. Sen 49, S. Senkin 38, C. Serfon 76, L. Serin 132, L. Serkin 66a,66b, M. Sessa 60a, H. Severini 128, T. Šﬁligoj 91, F. Sforza 55b,55a, A. Sfyrla 54, E. Shabalina 53, J.D. Shahinian 146, N.W. Shaikh 45a,45b, D. Shaked Renous 180, L.Y. Shan 15a, R. Shang 173, J.T. Shank 25, M. Shapiro 18, A. Sharma 135, A.S. Sharma 1, P.B. Shatalov 123, K. Shaw 156, S.M. Shaw 100, A. Shcherbakova 138, M. Shehade 180, Y. Shen 128, N. Sherafati 34, A.D. Sherman 25, P. Sherwood 94, L. Shi 158,at, S. Shimizu 81, C.O. Shimmin 183, Y. Shimogama 179, M. Shimojima 115, I.P.J. Shipsey 135, S. Shirabe 87, M. Shiyakova 79,ac, J. Shlomi 180, A. Shmeleva 110, M.J. Shochet 37, J. Shojaii 104, D.R. Shope 128, S. Shrestha 126, E.M. Shrif 33c, E. Shulga 180, P. Sicho 141, A.M. Sickles 173, P.E. Sidebo 154, E. Sideras Haddad 33c, O. Sidiropoulou 36, A. Sidoti 23b,23a, F. Siegert 48, Dj. Sijacki 16, M.Jr. Silva 181, M.V. Silva Oliveira 80a, S.B. Silverstein 45a, 18 The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 S. Simion 132, E. Simioni 99, R. Simoniello 99, S. Simsek 12b, P. Sinervo 167, V. Sinetckii 112,110, N.B. Sinev 131, M. Sioli 23b,23a, I. Siral 105, S.Yu. Sivoklokov 112, J. Sjölin 45a,45b, E. Skorda 96, P. Skubic 128, M. Slawinska 84, K. Sliwa 170, R. Slovak 143, V. Smakhtin 180, B.H. Smart 144, J. Smiesko 28a, N. Smirnov 111 S.Yu. Smirnov 111, Y. Smirnov 111, L.N. Smirnova 112,v, O. Smirnova 96, J.W. Smith 53, M. Smizanska 89, K. Smolek 142, A. Smykiewicz 84, A.A. Snesarev 110, H.L. Snoek 119, I.M. Snyder 131, S. Snyder 29, R. Sobie 176,ae, A. Soffer 161, A. Søgaard 50, F. Sohns 53, C.A. Solans Sanchez 36, E.Yu. Soldatov 111, U. Soldevila 174, A.A. Solodkov 122, A. Soloshenko 79, O.V. Solovyanov 122, V. Solovyev 138, P. Sommer 149, H. Son 170, W. Song 144, W.Y. Song 168b, A. Sopczak 142, F. Sopkova 28b, C.L. Sotiropoulou 71a,71b, S. Sottocornola 70a,70b, R. Soualah 66a,66c,g, A.M. Soukharev 121b,121a, D. The ATLAS Collaboration South 46, S. Spagnolo 67a,67b, M. Spalla 114, M. Spangenberg 178, F. Spanò 93, D. Sperlich 52, T.M. Spieker 61a, R. Spighi 23b, G. Spigo 36, M. Spina 156, D.P. Spiteri 57, M. Spousta 143, A. Stabile 68a,68b, B.L. Stamas 120, R. Stamen 61a, M. Stamenkovic 119, E. Stanecka 84, B. Stanislaus 135, M.M. Stanitzki 46, M. Stankaityte 135, B. Stapf 119, E.A. Starchenko 122, G.H. Stark 146, J. Stark 58, S.H. Stark 40, P. Staroba 141, P. Starovoitov 61a, S. Stärz 103, R. Staszewski 84, G. Stavropoulos 44, M. Stegler 46, P. Steinberg 29, A.L. Steinhebel 131, B. Stelzer 152, H.J. Stelzer 139, O. Stelzer-Chilton 168a, H. Stenzel 56, T.J. Stevenson 156, G.A. Stewart 36, M.C. Stockton 36, G. Stoicea 27b, M. Stolarski 140a, S. Stonjek 114, A. Straessner 48, J. Strandberg 154, S. Strandberg 45a,45b, M. Strauss 128, P. Strizenec 28b, R. Ströhmer 177, D.M. Strom 131, R. Stroynowski 42, A. Strubig 50, S.A. Stucci 29, B. Stugu 17, J. Stupak 128, N.A. Styles 46, D. Su 153, S. Suchek 61a, V.V. Sulin 110, M.J. Sullivan 90, D.M.S. Sultan 54, S. Sultansoy 4c, T. Sumida 85, S. Sun 105, X. Sun 3, K. Suruliz 156, C.J.E. Suster 157, M.R. Sutton 156, S. Suzuki 81, M. Svatos 141, M. Swiatlowski 37, S.P. Swift 2, T. Swirski 177, A. Sydorenko 99, I. Sykora 28a, M. Sykora 143, T. Sykora 143, D. Ta 99, K. Tackmann 46,aa, J. Taenzer 161, A. Taffard 171, R. Taﬁrout 168a, H. Takai 29, R. Takashima 86, K. Takeda 82, T. Takeshita 150, E.P. Takeva 50, Y. Takubo 81, M. Talby 101, A.A. Talyshev 121b,121a, N.M. Tamir 161, J. Tanaka 163, M. Tanaka 165, R. Tanaka 132, S. Tapia Araya 173, S. Tapprogge 99, A. Tarek Abouelfadl Mohamed 136, S. Tarem 160, K. Tariq 60b, G. Tarna 27b,c, G.F. Tartarelli 68a, P. Tas 143, M. Tasevsky 141, T. Tashiro 85, E. Tassi 41b,41a, A. Tavares Delgado 140a,140b, Y. Tayalati 35e, A.J. Taylor 50, G.N. Taylor 104, W. Taylor 168b, A.S. Tee 89, R. Teixeira De Lima 153, P. Teixeira-Dias 93, H. Ten Kate 36, J.J. Teoh 119, S. Terada 81, K. Terashi 163, J. Terron 98, S. Terzo 14, M. Testa 51, R.J. Teuscher 167,ae, S.J. Thais 183, T. Theveneaux-Pelzer 46, F. Thiele 40, D.W. Thomas 93, J.O. Thomas 42, J.P. Thomas 21, A.S. Thompson 57, P.D. Thompson 21, L.A. Thomsen 183, E. Thomson 137, E.J. The ATLAS Collaboration Thorpe 92, R.E. Ticse Torres 53, V.O. Tikhomirov 110,ap, Yu.A. Tikhonov 121b,121a, S. Timoshenko 111, P. Tipton 183, S. Tisserant 101, K. Todome 23b,23a, S. Todorova-Nova 5, S. Todt 48, J. Tojo 87, S. Tokár 28a, K. Tokushuku 81, E. Tolley 126, K.G. Tomiwa 33c, M. Tomoto 116, L. Tompkins 153,q, B. Tong 59, P. Tornambe 102, E. Torrence 131, H. Torres 48, E. Torró Pastor 148, C. Tosciri 135, J. Toth 101,ad, D.R. Tovey 149, A. Traeet 17, C.J. Treado 124, T. Trefzger 177, F. Tresoldi 156, A. Tricoli 29, I.M. Trigger 168a, S. Trincaz-Duvoid 136, W. Trischuk 167, B. Trocmé 58, A. Trofymov 145, C. Troncon 68a, M. Trovatelli 176, F. Trovato 156, L. Truong 33b, M. Trzebinski 84, A. Trzupek 84, F. Tsai 46, J.C-L. Tseng 135, P.V. Tsiareshka 107,a A. Tsirigotis 162, V. Tsiskaridze 155, E.G. Tskhadadze 159a, M. Tsopoulou 162, I.I. Tsukerman 123, V. Tsulaia 18 S. Tsuno 81, D. Tsybychev 155, Y. Tu 63b, A. Tudorache 27b, V. Tudorache 27b, T.T. Tulbure 27a, A.N. Tuna 59, S. Turchikhin 79, D. Turgeman 180, I. Turk Cakir 4b,w, R.J. Turner 21, R.T. Turra 68a, P.M. Tuts 39, S. Tzamarias 162, E. Tzovara 99, G. Ucchielli 47, K. Uchida 163, I. Ueda 81, M. Ughetto 45a,45b, F. Ukegawa 169 G. Unal 36, A. Undrus 29, G. Unel 171, F.C. Ungaro 104, Y. Unno 81, K. Uno 163, J. Urban 28b, P. Urquijo 104, G. Usai 8, Z. Uysal 12d, L. Vacavant 101, V. Vacek 142, B. Vachon 103, K.O.H. Vadla 134, A. Vaidya 94, C. Valderanis 113, E. Valdes Santurio 45a,45b, M. Valente 54, S. Valentinetti 23b,23a, A. Valero 174, L. Valéry 46 R.A. Vallance 21, A. Vallier 36, J.A. Valls Ferrer 174, T.R. Van Daalen 14, P. Van Gemmeren 6, I. Van Vulpen 119, M. Vanadia 73a,73b, W. Vandelli 36, E.R. Vandewall 129, A. Vaniachine 166, D. Vannicola 72a,72b, R. Vari 72a, E.W. Varnes 7, C. Varni 55b,55a, T. Varol 158, D. Varouchas 132, K.E. Varvell 157, M.E. Vasile 27b, G.A. Vasquez 176, J.G. Vasquez 183, F. Vazeille 38, D. Vazquez Furelos 14, T. Vazquez Schroeder 36, J. Veatch 53, V. Vecchio 74a,74b, M.J. Veen 119, L.M. Veloce 167, F. Veloso 140a,140c, S. Veneziano 72a, A. Ventura 67a,67b, N. Venturi 36, A. Verbytskyi 114, V. Vercesi 70a, M. Verducci 71a,71b, C.M. Vergel Infante 78, C. Vergis 24, W. Verkerke 119, A.T. Vermeulen 119, J.C. The ATLAS Collaboration Zhang 15a,15d, Z. Zhang 6 Z. Zhang 132, P. Zhao 49, Y. Zhao 60b, Z. Zhao 60a, A. Zhemchugov 79, Z. Zheng 105, D. Zhong 173, B. Zhou 1 C. Zhou 181, M.S. Zhou 15a,15d, M. Zhou 155, N. Zhou 60c, Y. Zhou 7, C.G. Zhu 60b, C. Zhu 15a,15d, H.L. Zhu 6 H. Zhu 15a, J. Zhu 105, Y. Zhu 60a, X. Zhuang 15a, K. Zhukov 110, V. Zhulanov 121b,121a, D. Zieminska 65, N.I. Zimine 79, S. Zimmermann 52, Z. Zinonos 114, M. Ziolkowski 151, L. Živkovi´c 16, G. Zobernig 181, A. Zoccoli 23b,23a, K. Zoch 53, T.G. Zorbas 149, R. Zou 37, L. The ATLAS Collaboration Zaazoua 35e, B. Zabinski 84, G. Zacharis 10, E. Zaffaroni 54, J. Zahreddine 136, A.M. Zaitsev 122,ao, T. Zakareishvili 159b, N. Zakharchuk 34, S. Zambito 59, D. Zanzi 36, D.R. Zaripovas 57, S.V. Zeißner 47, C. Zeitnitz 182, G. Zemaityte 135, J.C. Zeng 173, O. Zenin 122, T. Ženiš 28a, D. Zerwas 132, M. Zgubiˇc 135, B. Zhang 15c, D.F. Zhang 15b, G. Zhang 15b, H. Zhang 15c, J. Zhang 6, L. Zhang 15c, L. Zhang 60a, M. Zhang 173, R. Zhang 24, X. Zhang 60b, Y. Zhang 15a,15d, Z. Zhang 63a, Z. Zhang 132, P. Zhao 49, Y. Zhao 60b, Z. Zhao 60a, A. Zhemchugov 79, Z. Zheng 105, D. Zhong 173, B. Zhou 105, C. Zhou 181, M.S. Zhou 15a,15d, M. Zhou 155, N. Zhou 60c, Y. Zhou 7, C.G. Zhu 60b, C. Zhu 15a,15d, H.L. Zhu 60a, H. Zhu 15a, J. Zhu 105, Y. Zhu 60a, X. Zhuang 15a, K. Zhukov 110, V. Zhulanov 121b,121a, D. Zieminska 65, N.I. Zimine 79, S. Zimmermann 52, Z. Zinonos 114, M. Ziolkowski 151, L. Živkovi´c 16, G. Zobernig 181, A. Zoccoli 23b,23a, K. Zoch 53, T.G. Zorbas 149, R. Zou 37, L. Zwalinski 36 M.G. Vincter 34, G.S. Virdee 21, A. Vishwakarma 46, C. Vittori 23b,23a, I. Vivarelli 156, M. Vogel 182, P. Vokac 142, S.E. von Buddenbrock 33c, E. Von Toerne 24, V. Vorobel 143, K. Vorobev 111, M. Vos 174, J.H. Vossebeld 90, M. Vozak 100, N. Vranjes 16, M. Vranjes Milosavljevic 16, V. Vrba 142, M. Vreeswijk 119, R. Vuillermet 36, I. Vukotic 37, P. Wagner 24, W. Wagner 182, J. Wagner-Kuhr 113, S. Wahdan 182, H. Wahlberg 88, V.M. Walbrecht 114, J. Walder 89, R. Walker 113, S.D. Walker 93, W. Walkowiak 151, V. Wallangen 45a,45b, A.M. Wang 59, C. Wang 60c, C. Wang 60b, F. Wang 181, H. Wang 18, H. Wang 3, J. Wang 63a, J. Wang 157, J. Wang 61b, P. Wang 42, Q. Wang 128, R.-J. Wang 99, R. Wang 60a, R. Wang 6, S.M. Wang 158, W.T. Wang 60a, W. Wang 15c,af , W.X. Wang 60a,af , Y. Wang 60a,am, Z. Wang 60c, C. Wanotayaroj 46, A. Warburton 103, C.P. Ward 32, D.R. Wardrope 94, N. Warrack 57, A. Washbrook 50, A.T. Watson 21, M.F. Watson 21, G. Watts 148, B.M. Waugh 94, A.F. Webb 11, S. Webb 99, C. Weber 183, M.S. Weber 20, S.A. Weber 34, S.M. The ATLAS Collaboration Weber 61a, A.R. Weidberg 135, J. Weingarten 47, M. Weirich 99, C. Weiser 52, P.S. Wells 36, T. Wenaus 29, T. Wengler 36, S. Wenig 36, N. Wermes 24, M.D. Werner 78, M. Wessels 61a, T.D. Weston 20, K. Whalen 131, N.L. Whallon 148, A.M. Wharton 89, A.S. White 105, A. White 8, M.J. White 1, D. Whiteson 171, B.W. Whitmore 89, W. Wiedenmann 181, M. Wielers 144, N. Wieseotte 99, C. Wiglesworth 40, L.A.M. Wiik-Fuchs 52, F. Wilk 100, H.G. Wilkens 36, L.J. Wilkins 93, H.H. Williams 137, S. Williams 32, C. Willis 106, S. Willocq 102, J.A. Wilson 21, I. Wingerter-Seez 5, E. Winkels 156, F. Winklmeier 131, O.J. Winston 156, B.T. Winter 52, M. Wittgen 153, M. Wobisch 95, A. Wolf 99, T.M.H. Wolf 119, R. Wolff 101, R.W. Wölker 135, J. Wollrath 52, M.W. Wolter 84, H. Wolters 140a,140c, V.W.S. Wong 175, N.L. Woods 146, S.D. Worm 21, B.K. Wosiek 84, K.W. Wo´zniak 84, K. Wraight 57, S.L. Wu 181, X. Wu 54, Y. Wu 60a, T.R. Wyatt 100, B.M. Wynne 50, S. Xella 40, Z. Xi 105, L. Xia 178, X. Xiao 105, I. Xiotidis 156, D. Xu 15a, H. Xu 60a,c, L. Xu 29, T. Xu 145, W. Xu 105, Z. Xu 60b, Z. Xu 1 B. Yabsley 157, S. Yacoob 33a, K. Yajima 133, D.P. Yallup 94, D. Yamaguchi 165, Y. Yamaguchi 165, A. Yamamoto 81, M. Yamatani 163, T. Yamazaki 163, Y. Yamazaki 82, Z. Yan 25, H.J. Yang 60c,60d, H.T. Yang 1 S. Yang 77, X. Yang 60b,58, Y. Yang 163, W-M. Yao 18, Y.C. Yap 46, Y. Yasu 81, E. Yatsenko 60c,60d, J. Ye 42, S. Ye 29, I. Yeletskikh 79, M.R. Yexley 89, E. Yigitbasi 25, K. Yorita 179, K. Yoshihara 137, C.J.S. Young 36, C. Young 153, J. Yu 78, R. Yuan 60b,i, X. Yue 61a, S.P.Y. Yuen 24, M. Zaazoua 35e, B. Zabinski 84, G. Zacharis 1 E. Zaffaroni 54, J. Zahreddine 136, A.M. Zaitsev 122,ao, T. Zakareishvili 159b, N. Zakharchuk 34, S. Zambito 5 D. Zanzi 36, D.R. Zaripovas 57, S.V. Zeißner 47, C. Zeitnitz 182, G. Zemaityte 135, J.C. Zeng 173, O. Zenin 122 T. Ženiš 28a, D. Zerwas 132, M. Zgubiˇc 135, B. Zhang 15c, D.F. Zhang 15b, G. Zhang 15b, H. Zhang 15c, J. Zhang 6, L. Zhang 15c, L. Zhang 60a, M. Zhang 173, R. Zhang 24, X. Zhang 60b, Y. The ATLAS Collaboration Ukegawa 169, G. Unal 36, A. Undrus 29, G. Unel 171, F.C. Ungaro 104, Y. Unno 81, K. Uno 163, J. Urban 28b, P. Urquijo 104, G. Usai 8, Z. Uysal 12d, L. Vacavant 101, V. Vacek 142, B. Vachon 103, K.O.H. Vadla 134, A. Vaidya 94, C. Valderanis 113, E. Valdes Santurio 45a,45b, M. Valente 54, S. Valentinetti 23b,23a, A. Valero 174, L. Valéry 46 R.A. Vallance 21, A. Vallier 36, J.A. Valls Ferrer 174, T.R. Van Daalen 14, P. Van Gemmeren 6, I. Van Vulpen 119, M. Vanadia 73a,73b, W. Vandelli 36, E.R. Vandewall 129, A. Vaniachine 166, D. Vannicola 72a,72b, R. Vari 72a, E.W. Varnes 7, C. Varni 55b,55a, T. Varol 158, D. Varouchas 132, K.E. Varvell 157, M.E. Vasile 27b, G.A. Vasquez 176, J.G. Vasquez 183, F. Vazeille 38, D. Vazquez Furelos 14, T. Vazquez Schroeder 36, J. Veatch 53, V. Vecchio 74a,74b, M.J. Veen 119, L.M. Veloce 167, F. Veloso 140a,140c, 72 67 67b 36 114 70 71 71b S. Simion , E. Simioni , R. Simoniello , S. Simsek , P. Sinervo , V. Sinetckii , N.B. Sinev 131, M. Sioli 23b,23a, I. Siral 105, S.Yu. Sivoklokov 112, J. Sjölin 45a,45b, E. Skorda 96, P. Skubic 128, M. Slawinska 84, K. Sliwa 170, R. Slovak 143, V. Smakhtin 180, B.H. Smart 144, J. Smiesko 28a, N. Smirnov 111, S.Yu. Smirnov 111, Y. Smirnov 111, L.N. Smirnova 112,v, O. Smirnova 96, J.W. Smith 53, M. Smizanska 89, K. Smolek 142, A. Smykiewicz 84, A.A. Snesarev 110, H.L. Snoek 119, I.M. Snyder 131, S. Snyder 29, 176 161 50 53 36 111 The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 19 / y ( ) M.G. Vincter 34, G.S. Virdee 21, A. Vishwakarma 46, C. Vittori 23b,23a, I. Vivarelli 156, M. Vogel 182, P. Vokac 142, S.E. von Buddenbrock 33c, E. Von Toerne 24, V. Vorobel 143, K. Vorobev 111, M. Vos 174, J.H. Vossebeld 90, M. Vozak 100, N. Vranjes 16, M. Vranjes Milosavljevic 16, V. Vrba 142, M. Vreeswijk 119, R. Vuillermet 36, I. Vukotic 37, P. Wagner 24, W. Wagner 182, J. Wagner-Kuhr 113, S. Wahdan 182, H. Wahlberg 88, V.M. Walbrecht 114, J. Walder 89, R. Walker 113, S.D. Walker 93, W. Walkowiak 151, V. Wallangen 45a,45b, A.M. Wang 59, C. Wang 60c, C. Wang 60b, F. Wang 181, H. Wang 18, H. Wang 3, J. Wang 63a, J. The ATLAS Collaboration Wang 157, J. Wang 61b, P. Wang 42, Q. Wang 128, R.-J. Wang 99, R. Wang 60a, R. Wang 6, S.M. Wang 158, W.T. Wang 60a, W. Wang 15c,af , W.X. Wang 60a,af , Y. Wang 60a,am, Z. Wang 60c, C. Wanotayaroj 46, A. Warburton 103, C.P. Ward 32, D.R. Wardrope 94, N. Warrack 57, A. Washbrook 50, A.T. Watson 21, M.F. Watson 21, G. Watts 148, B.M. Waugh 94, A.F. Webb 11, S. Webb 99, C. Weber 183, M.S. Weber 20, S.A. Weber 34, S.M. Weber 61a, A.R. Weidberg 135, J. Weingarten 47, M. Weirich 99, C. Weiser 52, P.S. Wells 36, T. Wenaus 29, T. Wengler 36, S. Wenig 36, N. Wermes 24, M.D. Werner 78, M. Wessels 61a, T.D. Weston 20, K. Whalen 131, N.L. Whallon 148, A.M. Wharton 89, A.S. White 105, A. White 8, M.J. White 1, D. Whiteson 171, B.W. Whitmore 89, W. Wiedenmann 181, M. Wielers 144, N. Wieseotte 99, C. Wiglesworth 40, L.A.M. Wiik-Fuchs 52, F. Wilk 100, H.G. Wilkens 36, L.J. Wilkins 93, H.H. Williams 137, S. Williams 32, C. Willis 106, S. Willocq 102, J.A. Wilson 21, I. Wingerter-Seez 5, E. Winkels 156, F. Winklmeier 131, O.J. Winston 156, B.T. Winter 52, M. Wittgen 153, M. Wobisch 95, A. Wolf 99, T.M.H. Wolf 119, R. Wolff 101, R.W. Wölker 135, J. Wollrath 52, M.W. Wolter 84, H. Wolters 140a,140c, V.W.S. Wong 175, N.L. Woods 146, S.D. Worm 21, B.K. Wosiek 84, K.W. Wo´zniak 84, K. Wraight 57, S.L. Wu 181, X. Wu 54, Y. Wu 60a, T.R. Wyatt 100, B.M. Wynne 50, S. Xella 40, Z. Xi 105, L. Xia 178, X. Xiao 105, I. Xiotidis 156, D. Xu 15a, H. Xu 60a,c, L. Xu 29, T. Xu 145, W. Xu 105, Z. Xu 60b, Z. Xu 153, B. Yabsley 157, S. Yacoob 33a, K. Yajima 133, D.P. Yallup 94, D. Yamaguchi 165, Y. Yamaguchi 165, A. Yamamoto 81, M. Yamatani 163, T. Yamazaki 163, Y. Yamazaki 82, Z. Yan 25, H.J. Yang 60c,60d, H.T. Yang 18, S. Yang 77, X. Yang 60b,58, Y. Yang 163, W-M. Yao 18, Y.C. Yap 46, Y. Yasu 81, E. Yatsenko 60c,60d, J. Ye 42, S. Ye 29, I. Yeletskikh 79, M.R. Yexley 89, E. Yigitbasi 25, K. Yorita 179, K. Yoshihara 137, C.J.S. Young 36, C. Young 153, J. Yu 78, R. Yuan 60b,i, X. Yue 61a, S.P.Y. Yuen 24, M. The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 Fermi, Università di Pisa, Pisa, Italy 72 (a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy 72 (a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Università 73 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Università di Roma Tor Verg 74 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Università Roma Tre, Roma, Italy b 4 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e F 74 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e F 74 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Università Roma Tre, R 75 (a) INFN-TIFPA; (b) Università degli Studi di Trento, Trento, Italy 75 (a) INFN-TIFPA; (b) Università degli Studi di Trento, Trento, Italy 76 Institut für Astro- und Teilchenphysik, Leopold-Franzens-Uni 76 Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria 77 University of Iowa, Iowa City, IA, United States of America 78 Department of Physics and Astronomy, Iowa State University, Ames, IA, United States of America 79 and Astronomy, Iowa State University, Ames, IA, United States of Americ 78 Department of Physics and Astronomy, Iowa State U 78 Department of Physics and Astronomy, Iowa State University, Ames, IA, United States of America 79 79 Joint Institute for Nuclear Research, Dubna, Russia 79 Joint Institute for Nuclear Research, Dubna, Russia b 79 Joint Institute for Nuclear Research, Dubna, Russia 80 (a) D t t d E h i Elét i U i id d F d l d J i d F (UFJF) J i d F (b) U i id d F d l d Ri D J i COPPE/EE/IF Ri d J i 79 Joint Institute for Nuclear Research, Dubna, Russia 80 (a) Departamento de Engenharia Elétrica, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora; (b) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; ( ) (d) Joint Institute for Nuclear Research, Dubna, Russia 80 (a) Departamento de Engenharia Elétrica, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora; (b) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (c) Universidade Federal de São João del Rei (UFSJ), São João del Rei; (d) Instituto de Física, Universidade de São Paulo, São Paulo, Brazil Joint Institute for Nuclear Research, Dubna, Russia 80 (a) Departamento de Engenharia Elétrica, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora; (b) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (c) Universidade Federal de São João del Rei (UFSJ), São João del Rei; (d) Instituto de Física, Universidade de São Paulo, São Paulo, Brazil 81 J f 80 (a) Departamento de Engenharia Elétrica, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora; (b) Universidade Federal do Rio De (c) Universidade Federal de São João del Rei (UFSJ), São João del Rei; (d) Instituto de Física, Universidade de São Paulo, São Paulo, Brazil 80 (a) Departamento de Engenharia Elétrica, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora; 0 (a) Departamento de Engenharia Elétrica, Universidade Federal de Juiz 80 (a) Departamento de Engenharia Elétrica, Universi (c) Universidade Federal de São João del Rei (UFSJ), São João del Rei; (d) 81 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 81 KEK, High Energy Accelerator Research Organization, Tsukub 82 Graduate School of Science, Kobe University, Kobe, Japan f y J p 83 (a) AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow; (b) Marian Smoluchowski Institut Poland The ATLAS Collaboration Zwalinski 36 1 Department of Physics, University of Adelaide, Adelaide, Australia 1 Department of Physics, University of Adelaide, Adelaide, Australia Department of Physics, University of Adelaide, Adelaide, Australia 2 Physics Department, SUNY Albany, Albany, NY, United States of America 3 2 Physics Department, SUNY Albany, Albany, NY, United States of Americ 3 3 Department of Physics, University of Alberta, Edmonton, AB, Canada 4 (a) Department of Physics, Ankara University, Ankara; (b) Istanbul Ayd 4 (a) Department of Physics, Ankara University, Ankara; (b) Istanbul Aydin University, Istanbul; (c) Div 5 4 (a) Department of Physics, Ankara University, Ankara; (b) Istanbul Aydin University, Istanbul; (c) Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey 5 LAPP, Université Grenoble Alpes, Université Savoie Mont Blanc, CNRS/IN2P3, Annecy, France 5 LAPP, Université Grenoble Alpes, Université Savoie Mont Blanc, CNRS/IN2P3, Annecy, France 5 LAPP, Université Grenoble Alpes, Université Savoie Mont Blanc, CNRS/IN2P3, 6 6 High Energy Physics Division, Argonne National Laboratory, Argonne, IL, United States of America 7 of Physics, University of Arizona, Tucson, AZ, United States of America 8 Department of Physics, University of Texas at Arlington, Arlington, TX, United States of America 9 Physics Department, National and Kapodistrian University of Athens, Athens, Greece hysics Department, National Technical University of Athens, Zografou, Gr 11 Department of Physics, University of Texas at Austin, Austin, TX, United States of America a) Bahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul; (b) Istanbul Bilgi University, Faculty of Engineering and Natural S sics Bogazici University Istanbul; (d) Department of Physics Engineering Gaziantep University Gaziantep Turkey 12 (a) Bahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul; (b) Istanbul Bilgi University, Faculty Physics Bogazici University Istanbul; (d) Department of Physics Engineering Gaziantep University Gaziantep Turkey 13 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 14 Institut de Física d’Altes Energies (IFAE), Barcelona Institute of Science and Technology, Barcelona, Spain 15 (a) Institute of High Energy Physics, Chinese Academy of Sciences, B (d) University of Chinese Academy of Science (UCAS), Beijing, Chin (d) University of Chinese Academy of Science (UCAS), Beijing, China 16 Institute of Physics, University of Belgrade, Belgrade, Serbia 17 Department for Physics and Technology, University of Bergen, Bergen 18 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, CA, United States of America 19 18 Physics Division, Lawrence Berkeley National Laboratory and Univers 19 Institut für Physik, Humboldt Universität zu Berlin, Berlin, Germany 21 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 22 22 Facultad de Ciencias y Centro de Investigaciónes, Universidad Antonio Nariño, Bogota, b y g g 23 (a) INFN Bologna and Università di Bologna, Dipartimento di Fisica; (b) INFN Sezione di Bologna, Italy The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 Physikalisches Institut, Georg-August-Universität Göttingen, Götting 54 Département de Physique Nucléaire et Corpusculaire, Université de Genève, Genève, Switzerland 54 Département de Physique Nucléaire et Corpusculaire, Université de Genève, Genève, Switzerland 4 Département de Physique Nucléaire et Corpusculaire, Université de Ge p y q p 55 (a) Dipartimento di Fisica, Università di Genova, Genova; (b) INFN Sezione di Genova, Italy 55 (a) Dipartimento di Fisica, Università di Genova, Genova; (b) INFN Sezione di Genova, Italy 5 (a) Dipartimento di Fisica, Università di Genova, Genova; (b) INFN Sezi Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germa 57 SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 57 SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, 58 LPSC, Université Grenoble Alpes, CNRS/IN2P3, Grenoble INP, Grenoble, France 58 LPSC, Université Grenoble Alpes, CNRS/IN2P3, Grenoble INP, Grenoble, France 9 Laboratory for Particle Physics and Cosmology, Harvard University, Cam 60 (a) Department of Modern Physics and State Key Laboratory of Particle Detection and Electronics, University of Science and Technology o ( ) 0 (a) Department of Modern Physics and State Key Laboratory of Particle Interdisciplinary Science and Key Laboratory of Particle Physics and Particle Irradiation (MOE), Shandong University, Qingdao; (c) School of Physics and Astronomy, Shanghai Jiao Tong University, KLPPAC-MoE, SKLPPC, Shanghai; (d) Tsung-Dao Lee Institute, S y g g g 61 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikalisches Institut, Ruprecht-Karls-Universität 61 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikal 61 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Phy 62 Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan 62 Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan y f pp f , f gy, , J p 63 (a) Department of Physics, Chinese University of Hong Kong, Shatin, N.T., Hong Kong; (b) Departm titute for Advanced Study, Hong Kong University of Science and Technolo Institute for Advanced Study, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China 64 Institute for Advanced Study, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon 64 Department of Physics, National Tsing Hua University, Hsinchu, Taiwan 4 Department of Physics, National Tsing Hua University, Hsinchu, Taiwan 65 Department of Physics, Indiana University, Bloomington, IN, United States of America 65 Department of Physics, Indiana University, Bloomington, IN, United States of America 5 Department of Physics, Indiana University, Bloomington, IN, United Sta p f y , y, g , , f 66 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento Politecnico di Ingegneria e Architettura, Università di Udine, Udine, Italy 67 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy p f y y g f 66 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento Politecnico di Ingegneria e Architettura, Università di Udine, Udine, Italy 67 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy 66 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento Politecnico di Ingegneria e Architettura, Un b 66 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento ( ) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; ( ) ICTP, Trieste; ( ) Dipartimento Politecnico di Ingegneria e Architettura, Università di Udine, Udine, Italy 67 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy pp g p g g 67 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy 68 (a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Università di Milano, Milano, Italy 68 (a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Università di Milano, Milano, Italy 69 (a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Università di Napoli, Napoli, Italy 69 (a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Università di Napoli, Napoli, Italy p p p 0 (a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Università di Pavi 70 (a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Università di Pavia, Pavia, Italy 71 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 20 24 Physikalisches Institut, Universität Bonn, Bonn, Germany 5 Department of Physics, Boston University, Boston, MA, United States of p f y y f 26 Department of Physics, Brandeis University, Waltham, MA, United States of America 26 Department of Physics, Brandeis University, Waltham, MA, United States of America p f y , y, , , f 27 (a) Transilvania University of Brasov, Brasov; (b) Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest; (c) Department of Physics, Alexandru Ioan Cuza University of Iasi, Iasi; (d) National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj-Napoca; (e) University Politehnica Bucharest, Bucharest; (f) West University in Timisoara, Timisoara, Romania p f y y f 27 (a) Transilvania University of Brasov, Brasov; (b) Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest; (c) Department of Physics, Alexandru Ioan Cuza University of Iasi, Iasi; (d) National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj-Napoca; (e) University Politehnica Bucharest, Bucharest; (f) West University in Timisoara, Timisoara, Romania 27 (a) Transilvania University of Brasov, Brasov; (b) Horia Hulubei National Institute of Physics and Nuclear 7 (a) Transilvania University of Brasov, Brasov; (b) Horia Hulubei Nation University of Iasi, Iasi; (d) National Institute for Research and Dev Bucharest; (f) West University in Timisoara, Timisoara, Romania y 28 (a) Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 29 Ph i D B kh N i l L b U NY U i d S f A i nd Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy o 28 (a) Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Exp Sciences Kosice Slovak Republic 28 (a) Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 29 28 (a) Faculty of Mathematics, Physics and Informatics, Comenius 28 (a) Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava; (b) Depa Sciences, Kosice, Slovak Republic 29 Physics Department, Brookhaven National Laboratory, Upton, NY, United States of America 29 Physics Department, Brookhaven National Laboratory, Upton, NY, United States of America 30 Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Argentina 30 Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Argentina 0 Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Ar 31 California State University, CA, United States of America 32 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 32 Cavendish Laboratory, University of Cambridge, Cambridg 32 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom b 33 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Mechanical Engineering Science, University of Johannesburg, Johannesburg; (c) School of Physics, University of the Witwatersrand, Johannesburg, South Africa 34 D t t f Ph i C l t U i it Ott ON C d 33 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Mechanical Engineering Science, University of Johannesburg, Johannesburg; (c) School of Physics, University of the Witwatersrand, Johannesburg, South Africa 34 D f Ph i C l U i i O ON C d 33 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Mechanical Engineering Science, University of Johannesburg, Johannesburg; (c) School of Physics, University of the Witwatersrand Johannesburg South Africa University of the Witwatersrand, Johannesburg, South Africa 34 Department of Physics, Carleton University, Ottawa, ON, Canada 34 Department of Physics, Carleton University, Ottawa, ON, Canada p f y , y, , , 35 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - Université Hassan II, Casablanca; (b) Faculté des Sciences, Université Ibn-Tofail, Kénitra; (c) Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA-Marrakech; (d) Faculté des Sciences, Université Mohamed Premier and LPTPM, Oujda; (e) Faculté des sciences, Université Mohammed V, Rabat, Morocco 36 35 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique 35 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - Université Hassan II, Casablanca; (b) Faculté des Sciences, Université Ibn-Tofail, Kénitra; (c) Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA-Marrakech; (d) Faculté des Sciences, Université Mohamed Premier and LPTPM, Oujda; (e) Faculté des sciences, Université Mohammed V, Rabat, Morocco 36 35 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - Université Hassan II, Casablanca; (b) Faculté des Sciences, Université Ibn-Tofail, Kénitra; (c) Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA-Marrakech; (d) Faculté des Sciences, Université Mohamed Premier and LPTPM, Oujda; (e) Faculté des sciences, Université Mohammed V, Rabat, Morocco 36 CERN, Geneva, Switzerland 37 Enrico Fermi Institute, University of Chicago, Chicago, IL, United States of America 37 Enrico Fermi Institute, University of Chicago, Chicago, IL, United States of America 38 38 LPC, Université Clermont Auvergne, CNRS/IN2P3, Clermont-Ferrand, France 38 LPC, Université Clermont Auvergne, CNRS/IN2P3, Clermont-Ferrand, France 39 Nevis Laboratory, Columbia University, Irvington, NY, United States o 39 Nevis Laboratory, Columbia University, Irvington, NY, United States of Ameri 40 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 40 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark y f p g p g 41 (a) Dipartimento di Fisica, Università della Calabria, Rende; (b) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati, Italy 42 41 (a) Dipartimento di Fisica, Università della Calabria, Rende; (b) INFN Gruppo Collegato di Cose 41 (a) Dipartimento di Fisica, Università della Calabria, Rende; (b) INFN Dipartimento di Fisica, Università della Calabria, Rende; INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati, 42 Physics Department, Southern Methodist University, Dallas, TX, United States of America p , , ; pp g 42 Physics Department, Southern Methodist University, Dallas, TX, United States of Ame p , , ; pp g , 42 Physics Department, Southern Methodist University, Dallas, TX, United States of America 42 Physics Department, Southern Methodist University, Dallas, TX, United States 2 Physics Department, Southern Methodist University, Dallas, TX, United 43 Physics Department, University of Texas at Dallas, Richardson, TX, United States of America 43 Physics Department, University of Texas at Dallas, Richardson, TX, United States of America 44 National Centre for Scientiﬁc Research “Demokritos”, Agia Paraskevi, Greece 44 National Centre for Scientiﬁc Research “Demokritos”, Agia Paraskevi, Greece 5 (a) Department of Physics, Stockholm University; (b) Oskar Klein Centr 45 (a) Department of Physics, Stockholm University; (b) Oskar Klein Centre, Stockho 46 Deutsches Elektronen-Synchrotron DESY, Hamburg and Zeuthen, Germany 47 46 Deutsches Elektronen-Synchrotron DESY, Hamburg and Zeuthen, Germany 7 Lehrstuhl für Experimentelle Physik IV, Technische Universität Dortmun f p y 48 Institut für Kern- und Teilchenphysik, Technische Universität Dresden, Dresden, Germany f p y y 48 Institut für Kern- und Teilchenphysik, Technische Universität Dresden, Dresden, Germany 8 Institut für Kern- und Teilchenphysik, Technische Universität Dresden, 49 Department of Physics, Duke University, Durham, NC, United States of America 49 Department of Physics, Duke University, Durham, NC, United States of America 50 SUPA - School of Physics and Astronomy, University of 51 INFN e Laboratori Nazionali di Frascati, Frascati, Italy 52 Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany 52 Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany 52 Physikalisches Institut, Albert-Ludwigs-Universität F 3 II. The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 Fermi, Università di Pisa, Pisa, Italy 71 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Poland Petersburg, Russia 138 Konstantinov Nuclear Physics Institute of National Research Centre “Kurchatov Institu 138 Konstantinov Nuclear Physics Institute of National Research Centre “ y f 139 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA, United States of America y f 139 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA, United States of America p f y y, y f g , g , , f 140 (a) Laboratório de Instrumentação e Física Experimental de Partículas - LIP, Lisboa; (b) Departamento de Física, Faculdade de Ciências, Universidade de Lisboa, Lisboa; (c) Departamento de Física, Universidade de Coimbra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de Física, Universidade do Minho, Braga; (f) Departamento de Física Teórica y del Cosmos, Universidad de Granada, Granada (Spain); (g) Dep Física and CEFITEC of Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica; (h) Instituto Superior Técnico Universidade de Lisboa Lisboa Portugal p f y y y f g g f 140 (a) Laboratório de Instrumentação e Física Experimental de Partículas - LIP, Lisboa; (b) Departamento de Física, Faculdade de Ciências, Uni d 140 (a) Laboratório de Instrumentação e Física Experimental de Partícula 140 (a) Laboratório de Instrumentação e Física Experimental de Partículas - LIP, Lisboa; (b) Departamento de F 140 (a) Laboratório de Instrumentação e Física Experimental de Partículas - LIP, Lisboa; (b) Departamento de Física, Faculdade de Ciências, Universidade de Lisboa, Lisboa; (c) Departamento de Física, Universidade de Coimbra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de Física, Universidade do Minho, Braga; (f) Departamento de Física Teórica y del Cosmos, Universidad de Granada, Granada (Spain); (g) Dep Física and CEFITEC of Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica; Laboratório de Instrumentação e Física Experimental de Partículas LIP, Lisboa; Departamento de Física, Faculdade de Ciências, Universidade de Lisboa, Lisboa; Departamento de Física, Universidade de Coimbra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de Física, Universidade do Minho, Braga; (f) Departamento d Fí i T ó i d l C U i id d d G d G d (S i ) (g) D Fí i d CEFITEC f F ld d d Ciê i T l i U i id d N d Li b C i de Física, Universidade de Coimbra, Coimbra; (d) Centro de Física Nuclear da Univers de Física, Universidade de Coimbra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de Física, Universidade do Minho, Braga; (f) Depa ( ) de Física, Universidade de Coimbra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) De ( ) de Física, Universidade de Coimbra, Coimbra; (d) Centro de Física Nuc de Física Teórica y del Cosmos, Universidad de Granada, Granada (Spain); (g) Dep Física and CEFITEC of Facu de Física Teórica y del Cosmos, Universidad de Granada, Granada (Spain); (g) Dep Física and CEFITEC of Faculdade d de Física Teórica y del Cosmos, Universidad de Granada, Granada (Spain (h) Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal (h) Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal 141 Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic 141 Institute of Physics of the Czech Academy of Sciences, Prague, Cz 142 Czech Technical University in Prague, Prague, Czech Republic 142 Czech Technical University in Prague, Prague, Czech Republic 143 Charles University, Faculty of Mathematics and Physics, Prague, Czech Republic 143 Charles University, Faculty of Mathematics and Physics, Prague, Czech Republic 144 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United 144 Particle Physics Department, Rutherford Appleton Laborato 145 IRFU, CEA, Université Paris-Saclay, Gif-sur-Yvette, France y f 146 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, CA, United States of America 147 ( ) (b) 146 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, CA, United States of Americ b 146 Santa Cruz Institute for Particle Physics, University of Ca 147 (a) Departamento de Física, Pontiﬁcia Universidad Católic 147 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago 147 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Universidad Andres Bello, Depa 147 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Universidad Andres Bello, Department of Physics, Santiago; (c) Departamento de Física, Universidad Té i F d i S M í V l í Chil 147 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Universidad Andres Bello, Department of Physics, Santiago; (c) Departamento de Física, Universidad Técnica Federico Santa María Valparaíso Chile 147 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Universidad Andres Bello, Department of Physics, Santiago; (c) Departamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile Técnica Federico Santa María, Valparaíso, Chile Técnica Federico Santa María, Valparaíso, Chile 148 Department of Physics, University of Washington, Seattle, WA, United States of America 148 Department of Physics, University of Washington, Seattle, WA, United States of America 149 Department of Physics and Astronomy, University of Sheﬃeld, S 149 Department of Physics and Astronomy, University of Sheﬃeld, Sheﬃeld, Unite 150 Department of Physics, Shinshu University, Nagano, Japan 150 Department of Physics, Shinshu University, Nagano, Japan 151 Department Physik, Universität Siegen, Siegen, Germany 152 Department of Physics, Simon Fraser University, Burnaby, BC, Canad 153 SLAC National Accelerator Laboratory, Stanford, CA, United States of America 153 SLAC National Accelerator Laboratory, Stanford, CA, United States of America 4 Physics Department, Royal Institute of Technology, Stockholm, Sweden y p y f gy 155 Departments of Physics and Astronomy, Stony Brook University, Stony Brook, NY, United States of America 155 Departments of Physics and Astronomy, Stony Brook University, Stony Brook, NY, United States of America 155 Departments of Physics and Astronomy, Stony Brook University, Ston 156 Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 156 Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 156 Department of Physics and Astronomy, University of Sussex, Br 157 School of Physics, University of Sydney, Sydney, Australia 158 Institute of Physics, Academia Sinica, Taipei, Taiwan nstitute of hysics, cademia Sinica, aipei, ai an 159 (a) E. Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi; (b) High Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia f y , , p , 159 (a) E. Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi; (b) High Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 160 Department of Physics Technion Israel Institute of Technology Haifa Israel 159 (a) E. Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi; (b) High Energy Physics Institute, Tbilisi State 159 (a) E. Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi; (b) High En 159 (a) E. Andronikashvili Institute of Physics, Iv. Poland Stepanov Institute of Physics, National Academy of Sciences of Belarus, Min 108 Research Institute for Nuclear Problems of Byelorussian State University, Minsk, Belarus 108 Research Institute for Nuclear Problems of Byelorussian State University, Minsk, Belarus 109 Group of Particle Physics, University of Montreal, Montreal, QC, Canada 110 P.N. Lebedev Physical Institute of the Russian Academy of Sciences, 111 National Research Nuclear University MEPhI, Moscow, Russia 112 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Mosc 112 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov M 113 Fakultät für Physik, Ludwig-Maximilians-Universität München, Mü 113 Fakultät für Physik, Ludwig-Maximilians-Universität München, Mün 114 Max-Planck-Institut für Physik (Werner-Heisenberg-Inst 114 Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 115 Nagasaki Institute of Applied Science, Nagasaki, Japan 116 Graduate School of Science and Kobayashi-Maskawa In 117 Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM, United St 118 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhe 119 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Ne 119 Nikhef National Institute for Subatomic Physics and University of Am 119 Nikhef National Institute for Subatomic Physics and University 120 Department of Physics, Northern Illinois University, DeKalb, IL, United States of America f y y 122 Institute for High Energy Physics of the National Research Centre Kurchatov Institute, Protvino, Russia 123 Institute for Theoretical and Experimental Physics named by A.I. Alikhanov of National Re 124 Department of Physics, New York University, New York, NY, United 125 Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan 126 Ohio State University, Columbus, OH, United States of America 127 Faculty of Science, Okayama University, Okayama, Japan y f y y y J p 128 Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, OK, United States of America 129 128 Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, OK 128 Homer L. Poland Dodge Department of Physics and Astronomy, 129 Department of Physics, Oklahoma State University, Stillwater, OK, United States of America 129 Department of Physics, Oklahoma State University, Stillwater, OK, United States of America Palacký University, RCPTM, Joint Laboratory of Optics, Olomouc, Czech Re 131 Center for High Energy Physics, University of Oregon, Eugene, OR, United States of America 131 Center for High Energy Physics, University of Oregon, Eugene, OR, United States of Americ 132 LAL, Université Paris-Sud, CNRS/IN2P3, Université Paris-Saclay, Orsay, France 132 LAL, Université Paris-Sud, CNRS/IN2P3, Université Paris-Saclay, Orsay, France 133 Graduate School of Science, Osaka University, Osaka, Japan 134 Department of Physics, University of Oslo, Oslo, Norway 135 Department of Physics, Oxford University, Oxford, United Kingdom 136 LPNHE, Sorbonne Université, Université de Paris, CNRS/IN2P3, Paris, / 137 Department of Physics, University of Pennsylvania, Philadelphia, PA, United States of America 137 Department of Physics, University of Pennsylvania, Philadelphia, PA, United States of America 138 Konstantinov Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, PNPI, St. Poland 84 Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland 84 Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland 85 Faculty of Science, Kyoto University, Kyoto, Japan 86 Kyoto University of Education, Kyoto, Japan y y f , y , J p 87 Research Center for Advanced Particle Physics and Department of Physics, Kyushu University, Fukuoka , Japan 88 87 Research Center for Advanced Particle Physics and Department 87 Research Center for Advanced Particle Physics and Department of Physics, Kyushu University, Fuku 8 Instituto de Física La Plata, Universidad Nacional de La Plata and CONI 89 Physics Department, Lancaster University, Lancaster, United Kingdom 89 Physics Department, Lancaster University, Lancaster, United Kingdom 90 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 90 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 1 Department of Experimental Particle Physics, Jožef Stefan Institute and 21 The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 92 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 92 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom Department of Physics, Royal Holloway University of London, Egham, Un p f y y y y f g g 94 Department of Physics and Astronomy, University College London, London, United Kingdom 94 Department of Physics and Astronomy, University College London, London, United Kingdom 94 Department of Physics and Astronomy, University College London p f y y y g 95 Louisiana Tech University, Ruston, LA, United States of America 95 Louisiana Tech University, Ruston, LA, United States of America 96 Fysiska institutionen, Lunds universitet, Lund, Sweden y 97 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France 98 97 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), 97 Centre de Calcul de l’Institut National de Physique Nuc 98 Departamento de Física Teorica C-15 and CIAFF, Universidad Autónoma de Madrid, Madrid, Spain ica C-15 and CIAFF, Universidad Autónoma de Madrid, Madrid, Spain 98 Departamento de Física Teorica C-15 and CIAFF, Universidad Autónoma de Madrid, Madrid, Spai 98 Departamento de Física Teorica C-15 and CIAFF, Unive 99 Institut für Physik, Universität Mainz, Mainz, Germany f y y 100 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 100 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 100 School of Physics and Astronomy, University of Manchester, Manc 101 CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France , , / , , 102 Department of Physics, University of Massachusetts, Amherst, MA, United States of America 102 Department of Physics, University of Massachusetts, Amherst, MA 103 Department of Physics, McGill University, Montreal, QC, Canada 103 Department of Physics, McGill University, Montreal, QC, Canada 104 School of Physics, University of Melbourne, Victoria, Australia 105 Department of Physics, University of Michigan, Ann Arbor, MI, United States of America 105 Department of Physics, University of Michigan, Ann Arbor, MI, United States of America 105 Department of Physics, University of Michigan, Ann Arbor, MI, U 106 Department of Physics and Astronomy, Michigan State University, East Lansing, MI, United p f y y g y g 107 B.I. The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 22 167 Department of Physics, University of Toronto, Toronto, ON, Canada 168 (a) TRIUMF, Vancouver, BC; (b) Department of Physics and Astronom p f y y y 169 Division of Physics and Tomonaga Center for the History of the Universe, Faculty of Pure and Appli 170 Department of Physics and Astronomy, Tufts University, Medford, MA, United States of America 0 Department of Physics and Astronomy, Tufts University, Medford, MA, United States of America Department of Physics and Astronomy, University of California Irvine, Ir 172 Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 173 Department of Physics, University of Illinois, Urbana, IL, United States of America Instituto de Física Corpuscular (IFIC), Centro Mixto Universidad de Valen 175 Department of Physics, University of British Columbia, Vancouver, BC, Canada 176 Department of Physics and Astronomy, University of Victoria, Victoria, BC, C Fakultät für Physik und Astronomie, Julius-Maximilians-Universität Wü 178 Department of Physics, University of Warwick, Coventry, United Kingdom 179 Waseda University, Tokyo, Japan 180 Department of Particle Physics, Weizmann Institute of Science, Rehovot, Israel 181 Department of Physics, University of Wisconsin, Madison, WI, United States of America 181 Department of Physics, University of Wisconsin, Madison, WI, United States of America 182 Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal, Germany 182 Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal, Germany 183 182 Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Phy 182 Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal, Germany 183 Department of Physics, Yale University, New Haven, CT, United States of America 183 Department of Physics, Yale University, New Haven, CT, United States of America 184 Yerevan Physics Institute, Yerevan, Armenia 184 Yerevan Physics Institute, Yerevan, Armenia a Also at Borough of Manhattan Community College, City University of New York, New York, NY; United States of America. b Also at CERN Geneva; Switzerland a Also at Borough of Manhattan Community College, City University of New York, New York, NY; United States of America. b Al CERN G S i l d a Also at Borough of Manhattan Community College, City University of New York, New York, NY; United States of America. b Also at CERN, Geneva; Switzerland. g y g y y b Also at CERN, Geneva; Switzerland. b Also at CERN, Geneva; Switzerland. Técnica Federico Santa María, Valparaíso, Chile Javakhishvili Tbilisi S 0 Department of Physics, Technion, Israel Institute of Technology, Haifa, I p f y f gy f 161 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 161 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 162 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 162 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 163 International Center for Elementary Particle Physics and Departm 164 Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 164 Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 4 Graduate School of Science and Technology, Tokyo Metropolitan Univ 164 Graduate School of Science and Technology, Tokyo Metropolitan U 165 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 165 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 166 Tomsk State University, Tomsk, Russia The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 q Also at Department of Physics, Stanford University, Stanford, CA r Also at Department of Physics, University of Adelaide, Adelaide s Also at Department of Physics, University of Fribourg, Fribourg; Switzerland. s Also at Department of Physics, University of Fribourg, Fribourg; Switzerland. t Also at Department of Physics, University of Michigan, Ann Arbor, MI; United States of America. t Also at Department of Physics, University of Michigan, Ann Arbor, MI; United States of Amer t Also at Department of Physics, University of Michigan, Ann Arb u Also at Dipartimento di Matematica, Informatica e Fisica, Unive v Also at Faculty of Physics, M.V. Lomonosov Moscow State University, Moscow; Russia. v Also at Faculty of Physics, M.V. Lomonosov Moscow State University, Moscow; Russia. v Also at Faculty of Physics, M.V. Lomonosov Moscow State Unive w Also at Giresun University, Faculty of Engineering, Giresun; Turkey. w Also at Giresun University, Faculty of Engineering, Giresun; Turkey. x Also at Graduate School of Science, Osaka Univers y Also at Hellenic Open University, Patras; Greece. p y z Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona; Spain. z Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona; Spain. z Also at Institucio Catalana de Recerca i Estudis A aa Also at Institut für Experimentalphysik, Universität Hamburg, H ab Also at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen; Netherlan ab Also at Institute for Mathematics, Astrophysics and Particle Physics, Radboud Universi ac Also at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Acade ac Also at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academy of Sciences, Soﬁa; B ad Also at Institute for Particle and Nuclear Physics, Wigner Resea ae Also at Institute of Particle Physics (IPP), Vancouver; Canada. ae Also at Institute of Particle Physics (IPP), Vancouver; Canada. af Also at Institute of Physics, Academia Sinica, Taipei; Taiwan. af Also at Institute of Physics, Academia Sinica, Taipei; Taiwan. ag Also at Institute of Physics, Azerbaijan Academy of Sciences, B ah Also at Institute of Theoretical Physics, Ilia State University, Tbilisi; Georgia. ah Also at Institute of Theoretical Physics, Ilia State University, T ah Also at Institute of Theoretical Physics, Ilia State University, Tbilisi; Georgia. ai Also at Instituto de Fisica Teorica, IFT-UAM/CSIC, Madrid; Spain. The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 , ; c Also at CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille; France. c Also at CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille; France. d Also at Département de Physique Nucléaire et Corpusculaire, Université de Genève, Genève; Switzerland. d Also at Département de Physique Nucléaire et Corpusculaire, Université de Genève, Genève; d Also at Département de Physique Nucléaire et Corpusculaire, Un e Also at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona; Spain. Also at Departament de Fisica de la Universitat Autonoma de Barc f Also at Departamento de Física, Instituto Superior Técnico, Universidade de Lisboa, Lisboa; Portugal. f Also at Departamento de Física, Instituto Superior Técnico, Universidade de Lisboa, Lisboa; Po Also at Department of Applied Physics and Astronomy, University g Also at Department of Applied Physics and Astronomy, University of Sharjah, Sharjah; United Arab Emirates. g Also at Department of Applied Physics and Astronomy, University of Sharjah, Sharjah; United Arab Emirate h Also at Department of Financial and Management Engineering, University of the Aegean, Chios; Greece. h Also at Department of Financial and Management Engineering, University of the Aegean, Chios; Gre Also at Department of Physics and Astronomy, Michigan State Uni j Also at Department of Physics and Astronomy, University of Louisville, Louisville, KY; United States of America. j Also at Department of Physics and Astronomy, University of Louisville, Louisville, KY; Uni k Also at Department of Physics, Ben Gurion University of the Negev, Beer Sheva; Israel. k Also at Department of Physics, Ben Gurion University of the Negev, Beer Sheva; Israel. o at Department of Physics, California State University, East Bay; Un p y y y m Also at Department of Physics, California State University, Fresno; United States of America. m Also at Department of Physics, California State University, Fresno; United States of America. n Also at Department of Physics, California State University, Sacramento; United States of America. n Also at Department of Physics, California State University, Sacra n Also at Department of Physics, California State University, Sacramento; United States of America. o at Department of Physics, King’s College London, London; United p Also at Department of Physics, St. Petersburg State Polytechnical University, St. Petersburg; q Also at Department of Physics, Stanford University, Stanford, CA; United States of America. q Also at Department of Physics, Stanford University, Stanford, CA; United States of America. The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 ai Also at Instituto de Fisica Teorica, IFT-UAM/CSIC, Madrid; Spain aj Also at Joint Institute for Nuclear Research, Dubna; Russia. aj Also at Joint Institute for Nuclear Research, Dubna; Russia. ak Also at LAL, Université Paris-Sud, CNRS/IN2P3, Université Paris-Saclay, Orsay; ak Also at LAL, Université Paris-Sud, CNRS/IN2P3, Université Paris-Saclay, Orsay; Fra ak Also at LAL, Université Paris-Sud, CNRS/IN2P3, Université Par al Also at Louisiana Tech University, Ruston, LA; United States of America. al Also at Louisiana Tech University, Ruston, LA; United States of America am Also at LPNHE, Sorbonne Université, Université de Paris, CNRS/IN2P3, Paris; France. am Also at LPNHE, Sorbonne Université, Université de Paris, CNRS/IN2P3, Paris; France an Also at Manhattan College, New York, NY; United States of Ame an Also at Manhattan College, New York, NY; United States of America. g ao Also at Moscow Institute of Physics and Technology State University, Dolgoprudny; Russia. ao Also at Moscow Institute of Physics and Technology State University, Dolgop ap Also at National Research Nuclear University MEPhI, Moscow; Russia. ap Also at National Research Nuclear University MEPhI, Moscow; Russia. aq Also at Physics Department, An-Najah National University, Nablus; Palestine. aq Also at Physics Department, An-Najah National University, Nablus; Palestine aq Also at Physics Department, An-Najah National University, Nabl ar Also at Physics Dept, University of South Africa, Pretoria; South Africa. ar Also at Physics Dept, University of South Africa, Pretoria; South Africa. as Also at Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg; Germany. as Also at Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg; Germa y g g g at Also at School of Physics, Sun Yat-sen University, Guangzhou; China. y g g g at Also at School of Physics, Sun Yat-sen University, Guangzhou; China. y y g au Also at The City College of New York, New York, NY; United States of America. au Also at The City College of New York, New York, NY; United States of America. av Also at The Collaborative Innovation Center of Quantum Matter (CICQM), Beijing; China. av Also at The Collaborative Innovation Center of Quantum Matter av Also at The Collaborative Innovation Center of Quantum Matter (CICQM), Beijing; China. aw Also at Tomsk State University, Tomsk, and Moscow Institute of Physics and Technology State University, Dolgoprudny; Russi aw Also at Tomsk State University, Tomsk, and Moscow Institute of Physics and Technology State University, Dolgoprudny; Russia. The ATLAS Collaboration / Physics Letters B 801 (2020) 135145 aw Also at Tomsk State University, Tomsk, and Moscow Institute aw Also at Tomsk State University, Tomsk, and Moscow Institute of Physics and Technology State Un ax Also at TRIUMF, Vancouver, BC; Canada. ay Also at Universita di Napoli Parthenope, Napoli; Italy. ∗Deceased. ∗Deceased.
https://openalex.org/W4212820465	https://journal.uinmataram.ac.id/index.php/tatsqif/article/download/3260/1666	English	null	TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS	Jurnal Tatsqif	2,021	cc-by-sa	8,426	P ISSN: 1829-5940 E ISSN: 2503-4510 P ISSN: 1829-5940 E ISSN: 2503-4510 J U R N A L T A T S Q I F P ISSN: 1829-5940 Jurnal Pemikiran dan Penelitian Pendidikan E ISSN: 2503-4510 Volume 19, No. 1 (June) 2021 Site: http://journal.uinmataram.ac.id/index.php/tatsqif Email: jurnaltatsqif@uinmataram.ac.id Abstract This study examined aggressive behavior (verbal aggression, physical assault, socially unacceptable and destructive behavior) among learners with moderate intellectual disability in south- western Nigeria, through teachers’ perceptions. The study used a descriptive survey design. Convenient sampling technique was used to select respondents (87 teachers of pupils with intellectual ability in private and public schools in south West Nigeria). The aggressive behavior scale (ABS) was used to elicit responses. Data retrieved was analysed using descriptive and inferential statistics. Results showed that teachers’ perception of the developmental trend of aggressive behavior among learners with moderate intellectual disability was significant (T = 44.385, Mean Difference = 3.115 while p < .05). Also, there was no significant gender difference in verbal aggressive (F=0.997, p>.05), physical aggressive (F=3.628, p>.05), socially unacceptable aggressive (F=1.233, p>.05), and destructive behavior (F=0.571, p>.05). Interventions should be in line with these. Keywords: aggressive behavior, developmental trend, learners with moderate intellectual disability, teachers’ perception, TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS Udeme Samuel Jacob 1a, Jace Pillay 1b, Esther Olufunke Oyefeso 2c, 1 University of Johannesburg, South Africa; 2 University of Ibadan, Nigeria a udeme01@gmail.com, b jacep@uj.ac.za, c estheroyefeso@gmail.com INTRODUCTION Displaying aggressive behavior is a major obstacle to the social integration of individuals with intellectual disability. It affects their access to certain residential environments, educational and occupational programmes, and their general social acceptability (Harris, 1993; Bruininks et al., 1994). Due to the magnitude of the issue of aggressive behavior and the negative impact it has on the health of the children involved, it transcended institutional contexts to become a major public health problem (Shetgiri, 2013). Thus, to maintain a peaceful and safe school environment, stakeholders in education have tended to concern themselves with the problem of violence in our schools (Oyaziwo & 41 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Adegoke, 2011). Moeller (2001) argued that aggression and aggressive behavior are two terms that are used interchangeably to describe negative feelings and behaviors. He added that aggressive behaviors are anti-social, and are viewed as morally, ethically, and legally unacceptable. Aggression is described as an undesired attitude of individuals who force other people to accept their requests and exhibit hostile behaviors that are intended to harm or hurt others, or that causes fear in them (Başaran, 2000; Dodge et al., 2006). Such threats either results in, or have a high likelihood of resulting in injury, death, psychological harm, mal- development, or deprivation. Individuals who engage in aggressive behavior are at increased risk of victimization (Rusch et al., 1986) and criminalization (Lund, 1990; Crocker & Hodgins, 1997; Logan-Greene et al., 2010). According to Wood et al. (2005), aggressive behavior is the intentional plan to harm others either physically or psychologically. De Almeida et al. (2015) view aggressive behavior as overt, but mostly harmful, social interaction with the intention to inflict damage or other unpleasantness upon another individual. Ferguson and Beaver (2009) added that aggressive behaviors are those which increase the social dominance of an organism relative to the dominance position of other organisms. Different types and expressions of aggressive behaviour must be observed because the causes and consequences of aggressive behaviour vary. Aggression in Schools Aggression directed at self or others is often a reason for referral to inpatient mental health services and poses a major obstacle to proper treatment (Gardner & Moffat 1990; Cowley et al. 2005). Aggressive behavior should be taken seriously as it can have adverse consequences for the aggressor, such as seclusion or sedation, as well as for staff. In fact, 42 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 aggression has been linked to burnout and stress among staff and it can affect the therapeutic environment negatively (Edwards & Miltenberger 1991; Hunter & Carmel 1992). These consequences warrant a closer examination of aggressive behaviors in treatment facilities for persons with intellectual disability. Knowledge and understanding of their nature, extent and circumstances is an important first step towards better tailored services and will help staff to better cope with these behaviors. Aggressive behavior in persons with intellectual disability and its correlates have been examined in several studies (McClintock et al., 2003). These studies showed, although not consistently, that men (Sigafoos et al., 1994), persons aged between 20 and 35 years (Tyrer et al., 2006), persons with more severe levels of intellectual disability (Crocker et al., 2006; Tyrer et al., 2006), and persons with a history of violence (Davidson et al., 1994; Linaker, 1994), are most likely to show aggression. Besides, aggressive behavior appears to be associated with an increased prevalence of psychopathology (Moss et al. 2000; Hemmings et al., 2006). Furthermore, aggressive behavior is often assessed in samples that are heterogeneous in terms of the level of intellectual disability, age, or setting (Sigafoos et al., 1994; Crocker et al., 2006; Tyrer et al., 2006). Once children attend pre-school education institutions and begin to interact with their peers extensively, aggression becomes an important issue. Children adopt different compliance strategies when confronted with difficult situations, among them being aggressive behavior (Chen et al., 2010; Hockenberry et al., 2016). Aggression in Schools It is crucial to find ways to effectively manage early childhood conduct problems so that they do not lead to violence and different types of aggression later in life (Farrington & Welsh, 2007; Karaca & İkiz, 2014). 43 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Types of Aggressive behavior According to Poggenpoel and Myburgh (2002), aggression can be expressed through behaviors that are both constructive and destructive. Constructive aggression can be used for individual and collective welfare. Destructive aggression is disturbing for individuals and groups and is disruptive. Essau and Conradt (2004) developed a typology of aggressive behavior explaining different types of aggression, such as open aggression, hidden aggression, reactive aggression, proactive aggression, relational aggression, and instrumental or hostile aggressions. Although studies on the prevalence of aggressive behavior have been conducted, there remains a dearth of knowledge on aggressive behavior in school settings. In most reported studies, aggressive behavior and its characteristics were assessed retrospectively (Crocker et al., 2006; Tyrer et al., 2006). Investigation of various forms of aggressive behavior by Whitney and Smith (1993) and Owens et al. (2000), revealed that the most common and frequent forms are insults, name-calling, yelling at others, theft, hitting, threats, and social exclusion. Different studies have revealed that male adolescents display higher aggressive behavior than their female counterparts, whereas other studies have failed to find gender differences in aggressive behavior (Archer, 2004; Leenaars & Rinaldi, 2010). Concerning gender differences, Crick (1996) and Crick and Grotpeter (1995), argued that aggressive behaviors in girls have been overlooked because they are seen from a different perspective than those of their male counterparts. Imhonde (2013) argued that aggression is exhibited in different forms among individuals, such as physical, mental, or verbal manifestations. Physical abuse has repeatedly been linked to delinquency and interpersonal violence in adolescence (Manly et al., 2001; Smith & Thornberry, 1995). Youth who are victims of abuse are at greater risk of 44 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 themselves becoming abusive in adulthood (Fang & Corso, 2007). Goodenough (1931) asked mothers of 2- to 5-year-olds to record the details of their children’s angry outbursts in a diary. When he examined the data, Goodenough found that unfocused temper tantrums become less and less common between the ages of two and three as children began to physically retaliate (by hitting or kicking) when playmates frustrated or attacked them. Physical aggressive behavior Children often have heightened sensitivity to rejection derived from early experiences of physical abuse or emotional neglect that then triggers anxiety or angry states (Dodge et al., 1990). Previous studies identified serious risk factors among pupils with impaired development as aggressive behavior, victimization, and perpetration (Barnow et al., 2005; Smith et al., 2003) with effects extending into adult roles and functioning (Brown et al., 2007; Liu & Kaplan, 2004). Lochman et al. (1990), examined social cognitive variables in aggressive and non- aggressive boys at pre-adolescent and early adolescent developmental points. They found that aggressive children often misread interpersonal cues by interpreting ambiguous or pro social communication as hostile and, therefore, reacted aggressively. Slaby and Guerra (1988) elaborated on the cognitive profile of these aggressive adolescents who believed that there were limited consequences for aggression, that aggression had concrete benefits, and that it was a legitimate response. These findings are exceedingly important as understanding the impact of impaired social communication can assist families in dealing with violent outbursts and serve as the basis for developing potential interventions. Within the context of supportive relationships with adults who act as a buffer against any negative effects of instability, children learn how to cope with adversity, adapt to their 45 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 surroundings, and regulate their emotions (National Scientific Council on the Developing Child, 2007). Extreme levels of stress can be detrimental to children’s mental health and cognitive functioning (Evans et al., 2011; Shonkoff & Garner, 2011). Instability has been studied from various angles, with the underlying theme that certain kinds of change, and changes at certain points in their lives, predict negative outcomes for children (Moore et al., 2000). These changes do not occur in isolation. Developmental trend based on aggressive behavior Various scientific studies have revealed different perspectives on the causes of violence. Despite claims that people's aggression motivation is innate (Lorenz, 2008; Yörükoğlu, 2002), Bandura (1977) argued that children learn both aggressive and non-aggressive behaviors by observing and modeling others. Victimization and perpetration of aggression have been repeatedly linked (Maas et al., 2008) across multiple types of victimization. The character of children’s aggression changes dramatically with age. Physical aggression, however, gradually declined between ages three and five, only to be replaced by teasing, tattling, name-calling, and other forms of verbal aggression. Adolescents who witness violence in their homes are more likely to become violent or aggressive themselves (Kracke, 2001; Reppucci et al., 2002). A more recent study sought to characterize developmental change in physical aggression from toddlerhood to middle childhood (NICHD Early Child Care Research Network, 2004). This study of 1,195 children used mothers’ reports of the children’s levels of physical aggression, assessed each year from when the children were aged 2 to when they were 9. There are also links between victimization and later revictimization (Desai et al., 2002; Finkelhor et al., 2007). Negative outcomes of victimization (Arata, 2002; Classen et al., 2001) indicate that victimization and trauma play a central role in the development and persistence of 46 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 violent perpetration, although multiple mechanisms for this relationship have been proposed (Falshaw et al., 1996). Coupled with recent advances in the study of toxic stress and its adverse effects on child development (National Scientific Council on the Developing Child, 2007), there is a growing need to understand what it means for children to experience instability and how negative effects can be prevented. METHOD The present study used a descriptive survey design through which the researchers attempt to quantitatively describe how teachers perceive types of aggressive behavior of learners with moderate intellectual disability. This was achieved by administering a questionnaire to determine how teachers of learners with moderate intellectual disability depict aggressive behavior. Gender differences and aggressive behavior Gender is said to have an impact on various levels of adolescence and can also affect reaction to the disposition of others, leading to aggression. Although higher rates have generally been found among men than among women (Harris, 1992; Harris, 1993; Borthwick-Duffy, 1994; Davidson et al., 1994; Maccoby & Jacklin, 1974), research on this showed that girls’ aggressive tactics included gossip, ostracism, breaking confidences, and criticism of victim’s clothing, appearance, or personality (Owens et al., 2000). Girls rate such aggression as more hurtful than boys, suggesting their heightened sensitivity to it (Galen & Underwood, 1997). It is not until the age of 2½ to 3 that sex differences in aggression are reliable, and this is clearly enough time for gender typing to have steered boys and girls in different directions (Fagot et al., 1992). The reason for this could be that parents’ play with boys is rougher than with girls, and they react more negatively to the aggressive behaviors of daughters than to those of sons (Mills & Rubin, 1990; Parke & Slaby, 1983). During their preschool years, children come to view aggression as a male attribute in their gender schemas, and by middle childhood, boys expect aggressive acts to provide them with benefits that are more tangible and which will elicit less disapproval from either parents or peers than girls’ experience (Herzberger & Hall, 1993; Perry et al., 1989). In their literature review, McClintock et al. (2003) found that males were more likely than females to display aggression. This result was, however, 47 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 based on only two studies. Moreover, most studies have focused on more severe forms of aggression, and gender differences or similarities are unknown for lower levels of aggression. In a study on the influence of gender and personality features of violent behavior among adolescents in Nigeria, it was found that gender had no significant influence on violent behavior and personality traits (agreeableness, neuroticism, openness to experience, extraversion and conscientiousness), but had a significant influence on violent behavior among adolescents (Mokolapo & Dele, 2014). Purpose of the Study The goal of developing appropriate intervention strategies that would assist children with intellectual disability that have been exposed to one form of aggression or another should be done after the type and magnitude of aggressive behavior has been determined. The purpose of the study is, therefore, to examine aggressive behavior among learners with moderate intellectual disability in South-western Nigeria using teachers’ perceptions. Research questions 1. Is teachers’ perception of physical aggression among learners with moderate intellectual disability significantly different? 1. Is teachers’ perception of physical aggression among learners with moderate intellectual disability significantly different? 48 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 2. Is teachers’ perception of developmental trend of aggressive behavior among learners with moderate intellectual disability significant? 3. Is there any significant difference in teachers’ perception of gender and types of aggressive behavior among learners with moderate intellectual disability? 4. Is there any significant difference between teachers’ perception of gender based aggressive behavior and school type among learners with moderate intellectual disability? Participants The study respondents were teachers who taught pupils with intellectual disability in 8 private and 14 public schools in South West Nigeria (male = 36; female = 51; age range = 24-60 years). Three states were randomly selected out of the six states in South West Nigeria. The convenient sampling technique was used for the selection of teachers from schools for learners with moderate intellectual disability to ensure geographical coverage. Written consent forms were utilized. All responses were anonymous and confidential. Ethical Considerations Respondents were duly informed about the objectives of the study. After adequate explanation of the purpose of the research in line with the ethics of research, a consent form written in English was printed and given to each respondent. These needed to be signed before they could complete the online research instrument on Google form. Instrument The aggressive behavior scale (ABS) was a 4-item summary that measured verbal aggression (screaming at others), physical assault (hitting others), socially unacceptable and destructive behavior (throwing things), and resistance to care (shoving of caregivers). The frequency of ABS items was coded over 7 days as: not exhibited (0); behavior occurred 1 to 3 days over the past 7 days (1); behavior occurred 4 to 6 days over the past 7 days, but not more than daily (2); or behavior occurred daily (3) (Andres et al., 2017). ABS scores ranged from 0 to 12, with higher scores suggesting most behaviors occurred at a higher frequency. The average inter-rater reliability estimates for all behavior items on the MDS 49 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 2.0 was excellent, with a kappa of 0.72 (Morris et al., 1997). This instrument was used because of its ability to capture the constructs of interest (i.e., teachers’ perceptions and the presence of aggressive behavior) and age appropriateness (6-15 years). Method of Data Analysis The data retrieved was analysed using descriptive and inferential statistics. The descriptive statistics (percentage, mean and standard deviation) was used to analyse the demographic data of the respondents. Inferential statistics (t-test) was used to analyse the hypotheses at 0.05 level of significance. RESULTS Table 1 shows the demographic characteristic of the teachers who responded to the questionnaire about their perception of aggressive behavior among learners with moderate intellectual disability. Table 1: Teacher Demographics N Percentage Mean Std. Deviation Age range 87 4.26 1.985 below 24 years 10 11.5 25 - 29 years 11 12.6 30 -34 years 10 11.5 35 - 39 years 15 17.2 40 - 44 years 11 12.6 Table 1: Teacher Demographics 50 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 45 - 49 years 16 18.4 50 years and above 14 16.1 School type 87 1.56 .499 Private school Public school Gender Male Female 38 49 87 36 51 43.7 56.3 41.4 58.6 1.59 .495 Valid N (listwise) 87 Descriptive statistics of frequency counts and simple percentage was used to analyse the demographic data collected from the respondents. The analysis showed that 10 of the teachers (11.5%) were 24 and younger, 11 (12.6%) were 25–29, 10 (11.5%) were 30–34, 15 (17.2%) were 35–39, 11 (12.5%) were 40–44, 16 (18.4%) were 45–49, and 14 (16.1%) were 50 and above. This showed that most of the respondents were between 45 and 49. Furthermore, the table revealed that 38 (43.7%) of the teachers worked in private schools, while 49 (56.3%) were employed in public schools. Information based on gender distribution revealed that 51 (58.8%) of the respondents were female and 36 (41.4%) were male. This indicated that there were more females than males employed as teachers. Is teachers’ perception of physical aggression among learners with moderate intellectual disability significantly different? Table 2. One-sample t-test comparing the test assumed mean with the mean of teachers perception on items of physical aggression Test Value = 0 T df Sig. (2- tailed) Mean Difference 95% Confidence Interval of the Difference Lower Upper 47.115 86 .000 3.368 3.23 3.51 Is teachers’ perception of physical aggression among learners with moderate intellectual disability significantly different? Table 2. One-sample t-test comparing the test assumed mean with the mean of teachers perception on items of physical aggression Test Value = 0 T df Sig. RESULTS (2- tailed) Mean Difference 95% Confidence Interval of the Difference Lower Upper 47.115 86 .000 3.368 3.23 3.51 Table 2. One-sample t-test comparing the test assumed mean with the mean of teachers perception on items of physical aggression 51 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Table 2 shows the one-sample t-test comparing the test assumed mean with the mean of teachers’ perceptions on items of physical aggression (where t = 47.115, Mean Difference = 3.368, while p > .05). The result shows that teachers’ perceptions of physical aggression among learners with moderate intellectual disability is significant. Teachers’ perceptions of physical aggressive behavior among learners with moderate intellectual disability is significant. Is teachers’ perception of the developmental trend of aggressive behavior among learners with moderate intellectual disability significant? Table 3. One-sample t-test comparing the test assumed mean with the mean of teachers perception of developmental trend of aggressive behavior items Test Value = 0 T df Sig. (2- tailed) Mean Difference 95% Confidence Interval of the Difference Lower Upper 44.385 86 .000 3.115 2.98 3.25 Table 3. One-sample t-test comparing the test assumed mean with the mean of teachers perception of developmental trend of aggressive behavior items Table 3. One-sample t-test comparing the test assumed mean with the mean of teachers perception of developmental trend of aggressive behavior items Table 3 shows the one-sample t-test comparing the test assumed mean with the mean of teachers’ perceptions on items of the developmental trend of aggressive behavior, where (t= 44.385, Mean Difference = 3.115 while p > .05). The result shows that teachers’ perception of the developmental trend of aggressive behavior among learners with moderate intellectual disability is significant. Is there any significant difference in teachers’ perception of gender and types of aggressive behavior among learners with moderate intellectual disability? Is there any significant difference in teachers’ perception of gender and types of aggressive behavior among learners with moderate intellectual disability? Is there any significant difference in teachers’ perception of gender and types of aggressive behavior among learners with moderate intellectual disability? Table 4. Teachers’ perception of gender according to T-Test Results for aggressive behavior types Gender df Mean t F Sig. Developmental trend Male 2.97 -1.795 Female 85 3.22 -1.776 .997 .321 Physical Male 3.39 .330 Female 85 3.35 .320 3.628 .060 Socially unacceptable Male 2.97 .445 Female 85 3.22 .466 1.233 .270 Destructive Male 3.39 .082 Female 85 3.35 .085 .324 .571 Table 4. Teachers’ perception of gender according to T-Test Results for aggressive behavior types 52 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Table 4 shows no significant gender-based difference in verbal aggressive behavior (F = 0.997, p < .05). Nevertheless, the table also revealed that females had a mean of 3.22, which was higher than that of the mean of their male counterparts at 2.97, indicating that females had more verbal aggressive behavior than males. The hypothesis was, therefore, accepted. While Table 4 also shows no significant gender difference in physical aggressive behavior (F = 3.628, p < .05), males had a higher mean of 3.39 than that of their female counterparts at 3.35, indicating that males had more physical aggressive behavior than females. Therefore, the hypothesis is accepted. Table 4 also shows no significant gender difference in socially unacceptable aggressive behavior (F = 1.233, p < .05), however, females had a higher mean of 3.22 than their male counterparts with a mean of 2.97, indicating that females exhibited more socially unacceptable aggressive behavior. Therefore, the hypothesis is accepted. It was also revealed that while there was no gender difference in destructive behavior (F = 0.571, p > .05), Table 4 shows that males had a higher mean of 3.39 than their female counterparts, who had a mean of 3.35, indicating that males exhibited more destructive aggressive behavior than females. Is there any significant difference in teachers’ perception of gender and types of aggressive behavior among learners with moderate intellectual disability? Therefore, gender based aggressive behavior is not significantly different. Is there any significant difference between teachers’ perception of gender based aggressive behavior and school type among learners with moderate intellectual disability? Table 5. Perception of aggressive types According to T-Test Results for gender Table 5. Perception of aggressive types According to T-Test Results for gender Independent Samples Test Aggressive behavior School type df Mean t F Sig. Verbal Private schools 85 2.89 -2.813 .037 .847 Public schools 3.27 -2.709 Physical Private schools 85 3.42 .572 3.569 .062 Public schools 85 2.89 -2.813 .037 Socially unacceptable Private schools 3.27 .588 53 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Public school 85 3.42 .577 .583 .447 Destructive behavior Private schools 3.33 .218 Public school 85 2.89 .215 .805 .372 Table 5 shows that there was no significant difference in school type and verbal aggressive behavior (F = 0.037, p < .05). It also showed that public schools had a higher mean of 3.27 than their private school counterparts, which had a mean of 2.89, indicating that public schools had more verbal aggressive behavior than private schools. Table 5 showed no significant difference in school type in physical aggressive behavior (F = 3.569, p < .05). It was, however, revealed that private schools had a mean of 3.42 and public schools a mean of 2.89, indicating that private schools had more physical aggression than public schools. In addition, while no significant difference in socially unacceptable behavior by school type was found (F = 0.447, p < .05), public schools had a higher mean of 3.42 than that of their counterparts in private schools with a mean of 3.27. This indicated that public schools had more socially unacceptable aggressive behavior than private schools. Table 5 also revealed that there is no significant difference in school type in destructive aggressive behavior (F=0.805, p>.05). Private schools, however, had a higher mean of 3.33 than their public school counterparts with a mean of 2.89, indicating that private schools had more destructive aggressive behavior than public schools. Is there any significant difference in teachers’ perception of gender and types of aggressive behavior among learners with moderate intellectual disability? Therefore, gender based aggressive behavior is not significant with school type among adolescent with intellectual disability. DISCUSSION OF FINDINGS DISCUSSION OF FINDINGS The results show that teachers’ perception of physical aggression among learners with moderate intellectual disability is significant. This is consistent with the submission of Imhonde (2013), who argued that aggression could be exhibited in different forms among individuals, such as by physical, mental, or verbal manifestations. Dodge et al. (1990), asserted that children often have heightened sensitivity to rejection 54 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 derived from early experiences of physical abuse or emotional neglect, which then triggers anxiety or angry states. Aggression can also be expressed through both constructive and destructive behaviors (Poggenpoel & Myburgh, 2002). The reasons for this are that children often misread interpersonal cues and interpret ambiguous or prosaic communication as hostile and react aggressively. These findings align with the assertion of Manly et al. (2001), and Smith and Thornberry (1995) that physical abuse has repeatedly been linked to delinquency and interpersonal violence in adolescence. The result also reveals that teachers’ perception of the developmental trend of aggressive behavior among learners with moderate intellectual disability is significant. This is supported by the notion that victimization and perpetration of aggression have been repeatedly linked across multiple types of victimization (Maas et al., 2008). The character of children’s aggression changes dramatically with age. To explain these findings, one starts with the preschool period during which learners with moderate intellectual disability have not reached a level of cognitive and emotional development to achieve their goals using negotiation strategies or other less aggressive verbal methods of conflict resolution. By the time pupils reached school-going age, however, the expectation of researchers was that emotional self-regulation and language skills – which could be used in problem solving strategies – would already be developed. This was not so, which may be due to the pupils’ intellectual disability that had impacted on their cognitive development and their use of adaptive skills. There was no significant difference in teachers’ perception between gender and types of aggressive behavior among learners with moderate intellectual disability. DISCUSSION OF FINDINGS The reason for this, as argued by Crick (1996) and Crick and Grotpeter (1995), is that aggressive behaviors in girls have been overlooked because they are seen from a different perspective than those of their male counterparts. The finding does, however, align with 55 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 previous studies that failed to discern gender differences in aggressive behavior (Archer, 2004; Leenaars & Rinaldi, 2010). McClintock et al. (2003), disagree, finding that males were more likely than females to display aggression. There was no significant difference in teachers’ perception based on school type and aggressive behavior of learners with moderate intellectual disability. The findings do not align with the submission of Moore et al (2000), who noted that changes at certain points in the lives of persons with intellectual disability had negative outcomes for the children. This is because school type did not change the teachers’ perception of aggressive behavior among the study sample. Based on the findings it is, therefore, recommended that interventions to curb aggressive behavior in learners with moderate intellectual disability should cut across males and females as they are equally affected. Moreover, since learners with moderate intellectual disability in both public and private schools have the tendency to display aggressive behavior, appropriate management techniques should be adopted. CONFLICT OF INTEREST The authors confirm that there is no conflict of interest with respect to the data presented in this paper. FUNDING This work was supported by the South African Research Chairs Initiative of the Department of Science and Innovation and National Research Foundation of South Africa. South African Research Chair: Education and Care in Childhood: Faculty of Education: University of Johannesburg South Africa [grant number: 87300, 2017]. CONCLUSION This study concludes that there was significant difference between teachers’ perception and physical aggression among learners with moderate intellectual disability. The reason may be due to instability that this group of pupils generally experienced, both cognitively and emotionally. The study revealed that teachers’ perceptions of the developmental trend of aggressive behavior among learners with moderate intellectual disability is significant. It is, therefore, important to provide appropriate intervention focused on curbing aggressive behavior among learners with moderate intellectual disability. There was no significant gender difference in verbal aggressive behavior, physical aggressive behavior, socially unacceptable aggressive behavior, and 56 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 Jacob, U.S., Pillay, J., Oyefeso, E.O., (2021). TYPES OF AGGRESSIVE BEHAVIOR AMONG LEARNERS WITH MODERATE INTELLECTUAL DISABILITY IN NIGERIA AS PERCEIVED BY TEACHERS. Jurnal Tatsqif, 19 (1), 41-66. https://doi.org/10.20414/jtq.v19i1.3260 destructive behavior. Therefore, appropriate interventions must focus on both male and female learners with moderate intellectual disability. Also, it was shown that there was no significant difference in school type regarding verbal aggressive behavior, physical aggressive behavior, socially unacceptable behavior, and destructive aggressive behavior. School-based interventions to curb aggressive behavior must be provided at both public and private schools. ACKNOWLEDGMENTS The authors acknowledge the students who responded to their instruments and the authors whose materials they consulted. REFERENCES Andres, E., Kerling, F., Hamer, H., Kasper, B., & Winterholler, M. (2017). 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https://openalex.org/W1138184032	https://durham-repository.worktribe.com/preview/1434639/15031.pdf	English	null	The Muḍāraba Facility: Evolution, Stasis and Contemporary Revival	Arab law quarterly	2,015	cc-by	6,159	Muhammed-Shahid Ebrahim Durham University, UK Muhammed-Shahid Ebrahim Durham University, UK Mustapha Sheikh Leeds University, UK y , Q y ( ) 2 Mohammad O. Farooq, “Partnership, Equity-Financing and Islamic Finance: Whither Profit– Loss Sharing?”, Review of Islamic Economics 11 (2007): 68. The Muḍāraba Facility: Evolution, Stasis and Contemporary Revival Muhammed-Shahid Ebrahim Durham University, UK Mustapha Sheikh Leeds University, UK Abstract The age-old concept of partnership was seen by Muslim jurists from the 8th century Hijra onwards as a sacrosanct commercial arrangement – and, therefore, subjected to a form of fixity which is unparalleled in any other religious tradition. Since the formative period of Islamic law, the limited-liability partnership, or muḍāraba, a specific variation of the over-arching mushāraka partnership, has continued to hold central importance for Muslims. Yet, despite this centrality, it has not been examined with a view to reformulating it for contemporary Islamic banking and finance. This has led to its virtual neglect in modern Islamic banking operations. This article suggests that the revival of the muḍāraba facility requires the overcoming of key disadvantages inherent in its structure and that a restructuring on the basis of the hybrid facility called participating preferred ijāra is one possible way of achieving such an outcome. 1 Hamid Harasani, “Analysing the Islamic Prohibition on Ribā: A Prohibition on Substance or Form?”, Arab Law Quarterly 27 (2013): 289–296; Muhammad M. Iqbal, “Prohibition of Interest and Economic Rationality”, Arab Law Quarterly 24 (2010): 293–308. 1 Hamid Harasani, “Analysing the Islamic Prohibition on Ribā: A Prohibition on Substance or Form?”, Arab Law Quarterly 27 (2013): 289–296; Muhammad M. Iqbal, “Prohibition of Interest and Economic Rationality”, Arab Law Quarterly 24 (2010): 293–308. 2 Mohammad O. Farooq, “Partnership, Equity-Financing and Islamic Finance: Whither Profit– Loss Sharing?”, Review of Islamic Economics 11 (2007): 68. Keywords Keywords muḍāraba, mushāraka, sunna, rabb al-māl, participating preferred ijāra 3 Ibid. 4 Taqi Usmani, An Introduction to Islamic Finance (The Hague: Kluwer Law International, 2002). 5 Supra note 2. 6 See Muhammad Taqi Usmani, “The Concept of Musharakah and Its Application as an Islamic Method of Financing”, Arab Law Quarterly 14 (1999): 203–220. 7 Abraham L. Udovitch, “Partnership in Medieval Islamic Law’‘, PhD Dissertation, (Yale University, 1965). 8 Supra note 6 at 205. Introduction 1 Since their appearance in the 1970s, Islamic financial institutions (IFI) have been fixated, at least theoretically, on financial transactions which are designed to conform to the profit-loss sharing mode (PLS). The reason for this is two-fold: firstly, they are determined to adhere to the view of Muslim orthodoxy, which holds that interest is akin to the prohibited ribā and therefore is absolutely prohibited;1 secondly, they seek to differentiate themselves from their conventional counterparts as “Islamic”.2 Furthermore, Farooq describes IFIs as having idealised the PLS mode because of their determination to avoid debt- financing and use partnership and equity-financing, similar to venture 2 2 capitalism.3 This idealisation has its roots in classical Muslim jurisprudence, which gave centrality to two principal modes of PLS contract practiced by pre- Islamic Arabs and the early Muslims: shirka/mushāraka (partnership) and muḍāraba (limited liability partnership). According to Taqi Usmani, the globally renowned Ḥanafī scholar and pioneer of Islamic Banking in Pakistan, these are the “real and ideal instruments of financing in Sharīʿah”.4 Paradoxically, while the pertinent literature in the Islamic Banking and Finance (IBF) movement continues to emphasise PLS as the main financial mode (and simultaneously continues to idealise mushāraka and muḍāraba), in practice IFIs have deliberately and systematically avoided them, as Farooq has shown.5 This is unsurprising since the parameters for the PLS modes in classical Islamic law are not well-thought out and therefore have little appeal to investors/lenders. This article will highlight the aforementioned parameters with specific regard to the muḍāraba facility and suggest ways of restructuring it based on Islamic legal theory. It is hoped that the outcome will imply that the modified forms of PLS financing can once again take their place as central in IBF. Consistent with the study of Usmani,6 this article is divided into two parts: the first part undertakes a historical evaluation of mushāraka, which is a useful preamble to understanding muḍāraba, a subsidiary financial arrangement. Thereafter, we commence with a study of muḍāraba as a central financial instrument in the history of Islamic commercial law. Here we discuss the historical precedent of this financial instrument along with how the gradual onset of rigidity in Islamic legal reasoning might have been a central factor in jeopardising the underlying real economy in the Muslim world. Introduction The final section elaborates on one of the ways to revive the economy of the Muslim world – the adaptation of the muḍāraba facility to a hybrid form. 2 3 In the era of the Prophet (PBUH), and for several generations after his death (considered the golden-age of Islam, including the period of the Rightly-Guided Caliphs and the Successors), Arab society was commercially savvy, apparently employing several forms of mushāraka structures, as described in the fiqh literature: (1) Partnership in ownership (shirkat al-milk), a basic form of partnership ensuing when two or more individuals jointly own a particular asset without a commercial aim. This joint ownership may take place at their discretion. For example, if two persons jointly buy a car, their relationship is termed as shirkat al-milk. In other cases, the joint ownership may compulsorily ensue without any action taken by either parties, for example, if two persons inherit a property jointly, they take ownership automatically. (2) Partnership by contract (shirkat al-ʿaqd) is a form of partnership undertaken by two or more persons for commercial purposes. This form of partnership can be further categorised into: (i) Shirkat al-amwāl, where two or more persons contribute capital in a commercial enterprise; (ii) Labour partnership (shirkat al-ʿabdān), where two or more persons undertake to deliver some services to their customers and share the income generated; (iii) Credit partnership (shirkat al-wujūh), where the partners provide neither capital nor labour to invest; they buy goods or services on credit for the purpose of selling them and distributing the profit in accordance with the ratio of individual liabilities;9 and (iv) Silent partnership (muḍāraba), where one party (the silent partner) provides funding while the other party (entrepreneur) provides labour. Profits are distributed in accordance with an agreed-upon ratio, where: (1) all monetary losses are borne by the silent partner; while (2) the entrepreneur loses his labour and time. It is unusual that the mushāraka facility should have acquired a quasi-divine status given that simple partnership forms were not an Islamic innovation, but rather existed in Arabia in the pre-Islamic era.10 In fact, we know from historical sources that partnerships were known and practiced in the Near East at least since the Babylonians, and they were discussed in the Talmud and in the Corpus Juris Civilis of Justinian.11 Yet Muslims are generally nostalgic about the mushāraka facility. Clearly it played an important role in the economy in early Islamic history, but this cannot account for the post-formative and contemporary fetish. The most plausible explanation is the attitude toward mushāraka expressed by Muslim jurists. 9 Mahmoud El-Gamal, Islamic Finance: Law, Economics, and Practice (New York: Cambridge University Press, 2006). 10 Supra note 7 at 7–8. 11 Ibid. 9 Mahmoud El-Gamal, Islamic Finance: Law, Economics, and Practice (New York: Cambridge University Press, 2006). 10 S 7 7 8 10 Supra note 7 at 7–8. Mushāraka Mushāraka (sharika, partnership) was a pre-Islamic organisational form of contract which by the 3rd century Hijra (9th century CE) became islamicised through its integration into the fiqh literature. In essence, it allowed the pooling of resources of economic agents in the form of cash, goods, skills, or a combination of the same,7 and was akin to a private equity firm in the context of conventional finance. The term sharika is generally employed in Islamic jurisprudential literature more so than the term mushāraka, the latter being a neologism recently introduced in the area of Islamic finance.8 Since mushāraka constitutes an equity contract, its payoffs are based on profit and loss sharing. This is in contrast to rigid debt contracts, i.e., ribawī contracts, which are prohibited in Islam. 3 For example, the 13th century Ḥanafī 4 jurist, al-Maḥmūd b. Mawdūd al-Mūṣilī (d. 1284), who cites in his commentary the following Prophetic traditions: The hand of God is above two partners so long as none of them betrays his partner; if one of them betrays his partner, [God] withdraws His hand; God is the third in a partnership of two as long as they do not betray one another; if they betray one another, the grace is erased.12 He also presents the Prophet’s involvement in a partnership with Qays b. Sā’ib as a normative act, following which he proclaims, The Prophet was sent at a time when people engaged in partnerships; he did not censure them from engaging in this arrangement and so it has been employed until our day. That there exists no censurer indicates the community’s consensus.13 It is unclear whether the consensus al-Mūṣilī invokes relates to mere permission to engage in a partnership based on customary practice or whether it relates to some sort of divine sanction. It seems that Muslims understood it to be the latter, since there is an extensive literature ensuing from both classical and contemporary jurists on the merits of mushāraka contracts. A question discussed in the modern Western literature is whether the exposition of mushāraka in the fiqh literature is sufficient proof that the instrument was practically employed by Muslims. For some decades the dominant view among Western scholars of Islamic Law has been that the fiqh had very little to do with actual practice. Fiqh was considered by these scholars to be only of theoretical significance, and was developed by the jurists according to a paradigm which they considered represented the golden age of Islam. According to these scholars, to assume that the fiqh literature reflected the institutions and practice in classical Muslim societies is perilous. Yet we have started to see a more nuanced approach to this subject, with Udovitch among those advancing a different hypothesis on the question of theory and practice. Udovitch has highlighted that: While recognising the ideal character of Islamic law, one cannot state a priori that any given institution had no relationship whatsoever to practice. This is especially so in the area of fiqh termed muʿāmalāt. Most of the material covered by these laws, for example, the contracts of partnership and commenda, does not involve any religious or moral principle. 12 Al-Maḥmūd b. Mawdūd al-Mūṣilī, Al-Ikhtiyār li-taʿlīl al-mukhtār (Beirut: Dār al-Arqam bin Abī al-Arqam, reprinted in 1999). 13 Ibid., p. 19. 14 Supra note 7. q 13 Ibid., p. 19. 15 Ibid., p. 13. 16 Timur Kuran, The Long Divergence. How Islamic Law Held Back The Middle East (Princeton: Princeton University Press, 2011), 60. 17 Ibid. 5 The chapters on partnership and commenda contain numerous instances in which systematic legal reasoning is suspended because of the ‘custom of the merchants’ or ‘because of the needs of the merchants.’ Other applications of juristic preference (istiḥsān), although not coupled with these phrases, reveal a clear tendency toward allowing a greater freedom of trade practice. In the later legal treatises, this leniency, which often provides valuable indications of actual practice, is replaced by imitation and rigidity.15 It has been highlighted above that mushāraka became enshrined within the Islamic legal literature as a result of a broader process of islamicisation which integrated within the Islamic legal system many cultural and customary practices that were prevalent at the birth of Islam. Furthermore, the fact that classical Muslim jurists were, in some ways, jacks of all trades, they perpetuated the idea that mushāraka was divinely ordained in their legal compendia. Kuran says: It has been highlighted above that mushāraka became enshrined within the Islamic legal literature as a result of a broader process of islamicisation which integrated within the Islamic legal system many cultural and customary practices that were prevalent at the birth of Islam. Furthermore, the fact that classical Muslim jurists were, in some ways, jacks of all trades, they perpetuated the idea that mushāraka was divinely ordained in their legal compendia. Kuran says: For at least a half-millenium after the birth of Islam, then, Islamic partnership law was adopted by peoples located in far corners of the world as the institutional basis for commercial cooperation […] Islamic partnership law presented limitations even by medieval standards. Most of its variants required a partnership’s principal to consist of currency; they prohibited investing merchandise directly, ostensibly to prevent unjust enrichment, more plausibly to forestall conflicts over the value of the initial investment and the division of profits. Moreover, the merchant’s mission was incomplete until he reconverted all merchandise bought on behalf of the partnership into the selected currency. When these rules were followed, they could drive partners to sell merchandise, or trade currencies, at an inopportune time or place.16 Kuran is bemused as to why modern Islamists want to restructure economies according to such rules: In truth, by modern standards Islamic partnerships are very simple organizations. They are meant to support ventures of finite duration, not to open-ended ventures without an expected settlement date. 3 No religious or ethical value is attached to them. In the earliest legal texts especially, there is no reason not to consider them as a reflection and partial description of the institutions as they existed at that time.14 Furthermore, according to Udovitch: Furthermore, according to Udovitch: 18 Joseph Schacht, “Pre-Islamic Background and Early Development of Jurisprudence: The Schools of Law and Later Developments of Jurisprudence", in Majid Khadduri and Herbert J. Liebesny (Eds.), Law in the Middle East, Vol. 1 (Washington, DC: Middle East Institute, 1955), 76–77. 19 See Andrew Winton, “Limitation of Liability and the Ownership Structure of the Firm", Journal of Finance 48 (1993): 487–512; Timur Kuran, “The Logic of Financial Westernization in the Middle East", Journal of Economic Behavior and Organization 56 (2005): 593–615. 20 Supra note 7. 20 Supra note 7. 21 See Bernard Jackson, Jewish Law in Legal History and the Modern World (Leiden: Brill, 1980). 5 They are poorly suited to projects requiring a huge sunk investment and delivering returns over many years. Because they lack legal personhood, before the law their members deal with third parties as individuals, rather than employees of a firm.17 Despite the extensive efforts of the scholars, the onset of modernity has handicapped them. This is because the venturing of traditionally trained jurists into spheres of human life which demand technical knowledge has implications 6 on the development of Muslim societies. The economic decay of the Muslim world is described by Schacht as follows: Islamic law, which until the early ʿAbbāsid period had been adaptable and growing, from then onwards became increasingly rigid and set in its final mould ... . It was not all together immutable, but the changes which did take place were concerned more with legal theory and the systemic superstructure than with positive law ... . Taken as a whole, however, Islamic law reflects and fits the social and economic conditions of the early ʿAbbāsid period, but has become more and more out of touch with later developments of state and society.18 To elaborate the above further, we state that the mushāraka form has failed to evolve from the rudimentary stage of its inception in the medieval era to a form such as the corporate one which endows it the following comparative advantages: (i) limited liability; (ii) unlimited life; (iii) ease of transferability (liquidity) of shares (ensuing from a secondary market); and centralized management.19 Undoubtedly, this has had a corollary effect in the subsidiary form, known as muḍāraba. Following this overview of mushāraka, the over-arching partnership form in Islamic jurisprudence, our discussion can now move to muḍāraba. 7 Muḍāraba is a derivative of the Arabic infinitive, ḍarb (travel), as in the expression ḍarb fī l-arḍ, “to travel the land”. It is called muḍāraba because the agent earns his percentage of a venture’s profits as a consequence of the effort and labour he expends.23 The term muḍāraba is of Iraqi origin, and was the preferred term of Ḥanafī jurists to describe this particular form of arrangement because of its close semantic connection with the Qur’ānic verse, “... And others travelling (yaḍribūna) through the land (for the purposes of trade)”.24,25 Jurists of the Mālikī and Shāfiʿī Schools of Law refer to this contract as muqāraḍa (lit. “to surrender the profits”). According to the 11th century Ḥanafī jurist, al-Sarakhsī, the legality of this form of contract is known via the normative practice of the Prophet (sunna) and the consensus of the early Muslims (ijmāʿ). He cites several traditions in support of muḍāraba, the first two of which are given here: The practice of ʿAbbās ibn ʿAbd al-Muṭṭalib when he engaged in a muḍāraba arrangement was to stipulate in the contract that the agent (muḍārib) should not travel overseas or descend on a valley. If the agent failed to comply with this stipulation, ʿAbbās would demand compensation in the event of a loss. The news of this reached the Prophet, who deemed it acceptable. Another tradition tells of the two sons of ʿUmar, ʿAbd Allāh and ʿUbayd Allāh, who once came to stay with Abū Mūsā in Iraq. After lamenting the fact that he did not have any savings from which he could support the two men, he offered them instead some money from the treasury (bayt al-māl). He advised them to trade with the money and then, on their return to Medina, return the capital to the Leader of the Faithful (amīr al- muʾminīn) and retain the profits for themselves. When ʿUmar heard of the arrangement, he protested that the capital was from the public treasury and so any profits generated from it should be returned to the public fund. It was only after some companions advised ʿUmar to consider his two sons as agents (muḍārib), whereby they were to take half of the profits and the other half is returned to the treasury, that ʿUmar was willing to acquiesce.26 The remaining evidences which al-Sarakhsī furnishes in support of muḍaraba are of a similar ilk to these aforementioned traditions. 23 Shams al-Dīn Abū Muḥammad al-Sarakhsī (d. 945), Kitāb al-Mabsūṭ (Beirut: Dār al-Fikr, 2000), 17. 24 See Q73:20. 25 Supra note 23. 26 Ibid. 27 See Monzer Kahf and Tariqullah Khan, “Principles of Islamic Financing: A Survey", Research Paper No. 16, Islamic Research and Training Institute, Islamic Development Bank, Jeddah, Saudi Arabia (1992); M. Nejatullah Siddiqi, Partnership and Profit Sharing in Islamic Law (Leicester: The Islamic Foundation, 1985). Muḍāraba 3 Muḍāraba is a limited liability contract between a principal (ṣāḥib al-māl or rabb al-māl) and an agent (muḍārib), which constitutes a partnership of capital and entrepreneurship. The principal advances funds to an agent to be employed in a particular project, in return for a share in the profit (in a mutually pre- agreed ratio). The losses, if incurred, are borne only by the principal. This financial arrangement is a specific form of partnership (shirka or mushāraka), where capital owners (unable to participate in a trade) team up with those who have the necessary business skills (but are deficient in capital). This financial arrangement was known and practised in the Near East as early as the Babylonians,20 known and discussed in the Talmud and later Rabbinical literature,21 and its various forms were practiced by Meccan merchants preceding Islam,22 which explains its employment in early Islam. 7 Interestingly, in common with other jurists of the classical period, al-Sarakhsī made no attempt to provide a rational justification for this form of contract. Muḍāraba was probably employed because it (i) circumvented the problem of ribā and (ii) allowed the investor to overcome the problem of having to precisely fix a wage in an arrangement which was essentially the provision of capital in exchange for labour. These advantages would have been factors which led the Prophet Muhammad (PBUH) himself and his eminent companions to employ this facility in trade, either as a muḍārib or as a rabb al-māl.27 Yet there remained the inherent inefficiency associated with this arrangement, since the rabb al-māl was required to provide 100% of the capital, whilst at the same time 8 8 being the sole bearer of losses. This naturally created an incentive (agency) issue, as the muḍārib had none of his capital at stake.28 Another form of inefficiency was the denial of any form of control rights to the rabb al-māl. Both of these problems explain why the rabb al-māl tended to stipulate certain restrictions on the muḍārib, as can be seen in the above case of cAbbās, uncle of the Prophet, and in the long-term almost certainly led to the decline in the employment of muḍāraba as an instrument of financial intermediation. Despite its disadvantages, the Muslim schools of law generally endorsed the application of muḍāraba in commerce, with Ḥanafī jurists taking a more liberal stance in contrast to the more conservative Shāfiʿī, Mālikī and Ḥanbalī jurists, who advocated its employment only in trade, where hired labour was not feasible.29 For the latter schools, as explained by Hasanuz-Zaman,30 muḍāraba defied both the general law of hire as well as ran afoul of several Ḥadīths – ‘Profit goes with liability’ (al-kharāj bi-ḍ-ḍamān) and ‘… (no) profits without liability’ (… lā ribḥ mā lā yuḍman) both of which form the basis of the well- known legal maxim ‘profits are concomitant to risk’ (al-ghunum bi-l-ghurum) – since the working partner is entitled to profits without having to bear any risk. As for its defiance of the general laws of hire, Hasanuz-Zaman elaborates: Muḍārabah is a relationship between capital and labour in which the former utilizes the services and skill of the latter in return for a share in expected profits. Thus, it is essentially a contract of hire/ wage. p 32 See Siddiqi, Partnership and Profit Sharing; M. Umar Chapra, “Why has Islam prohibited interest? Rationale behind the prohibition of interest", in A.S. Thomas (Ed.), Interest in Islamic Economics: Understanding Ribā (London, New York: Routledge, 2006). g ( g ) 3 Mohammed Uzair, Interest-Free Banking (Karachi: Royal Book Company, 1978). 28 On this, see Humayon A. Dar and John R. Presley “Lack of Profit-Loss Sharing in Islamic Banking: Management and Control Imbalances", International Journal of Islamic Financial Services 2 (2000): 3–18. 29 See Syed M. Hasanuz-Zaman, “Muḍārabah in non-trade operations’‘, Journal of King Abdul Aziz University: Islamic Economics 2 (1990): 69–88. 30 Ibid p 81 31 Ibid. p. 70. 29 See Syed M. Hasanuz-Zaman, “Muḍārabah in non-trade operations’‘, Journal of King Abdul Aziz University: Islamic Economics 2 (1990): 69–88. b d 28 On this, see Humayon A. Dar and John R. Presley “Lack of Profit-Loss Sharing in Islamic Banking: Management and Control Imbalances", International Journal of Islamic Financial Services 2 (2000): 3–18. 7 But according to the basic rules of the Sharīʿah based on the Prophet's saying, a contract of hire/wage should precisely lay down the amount of hire/wage to be paid to the worker, failing which the contract becomes voidable and therefore the worker will have to be paid standard wage (ajr al-miṯl). In a contract of muḍārabah, on the other hand, the condition of precise fixation of wage to the worker does not exist. Thus the analogy of the law of wage demands that muḍārabah should be held unlawful. But the holy Prophet, in supersession of this rule, exempted this contract from the purview of the law of hire.31 The rather more liberal approach of the Ḥanafīs is the view promoted by the Jamāt-e-Islāmī, a political Islamist group of the Indo-Pak subcontinent.32 Its employment in the Islamic financial intermediation as a double muḍāraba is advanced by Uzair.33 In the first tier, the Islamic bank acts as a muḍārib, while the depositors serve as the rabb al-māl. In the second tier, the bank serves as a rabb-al-māl, thereby trusting an entrepreneur to conduct his/her business venture. Profits, if any, are shared between them, and then between the bank 9 9 and depositor. This structure is not observed in practice and is contested by the Traditionalist jurists (along with some Ḥanafīs), as elaborated below. 34 Murat Çizakça, A Comparative Evolution of Business Partnerships: The Islamic World and Europe, with Specific References to the Ottoman Archive (New York: Brill 1996). 35 Ibid. d h 34 Murat Çizakça, A Comparative Evolution of Business Partnerships: The Islamic World and Europe, with Specific References to the Ottoman Archive (New York: Brill 1996). 35 Ibid. 36 See Hasanuz-Zaman, supra note 29 at 70; Udovitch, supra note 7. 37 Supra note 18 at 76–77. 38 See Timur Kuran, “Why the Middle East is Economically Underdeveloped: Historical Mechanisms of Institutional Stagnation”, Journal of Economic Perspectives 18 (2004): 71– 90. p 38 See Timur Kuran, “Why the Middle East is Economically Underdeveloped: Historical Mechanisms of Institutional Stagnation”, Journal of Economic Perspectives 18 (2004): 71– 90. 36 See Hasanuz-Zaman, supra note 29 at 70; Udovitch, supra note 7. 37 Supra note 18 at 76–77. 36 See Hasanuz-Zaman, supra note 29 at 70; Udovitch, supra note 7. 39 Supra note 28 at 3. 40 See M. Shahid Ebrahim and Mustapha Sheikh, “Debt Instruments in Islamic Finance", Working Paper, Durham Business School (2014). 41 See Q2:280 39 Supra note 28 at 3. 40 See M. Shahid Ebrahim and Mustapha Sheikh, “Debt Instruments in Islamic Finance", Working Paper, Durham Business School (2014). 41 See Q2:280. 42 Supra note 28. 43 Ibid. 44 Ibid. 45 See Q2:275, Q2:278-279, Q2:281, Q3:130-132, Q4:161 and Q30:39. 42 Supra note 28. Muḍāraba and the Economic Decline of the Muslim World Nearly all Islamic banks, investment companies, and investment funds offer trade and project finance on mark- 10 up, commissioned manufacturing, or on leasing bases. PLS (i.e., Profit Loss Sharing based on muḍārabah or mushārakah) features marginally in the practice of Islamic banking and finance. up, commissioned manufacturing, or on leasing bases. PLS (i.e., Profit Loss Sharing based on muḍārabah or mushārakah) features marginally in the practice of Islamic banking and finance. Whatever is the degree of success of individual Islamic banks, they have so far failed in adopting PLS-based modes of financing in their business. Even specialised Islamic firms like Mudārabah Companies (MCos) in Pakistan, which are supposed to be functioning purely on a PLS basis, have negligible proportion of their funds invested on a muḍārabah or mushārakah basis. According to the International Association of Islamic Banks, PLS covered less than 20 percent of investments made by Islamic banks world-wide (1996 figures). Likewise, the Islamic Development Bank (IDB) has so far not used PLS in its financial business except in a few small projects.”39 In general, financial economists highlight the following advantages and disadvantages of the muḍāraba facility: In general, financial economists highlight the following advantages and disadvantages of the muḍāraba facility: In general, financial economists highlight the following advantages and disadvantages of the muḍāraba facility: Advantages: – It avoids fragility endowed in pure debt (or even traditional “Islamic” debt facilities of murābaḥa (cost plus mark-up), tawarruq (tripartite sale) and qarḍ ḥasan (benevolent loan).40 This enhances the robustness of the financial system and is consistent with the Qurʾānic injunction of giving respite to borrowers in times of difficulty.41 4 Ibid. 5 See Q2:275, Q2:278-279, Q2:281, Q3:130-132, Q4:161 and Q30:39. Muḍāraba and the Economic Decline of the Muslim World 4 Çizakça illustrates how the muḍāraba was adapted by the Italian merchants (trading with their Arab counterparts) in the form of partnership termed as ‘commenda’.34 Further adaption of this facility is said to have climaxed in the form of a convertible preferred stock employed by venture capitalists in Silicon Valley in California.35 The adaption of the muḍāraba from its classical structure, however, was precluded in the Muslim world due to constraints imposed by fiqh. The issue for the majority of jurists was that a thing established contrary to legal analogy (qiyās) cannot be used as an analogy for other things; since muḍāraba superseded basic rules of law and was in defiance of analogy, it could only be legalised on the grounds of social and economic necessity. It therefore had to suffer a legal limitation.36 In keeping with other financial instruments, and other aspects of Islamic law, muḍāraba remained undeveloped. As Schacht says: Islamic law, which until the early ʿAbbāsid period had been adaptable and growing, from then onwards became increasingly rigid and set in its final mold … . It was not all together immutable, but the changes which did take place were concerned more with legal theory and the systemic superstructure than with positive law…. Taken as a whole, however, Islamic law reflects and fits the social and economic conditions of the early ʿAbbāsid period, but has become more and more out of touch with later developments of state and society.37 It is this rigidity in Islamic law that led in part to the deterioration of the early Islamic financial instruments, institutions and markets, leading to the eventual economic decline of the Muslim world.38 It is this rigidity in Islamic law that led in part to the deterioration of the early Islamic financial instruments, institutions and markets, leading to the eventual economic decline of the Muslim world.38 It is therefore imperative to investigate the structure of Islamic financial intermediaries to realize what has gone awry with muḍāraba and how it can be revived. Dar and Presley state the following on the applicability of the muḍāraba in the real world. “Almost all theoretical models of Islamic banking are either based on muḍārabah or mushārakah or both, but to-date actual practice of Islamic banking is far from these modes. 39 Supra note 28 at 3. 46 See M. Shahid Ebrahim, Murizah O. Salleh and Mustapha Sheikh, “Rationalising Hybrid Financial Instruments from an Uṣūlī Perspective", Arab Law Quarterly 28 (2014). 47 Ibid arrangement to a requisite degree because he is made to bear all the risk, while the muḍārib, in contradistinction, bears no risk whatsoever. arrangement to a requisite degree because he is made to bear all the risk, while the muḍārib, in contradistinction, bears no risk whatsoever. arrangement to a requisite degree because he is made to bear all the risk, while the muḍārib, in contradistinction, bears no risk whatsoever. 48 See Q2:280 49 See M. Shahid Ebrahim, “Integrating Islamic and Conventional Project Finance", Thunderbird International Business Review 41 (1999): 583–609. 50 Supra note 46. Disadvantages: Disadvantages: – A restrictive requirement set by the dictates of the fiqh literature defines the sole responsibility of the rabb al-māl of providing 100% capital and solely being subject to losses. This constriction from creates an incentive (agency) issue, as the muḍārib has none of his/her capital at stake.42 – Another form of inefficiency endowed by fiqh is in denying any form of control rights to the rabb al-māl.43 – Muḍāraba suffers from the usual consequences of ex-ante and ex- post asymmetric information (i.e., adverse selection and moral hazard, respectively).44 It needs mechanisms to overcome these two problems. It is sometimes suggested that muḍāraba is advantageous insofar as it adheres to the Qurʾānic injunction of avoiding expropriation of a counterparty’s assets.45 The reality is that the rabb al-māl is not safeguarded in a classical muḍāraba 44 Ibid. 45 See Q2:275, Q2:278-279, Q2:281, Q3:130-132, Q4:161 and Q30:39. 48 See Q2:280 pp p y y , Q y ( ) 53 See Ronald H. Coase, “The Nature of the Firm", Economica (4) New Series (16) (1937): 386–405; and Armen A. Alchian, “Uncertainty, Evolution and Economic Theory", Journal of Political Economy 58 (1950): 211–221. 6 Tāha Jābir al-Alwānī, “The Role of Islamic Ijtihād in the Regulation and Correction of Capital Markets", American Journal of Islamic Social Sciences 14(3) (1997): 39–66. y ( ) 54 This is elaborated in M. Shahid Ebrahim and Ike Mathur, “On the Efficiency of the UPREIT Organizational Form: Implications for the Subprime Crisis and CDO'S", Journal of Economic Behavior and Organization 85 (2013): 286–305. p 52 See Abdulmajeed H. Bello, “Dynamic Approach to the Shari‘ah: A Case Study of Ijtihād and its Application in Contemporary Society”, Arab Law Quarterly 27 (2013): 259–270. g ( ) 55 Merton H. Miller, “Debt and Taxes’‘, Journal of Finance 32 (1977): 261–275. 51 Supra note 29 at 85. Reviving the Muḍāraba 5 The disadvantages of the muḍāraba (as stated above) necessitate its revival in spirit in the form of a hybrid facility called participating preferred ijāra (PPI).46 A PPI is structured by merging a special ijāra (leasing) facility with a muḍāraba one.47 A preferred ijāra is akin to a preferred stock but backed by leases whose payoffs are capped and reduced. Through this special ijāra component, investors are bestowed with limited income in return for a share in the risk of the project. This feature of sharing in the risk cures the preferred ijāra of the fragility endowed in its regular counterpart, making it compliant with the Qurʾānic injunction of giving respite to borrowers in times of difficulty.48 In contrast, the muḍāraba component bestows a share in operating income along with any appreciation at the terminal stage and mitigates fragility of the venture. The resulting hybrid facility (i.e., PPI) is basically a true Profit and Loss sharing one without the major disadvantages of the muḍāraba, as elaborated below. It confers the following benefits: – It is extremely malleable into family of financial instruments catering to the needs of a diverse clientele. That is, it ranges from a pure income bond (i.e., muqāraḍa bond) to a pure growth facility (like the classic muḍāraba).49 ) – The financier: (i) is ranked ahead of the owner of the firm (or project); and (ii) subsidises a 'capped' fixed portion (from the ijāra component of the facility) in return for a 'proportion' (or a fraction) of payoffs in the operating or terminal states of the economy (or both). – It resolves adverse selection, as it is asset-backed. – It alleviates the fragility of the financial system. This enhances the robustness of the financial system. – It allows an investor to retain control of the firm even in the good state of the economy, unlike convertible debt, where the conversion to equity dilutes these control rights. A PPI, however, needs to be priced to avoid expropriation of the assets of a counterparty. It also needs a mechanism to overcome moral hazard.50 A PPI, however, needs to be priced to avoid expropriation of the assets of a counterparty. It also needs a mechanism to overcome moral hazard.50 51 Supra note 29 at 85. 52 See Abdulmajeed H. Bello, “Dynamic Approach to the Shari‘ah: A Case Study of Ijtihād and its Application in Contemporary Society”, Arab Law Quarterly 27 (2013): 259–270. 53 See Ronald H. Coase, “The Nature of the Firm", Economica (4) New Series (16) (1937): 386–405; and Armen A. Alchian, “Uncertainty, Evolution and Economic Theory", Journal of Political Economy 58 (1950): 211–221. 54 This is elaborated in M. Shahid Ebrahim and Ike Mathur, “On the Efficiency of the UPREIT Organizational Form: Implications for the Subprime Crisis and CDO'S", Journal of Economic Behavior and Organization 85 (2013): 286–305. 55 Merton H. Miller, “Debt and Taxes’‘, Journal of Finance 32 (1977): 261–275. 56 Tāha Jābir al-Alwānī, “The Role of Islamic Ijtihād in the Regulation and Correction of Capital Markets", American Journal of Islamic Social Sciences 14(3) (1997): 39–66. 57 Supra note 46. 6. Conclusion Hasanuz-Zaman has argued that the importance of the muḍāraba facility in Islam has been overstated: “Shirkah and Muḍārabah are not sacrosanct nor are they end-all of financial or business relationship”.51 While the authors are minded to agree with this assessment, the reality on the ground runs counter to this; in fact, recent history demonstrates the widespread call from various Muslim quarters for a renewed implementation of these facilities, particularly the muḍāraba, in their classical form. Yet the failure to adapt facilities such as the muḍāraba has highlighted the uneasy relationship between intellectual rigidity and economic malaise, with consequences which have for centuries had a crippling effect on Muslim economies. This article has highlighted that static and flawed ijtihād (interpretation or deduction of the divine sources of law) stemming from the rigid rules of taqlīd (blindly imitating religious authority) has played a significant role in the perennial underdevelopment of the Muslim world. Muslim jurists failed to undertake a dynamic ijtihād52 by adapting organisational forms which would have allowed businesses to competitively deliver the products demanded by their customers at the lowest cost by mitigating transaction costs.53 Thus there is therefore a need to develop these organizational forms in conjunction with optimal financial instruments,54 a technical design based on the seminal study of Miller,55 which would link organizational forms with their underlying capital structure. In summary, there is an exigency to overhaul the fiqh literature developed in the classical period through the employment of a joint ijtihād (juristic interpretation), that galvanises the efforts of various experts, as encouraged by al-Alwānī.56 It is hoped that this paper demonstrates how this might be achieved vis-à-vis the classical muḍāraba facility, and its restructuring along the lines of a participating preferred ijāra.57
https://openalex.org/W4361873390	https://figshare.com/articles/journal_contribution/Supplementary_Figure_2_from_KLF5_Regulates_the_Integrity_and_Oncogenicity_of_Intestinal_Stem_Cells/22400129/1/files/39845885.pdf	Pashto, Pushto	null	Supplementary Figure 3 from KLF5 Regulates the Integrity and Oncogenicity of Intestinal Stem Cells	null	2,023	cc-by	114	Supplementary Figure S2. TA Lgr5 Cre+ Klf5∆/∆(Tamoxifen) vs Lgr5 Cre+ Klf5flox/flox (Oil) Stem Lgr5 Cre+ Klf5∆/∆(Tamoxifen)vs Lgr5 Cre+ Klf5flox/flox (Oil) Lgr5 Cre+ Klf5 ∆/∆(Tamoxifen) Lgr5 Cre+ Klf5 flox/flox (Oil) Lgr5 Cre+ Klf5∆/∆(Tamoxifen) Lgr5 Cre+ Klf5 flox/flox (Oil) g 5 C e lf5 (O ) g ( ) Lgr5 Cre r Klf5 K (Oil) A B Stem Lgr5 Cre+ Klf5∆/∆(Tamoxifen)vs Lgr5 Cre+ Klf5flox/flox (Oil) Lgr5 Cre+ Klf5 ∆/∆(Tamoxifen) Lgr5 Cre+ Klf5 flox/flox (Oil) g 5 C e lf5 (O ) g ( ) A TA Lgr5 Cre+ Klf5∆/∆(Tamoxifen) vs Lgr5 Cre+ Klf5flox/flox (Oil) Lgr5 Cre+ Klf5∆/∆(Tamoxifen) Lgr5 Cre+ Klf5 flox/flox (Oil) Lgr5 Cre r Klf5 K (Oil) B A B Lgr5 Cre+ Klf5 flox/flox (O
https://openalex.org/W4322495720	https://www.nature.com/articles/s41598-023-29077-y.pdf	English	null	Development of controlled nanosphere lithography technology	Scientific reports	2,023	cc-by	11,023	Development of controlled nanosphere lithography technology Artem A. Osipov 1,2, Alina E. Gagaeva 1, Anastasiya B. Speshilova 1, Ekaterina V. Endiiarova 1, Polina G. Bespalova 1, Armenak A. Osipov 2, Ilya A. Belyanov 1, Kirill S. Tyurikov 1, Irina A. Tyurikova 1 & Sergey E. Alexandrov 1 OPEN This work is devoted to the development of nanosphere lithography (NSL) technology, which is a low-cost and efficient method to form nanostructures for nanoelectronics, as well as optoelectronic, plasmonic and photovoltaic applications. Creating a nanosphere mask by spin-coating is a promising, but not sufficiently studied method, requiring a large experimental base for different sizes of nanospheres. So, in this work, we investigated the influence of the technological parameters of NSL by spin-coating on the substrate coverage area by a monolayer of nanospheres with a diameter of 300 nm. It was found that the coverage area increases with decreasing spin speed and time, isopropyl and propylene glycol content, and with increasing the content of nanospheres in solution. Moreover, the process of controllably reducing the size of nanospheres in inductively coupled oxygen plasma was studied in detail. It was determined that increasing the oxygen flow rate from 9 to 15 sccm does not change the polystyrene etching rate, whereas changing the high-frequency power from 250 to 500 W increases the etching rate and allows us to control the decreasing diameter with high accuracy. Based on the experimental data, the optimal technological parameters of NSL were selected and the nanosphere mask on Si substrate was created with coverage area of 97.8% and process reproducibility of 98.6%. Subsequently reducing the nanosphere diameter lets us obtain nanoneedles of various sizes, which can be used in field emission cathodes. In this work, the reduction of nanosphere size, silicon etching, and removal of polystyrene residues occurred in unified continuous process of plasma etching without sample unloading to atmosphere. The creation of ordered arrays of silicon nanostructures is of great interest to researchers due to their unique prop- erties and potential applications in various components of electronic1,2, plasmonic3,4, photonic5, photovoltaic6,7 devices, as well as surface-enhanced Raman spectroscopy (SERS)8. For example, the authors of9 created a pro- totype of vertically integrated nanowire field effect transistors (SiNW-FETs) based on vertically oriented silicon structures. In addition, SiNW-FETs are promising for biosensors due to their ultrasensitivity, selectivity, and label-free and real-time detection capabilities10. It is also known that the solar cells made on the basis of silicon vertically oriented nanostructures are promising in the solar energy industry. This is because of such advantages as increased efficiency of light trapping due to its multiple scattering inside the structure, with much lower mass and thickness of the cell compared to planar cells11. www.nature.com/scientificreports www.nature.com/scientificreports 1Peter the Great St. Petersburg Polytechnic University, St. Petersburg 195251, Russian Federation. 2Institute of Mineralogy of Southern-Urals Federal Research Center of Mineralogy and Geoecology of Ural Branch of RAS, Miass, Chelyabinsk Region 456317, Russian Federation. email: alina.gagaeva@mail.ru www.nature.com/scientificreports/ But in this study the focus was placed exclusively on achieving perfect orderliness on small-area samples (15 × 15 mm), and the effect of technological parameters on the total coverage area of the entire substrate was not investigated. In another study20, Chen et al. found a correlation between the area covered by a mono- and bilayer of nanospheres and parameters of the process such as spin speed and acceleration for four different sizes of nanospheres. However, in this work, the authors did not study the parameters related directly to colloidal solution preparation, which have a significant influence on the solvent evaporation process and hence on the quality of nanosphere self-assembly. In addition, it is worth noting that in some works21,23–25, mixtures of surfactants were additionally added to the solution with nanospheres to improve the wettability of the solution and slow down the evaporation rate of the suspension. But this fact can not only lead to additional contamination of the substrate with surfactants, but also significantly increase the cost of the nanosphere lithography process. It is important that in most works aimed at studying the spin-coating process, microphotographs demonstrating obtained nanosphere coatings cover the substrate area of no more than 100 × 100 µm219,20,22,23,26. Such magnification does not allow to objectively estimate the filling and ordering of nanospheres in larger areas, whereas high uniformity and monolayer coverage over the entire substrate area is a fundamental requirement for the nanosphere lithography process if it is used to create etching masks. Thus, a detailed investigation of the influence of technological parameters on the process f i l if h i i l k d i f ili h i l i solution with nanospheres to improve the wettability of the solution and slow down the evaporation rate of the suspension. But this fact can not only lead to additional contamination of the substrate with surfactants, but also significantly increase the cost of the nanosphere lithography process. It is important that in most works aimed at studying the spin-coating process, microphotographs demonstrating obtained nanosphere coatings cover the substrate area of no more than 100 × 100 µm219,20,22,23,26. Such magnification does not allow to objectively estimate i i the filling and ordering of nanospheres in larger areas, whereas high uniformity and monolayer coverage over the entire substrate area is a fundamental requirement for the nanosphere lithography process if it is used to create etching masks. www.nature.com/scientificreports/ Thus, a detailed investigation of the influence of technological parameters on the process of creating a monolayer uniform nanosphere coating is a relevant task, and it can facilitate the implementation of the nanosphere lithography by spin-coating method in both scientific and industrial fields.t g y y gii After obtaining a close-packed array of nanospheres on the substrate, it can be used immediately as a mask for the etching process or modified in a certain way to obtain the desired pattern. Plasma-chemical etching (PCE) in oxygen is widely used to reduce the size of nanospheres without changing their position on the substrate27–30. The size of nanospheres can be controlled by adjusting the duration and conditions of etching, and the distance between them depends on the choice of the initial size of the spheres. At the same time, the creation of silicon nanostructures through a mask of nanospheres in a single etching cycle is of practical interest. It implies reduc- tion of nanosphere size, etching of silicon and removal of nanosphere residues in a single process chamber without additional stages of sample loading and unloading to atmosphere. In this regard, this work is devoted to the development of the technology of nanosphere lithography (nanospheres with a diameter of 300 nm) using spin-coating method, the study of the influence of technological parameters on the process of reducing the size of spheres in plasma and approbation of the developed technology to create an array of silicon nanoneedles of a certain size in a single cycle for their potential use in the manufacture of field emission cathodes. www.nature.com/scientificreports/ of exposure made it possible to reduce the size of the obtained structures, these methods are characterized by significant financial and time expenditures, as well as technical complexity in their realization16. In this regard, the search and study of simpler, cheaper and more productive approaches to the formation of nanostructures with certain parameters has become of primary relevance and practical significance.h of exposure made it possible to reduce the size of the obtained structures, these methods are characterized by significant financial and time expenditures, as well as technical complexity in their realization16. In this regard, the search and study of simpler, cheaper and more productive approaches to the formation of nanostructures with certain parameters has become of primary relevance and practical significance.h p p y p gi The development of nanosphere lithography based on the phenomenon of self-organization has established itself as an alternative method for creating ordered nanostructures with such advantages as low cost, high pro- ductivity and lack of complex equipment17. The most common material for nanospheres, playing the role of a mask during subsequent etching, is polystyrene (PS), which is a monodisperse spherical particles in a wide range of sizes (from 20 nm to 10 μm). Self-assembly at the air/water interface (Langmuir–Blodgett (LB) method)18 and spin-coating method19 are considered to be the most promising techniques for the formation of nanosphere coatings. However, the authors of works19–21 note that low transfer rate of nanospheres to the substrate and dif- ficulty in obtaining uniform monolayer coating on a large area prevent wide application of LB method. Coating the substrate using spin-coating method involves deposition of a colloidal suspension to a hydrophilic substrate followed by an accelerated solvent evaporation process during spinning in a centrifuge. This method is currently considered the most efficient and flexibly controlled way for creating monolayers of self-assembled particles, which can be implemented in mass production. In addition, in the research22 authors studied nanospheres of size 500 and 1000 nm to compare spin-coating and LB methods, and they found that in the case of using nanospheres of small diameter the LB method does not let to obtain a close-packed monolayer on the whole substrate surface (the maximum coverage area was 54%). However, spin-coating method is not simple, because it requires optimization of a number of parameters that have an interdependent influence on the solvent evaporation process. www.nature.com/scientificreports/ In this regard, researchers have to find optimal technological parameters empirically, and usually the optimal mode varies depending on the size of nanospheres. To date, the application of spin-coating method for the formation of nanosphere coatings can be found in many papers. However, most of them either do not give any detailed information on the selection of optimal technological parameters, or study the influence of only some parameters on the quality of the resulting coating. For example, the authors of21 show how spin speed and concentration of the dispensed colloidal solu- tion affect the process of creating an ordered array of nanospheres with different diameters. But in this study the focus was placed exclusively on achieving perfect orderliness on small-area samples (15 × 15 mm), and the effect of technological parameters on the total coverage area of the entire substrate was not investigated. In another study20, Chen et al. found a correlation between the area covered by a mono- and bilayer of nanospheres and parameters of the process such as spin speed and acceleration for four different sizes of nanospheres. However, in this work, the authors did not study the parameters related directly to colloidal solution preparation, which have a significant influence on the solvent evaporation process and hence on the quality of nanosphere self-assembly. In addition, it is worth noting that in some works21,23–25, mixtures of surfactants were additionally added to the However, spin-coating method is not simple, because it requires optimization of a number of parameters that have an interdependent influence on the solvent evaporation process. In this regard, researchers have to find optimal technological parameters empirically, and usually the optimal mode varies depending on the size of nanospheres. To date, the application of spin-coating method for the formation of nanosphere coatings can be found in many papers. However, most of them either do not give any detailed information on the selection of optimal technological parameters, or study the influence of only some parameters on the quality of the resulting coating. For example, the authors of21 show how spin speed and concentration of the dispensed colloidal solu- tion affect the process of creating an ordered array of nanospheres with different diameters. Development of controlled nanosphere lithography technology Artem A. Osipov 1,2, Alina E. Gagaeva 1, Anastasiya B. Speshilova 1, Ekaterina V. Endiiarova 1, Polina G. Bespalova 1, Armenak A. Osipov 2, Ilya A. Belyanov 1, Kirill S. Tyurikov 1, Irina A. Tyurikova 1 & Sergey E. Alexandrov 1 OPEN Another promising application of silicon vertically oriented nanostructures, namely nanoneedles, is their use as field emission (cold) cathodes in electrovacuum devices. In contrast to thermionic cathodes, in this case electrons do not need to be pre-excited for their emission under the action of electric field12,13. i However, one of the main problems of technology development in this direction is the lack of simple methods for forming a pattern on the substrate surface to obtain the required topology. Typically, traditional lithographic techniques such as extreme ultraviolet (EUV) lithography and electron beam lithography (EBL) combined with a dry plasma etching process are used to create nanostructures with controllable size and shape. For example, in14 the authors demonstrate their refractive index sensor based on an array of resonant silicon nanodisks with diameter of 330 nm, which were fabricated using EBL and reactive ion etching. In another paper15, the authors present a process for fabricating an array of metallic nanowires smaller than 100 nm in diameter using EUV and plasma etching in oxygen. Despite the fact that the use of short-wavelength radiation and an alternative method 1Peter the Great St. Petersburg Polytechnic University, St. Petersburg 195251, Russian Federation. 2Institute of Mineralogy of Southern-Urals Federal Research Center of Mineralogy and Geoecology of Ural Branch of RAS, Miass, Chelyabinsk Region 456317, Russian Federation. email: alina.gagaeva@mail.ru \| https://doi.org/10.1038/s41598-023-29077-y Scientific Reports \| (2023) 13:3350 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ 1. Taking the required amount of aqueous solution of polystyrene nanospheres using a micropipette (JOAN- LAB) and placing it in a test tube for centrifugation. 1. Taking the required amount of aqueous solution of polystyrene nanospheres using a micropipette (JOAN- LAB) and placing it in a test tube for centrifugation. p g g . Centrifugation at 12,000 rpm for 8 min to separate the solution into fractions followed by removal of excess water from the test tube. 3. Adding the necessary amount of isopropyl alcohol and propylene glycol to the test tube to increase the viscosity of the solution. 3. Adding the necessary amount of isopropyl alcohol and propylene glycol to the test tube to increase the viscosity of the solution. y 4. Ultrasonic treatment of the resulting solution for 15 min at 50 ℃ for mixing the solution and uniform dis- tributing the nanospheres in the suspension to prevent their sticking together. g p p p g g 5. Transferring the prepared solution to the hydrophilic substrate using the micropipette and spin-coating at a certain speed and time. The acceleration of rotation was not regulated due to the lack of this function in the centrifuge. g . Drying the wafer on an oven at 60 ℃ for 5 min to finally evaporate the solvent and provide better contact between the nanospheres and the Si substrate. g 6. Drying the wafer on an oven at 60 ℃ for 5 min to finally evaporate the solvent and provide better con between the nanospheres and the Si substrate. Reducing the size of nanospheres and subsequent etching of silicon through the resulting mask was performed on a custom-built plasma-chemical etching system with a source of high-density inductively coupled plasma (ICP), described in details previously31. Oxygen (O2 class 6.0, 99.9999%, GOST TU 2114-001-05798345-2007) was used for isotropic etching of polystyrene nanosphere in ICP, first to reduce the diameter of nanospheres in the range from 300 to 15 nm, and at the end of the process to remove residual nanospheres. The Si substrate with a monolayer of polystyrene nanospheres as a mask was etched in ICP using a gas mixture of SF6 (99.998%, GOST TU 6-02-1249-83) and C4F8 (GOST TU 2412-128-05807960-96). Thus, the basic steps of the fabrication of Si ordered nanostructures using the nanosphere lithography process are shown in Fig. 1. Methods Monodisperse polystyrene nanospheres with a diameter of 300 nm in the form of an aqueous solution with a dry substance concentration of 10 wt % were purchased from the Institute of High-Molecular Compounds of the Russian Academy of Sciences and were used for the experiments. Monocrystalline silicon wafers (100) with a diameter of 76 mm and a thickness of 380 μm were purchased from OOO "Kremni" and were used as samples for depositing nanospheres. Before each experiment, the substrates were cleaned in acetone for 10 min at 80℃. Due to the fact that nanosphere lithography requires a hydrophilic surface, before nanosphere deposition, additional treatment of the sample surface in a piranha solution (1:3 H2O2:H2SO4) followed by washing in distilled water and drying in an air stream was applied.h Spin-coating process was conducted on a JOANLAB MC-12 Pro high-speed centrifuge. The process of nano- sphere lithography included the following steps: https://doi.org/10.1038/s41598-023-29077-y Scientific Reports \| (2023) 13:3350 \| www.nature.com/scientificreports/ where IO and IAr are the experimentally recorded emission intensities of oxygen (844.6 nm) and argon (750.4 nm) lines respectively, [Ar] is the argon density inside reactor without plasma discharge, KO is the oxygen excitation efficiency coefficient (0 07)32 where IO and IAr are the experimentally recorded emission intensities of oxygen (844.6 nm) and argon (750.4 nm) lines respectively, [Ar] is the argon density inside reactor without plasma discharge, KO is the oxygen excitation efficiency coefficient (0.07)32.t fifi After conducting the processes of nanosphere lithography and plasma-chemical etching, the resulting coatings of polystyrene nanospheres and Si nanostructures were analyzed using microphotographs made with a Supra 55VP (CarlZeiss) scanning electron microscope with an accuracy of ± 2.5%. As shown in the literature review, in most of the works, the coverage area of the substrate with mono- and bilayer nanospheres was estimated by microphotographs covering the substrate area not exceeding 100 × 100 μm219,20,22,23,26. The microphotographs in Fig. 2a–d show that high magnification does not give an objective result regarding the overall quality of the nanosphere coating. For example, a microphotograph at 1000 × magnification, covering an area of 105 × 75 μm2 (Fig. 2b), shows 100% filling of the substrate with a monolayer of nanospheres. However, if the area is enlarged to 560 × 400 µm2 (Fig. 2a), the presence of both voids (lightest regions) and double-layered areas (darkest regions) can be observed, shown separately in Fig. 2c,d. Therefore, in order to provide visual reliable information, in this work the analysis of the resulting coatings was conducted using microphotographs taken at a magnification of 200x (560 × 400 µm2). The coverage area of the substrate with nanospheres was estimated from a histogram of the binarized image, as shown in Fig. 2f (the nanospheres are painted black, the voids—white). Each experiment was carried out 3 times, and 5 points were measured on each substrate (Fig. 2e), after which the obtained values were averaged. Error bars have been set as follows: ± 2.5% for the dependencies of coverage areas on various parameters and ± 5% for the dependencies of nanosphere etching rates on various parameters. In addition to the desired close-packed monolayers with hexagonal structure, the obtained nanosphere arrays may contain voids (uncovered regions), locally arranged monolayers with random ordering, and borders between domains. Domains will be called regions of the array with high ordering and hexagonal symmetry. www.nature.com/scientificreports/ To understand the process of plasma-chemical etching of polystyrene, it is necessary to study the physico- chemical regularities of the processes occurring in plasma. One of the most promising methods for this purpose is in situ control using optical emission spectroscopy (OES). In addition, the OES method was used to deter- mine the end point of the process of removing the nanosphere residues in oxygen plasma by the disappearance of spectral lines specific for polystyrene from the spectra. Optical emission spectra were recorded using an OceanOptics HR 4000 spectrometer in the wavelength range of 200–1120 nm with a resolution of ~ 0.02 nm. The spectrometer was connected to the reaction chamber through a fiber-optic cable, which was integrated with the viewing window on the flange of the reaction chamber of the PCE system. Post-processing of the data was performed using SpectraGryph 1.2.14 software. t To determine the concentration of atomic oxygen in plasma, the method of optical actinometry was applied, for which the emission line of O (844.6 nm) and argon (750.4 nm) as the gas-actinometer were used. The equa- tion for the concentration of atomic oxygen can be written as follows: [O] = KO IO IAr [Ar], [O] = KO IO IAr [Ar], Figure 1. Schematic image of the basic steps for creating ordered silicon nanostructures through a mask of polystyrene nanospheres. Figure 1. Schematic image of the basic steps for creating ordered silicon nanostructures through a mask of polystyrene nanospheres. https://doi.org/10.1038/s41598-023-29077-y https://doi.org/10.1038/s41598-023-29077-y Scientific Reports \| (2023) 13:3350 \| www.nature.com/scientificreports/ Results and discussionh The influence of the time and temperature of ultrasonic mixing of the solution on the quality of the coating was also investigated, during which the best quality coatings were obtained at 15 min and 50 ℃, respectively. The ultrasonic treatment parameters were constant in all subsequent series of experiments.hil q p The first series of experiments was aimed at determining the nature of the influence of the spin speed on the quality of the coating, while other process parameters remained unchanged. It is known that the spin speed affect both the solvent evaporation rate and the centrifugal force, which moves the suspension to the edge of the substrate20. As can be seen from the plot (Fig. 3a), at a rotation speed below 3300 rpm, a significant num- ber of bilayer clusters are formed, shown in the microphotograph in Fig. 3b. This double-layered areas are not acceptable for the fabrication of nanosphere coatings used as masks for subsequent etching. This is probably due to the fact that at low spin speeds the balance of forces acting on the nanospheres is disturbed. Due to the low centrifugal force, the flow of nanospheres toward the edge of the substrate is not sufficient to compensate the capillary flow of the suspension into the central region. On the contrary, the high spin speed (> 3500 rpm), which facilitates the evaporation process and increases the centrifugal force, creates a large number of voids in the array (Fig. 3d), because most of the suspension is thrown away from the substrate surface. In addition, the solvent evaporates faster than the nanospheres have time to self-assemble into a hexagonal array22. The optimal spin speed of 3300 rpm was found experimentally, at which the coverage area was 98.6%, and there were almost no bilayers (0.7%) (Fig. 3c). With further increase of the spin speed, the bilayers completely disappear, but the total coverage area of the substrate decreases sharply. g y In the next series of experiments, we determined the optimal amount of aqueous solution of nanospheres, which was taken from a purchased solution and centrifuged. Then the water was removed from it, and finally the dry spheres were mixed with solvent in the form of isopropyl alcohol and propylene glycol. y p p py p py g y As can be seen from the plot (Fig. Results and discussionh Theoretical studies34,35 have demonstrated that interparticle capillary forces are the driving force for the order- ing of nanospheres on the substrate. Such forces occur as a result of increasing curvature of the liquid surface between the particles as the solvent evaporates. As a result of the action of capillary forces, the nanospheres are assembled into a hexagonal close-packed structure due to the fact that the system tends to the configuration with the lowest energy, and hence, to the maximum contact with neighboring particles. Thus, the evaporation rate of the solvent, consisting in our case of isopropyl alcohol and propylene glycol, is one of the main factors, which affects capillary forces and, as a result, the process of self-assembly of nanospheres36. In turn, the evaporation rate of the solvent is influenced by parameters such as rotation rate (N) and time (T), as well as the viscosity of the solution, which depends on the relative concentrations of each component in solution (V1—isopropyl volume, V2—propylene glycol volume, V3—volume of aqueous solution of nanospheres). In this regard, the nature of the influence of the above technological parameters on the area of the substrate covered by mono- and bilayer of nanospheres was studied in order to determine the optimal technological parameters for creating a monolayer close-packed nanosphere coating.h The content of each component in the solution applied to the substrate was considered as a percentage of the volume of one component to the total volumes of the other two components. This was done for the convenience of information perception, since in a series of experiments to determine the optimal contents of nanospheres, alcohol and propylene glycol in the solution, the total volume of the solution applied to the substrate varied according to the varying volume of the component under consideration, while the volumes of the other two components were unchanged.h The initial volume ratio of all solution components was determined during a preliminary series of experi- ments aimed at optimizing the parameters of the nanosphere lithography process based on the Taguchi matrix method37. The best result in terms of substrate coverage area was shown by the V1/V2/V3 = 80/56/50 μl solution, which was chosen as starting solution for further basic experiments. www.nature.com/scientificreports/ In this regard, to check the hexagonal symmetry of the resulting self-assembled monolayer, a 2D Fourier transform was applied to the SEM image in the NT-MDT NOVA software. In order to estimate the size of domains in the array obtained Figure 2. Methodology for estimating nanosphere coverage area: (a–d) demonstration of differences in the results obtained at different magnifications; (e) five-point measurement scheme for coverage area; (f) binarization of the image for coverage area estimation. Figure 2. Methodology for estimating nanosphere coverage area: (a–d) demonstration of differences in the results obtained at different magnifications; (e) five-point measurement scheme for coverage area; (f) binarization of the image for coverage area estimation. Scientific Reports \| (2023) 13:3350 \| https://doi.org/10.1038/s41598-023-29077-y www.nature.com/scientificreports/ using the developed technique, the HEXI software33 was used. It recognizes regions with hexagonal ordering in the loaded SEM image and marks the borders between them, as well as defects in the domains in the form of voids. Results and discussionh decreasing the total coverage area of the substrate, while the number of bilayer regions also decreases and is close to 0 at the content of isopropyl more than 83%. This is probably due to the fact that the small volume of isopropyl, which has a viscosity slightly higher than a viscosity of water, evaporates very quickly. Consequently, only propylene glycol with much higher viscosity remains in the solution, and it becomes more difficult for the nanospheres to move across the substrate surface with such excessive solution viscosity. As a result, more than one monolayer remains on the substrate (Fig. 5b). Increasing the content of isopropyl in the solution leads to a more rarefied and non-uniform coating (Fig. 5d). Due to decreasing the relative concentration of propylene glycol, the compaction forces are not capable to overcome the barrier of hydrophobic interactions to order the particles, as a result of which the nanospheres are randomly fixed on the substrate. Thus, the optimal value of the isopropyl content in the solution was found to be 83%, corresponding to the alcohol volume of 80 µl. At this chosen value, the substrate is filled with a close-packed array by 97.6%, and there are approximately 0.6% bilayer areas (Fig. 5c). It is worth noting that a purchased solution of nanospheres of a given size, although in small quantities, initially contains large polystyrene spheres. Therefore, in some cases, such as shown in the inset in Fig. 5c, this single large sphere can act as a nucleation center for multilayered areas of small spheres around it. Such areas have also been included in the total percentage of bilayer coverage of the substrate for a more objective study. p g y g j y It follows from the plot in Fig. 6a that the total lack of propylene glycol in the solution leads to a low total coverage area of the substrate (≈ 78%), with the formation of a completely non-monolayer coating. This is prob- ably related to the impossibility of self-assembly of nanospheres due to their poor wettability by isopropyl and the strong effect of hydrophobic interactions between the nanospheres in the solvent without adding propylene glycol to the solution. This assumption is confirmed by the microphotograph (Fig. 6b) demonstrating the forma- tion of multilayer disordered regions of nanospheres in the absence of propylene glycol in the solution. It can also be seen from the plot (Fig. Results and discussionh 4a), a good coverage area close to 100% is achieved at nanosphere content (in this case the percentage ratio of the volume of the aqueous nanosphere solution discussed above to the total volume of isopropyl and propylene glycol) starting from 29%. Smaller content values produce a large number of empty spaces on the substrate, as can be seen in Fig. 4b. However, the number of bilayers begins to increase sharply at nanosphere content of 37% and higher. This indicates that at the selected technological parameters of the spin speed and time, a larger amount of polystyrene remains on the substrate than is necessary for the close- packed monolayer coating (Fig. 4d). Therefore, a nanosphere content of 29% was selected for further experiments, corresponding to the volume of the initially sampled aqueous nanosphere solution of 40 µl. At this chosen value, the coverage area was equal to 95.3%, and there were no bilayers (Fig. 4c).hif g q y g The viscosity of the solution also has a significant effect on solvent evaporation and self-assembly of the nanospheres, and hence on the coverage area and the number of bilayers. Solution viscosity can be controlled by changing the relative concentrations of isopropyl alcohol and propylene glycol in the solution. Nanospheres without solvent are not capable to self-assemble into a dense hexagonal package, because there are electrostatic repulsive forces between them, which are overcome by surface tension forces arising from solvent carriers in the colloidal solution38. In addition, propylene glycol, which has two O–H groups and a small hydrophobic chain, wets the polystyrene surface more strongly than water and isopropyl alcohol, so it reduces the hydrophobic inter- actions of nanospheres in the solvent, which prevent the processes of reorientation and compaction of particles39. As can be seen from the plot (Fig. 5a), increasing the content of isopropyl alcohol (in this case the percentage ratio of the alcohol volume to the total volume of nanospheres and propylene glycol) from 23 to 103% leads to Scientific Reports \| (2023) 13:3350 \| https://doi.org/10.1038/s41598-023-29077-y www.nature.com/scientificreports/ Figure 3. (a) Plot of dependence of substrate coverage area by monolayer and bilayer of PS nanospheres on spin speed; (b–d) microphotographs of nanosphere coatings obtained at varying spin speed. Figure 3. (a) Plot of dependence of substrate coverage area by monolayer and bilayer of PS nanospheres on spin speed; (b–d) microphotographs of nanosphere coatings obtained at varying spin speed. Results and discussionh 6a) that with increasing the propylene glycol content (in this case the percentage ratio of the propylene glycol volume to the total volume of nanospheres and alcohol) from 16 to 33%, there is a slight decreasing coverage area of the substrate by a monolayer of nanospheres and almost complete disappear- ance of bilayer areas. In this regard, the content of 33% was chosen as the optimal value of the propylene glycol content in the solution, corresponding to the propylene glycol volume of 40 µl, at which the coverage area of the substrate with a close-packed monolayer of nanospheres is 98.5%, and there are approximately 0.8% of bilayer areas (Fig. 6c). As the propylene glycol content is further increased, solution viscosity probably becomes too high for the normal self-assembly process, and a significant number of voids appear, as shown in Fig. 6d. Scientific Reports \| (2023) 13:3350 \| https://doi.org/10.1038/s41598-023-29077-y www.nature.com/scientificreports/ Figure 4. (a) Plot of dependence of substrate coverage area by monolayer and bilayer of PS nanospheres on content of nanospheres in solution; (b–d) microphotographs of nanosphere coatings obtained at varying content of nanospheres in solution. Figure 4. (a) Plot of dependence of substrate coverage area by monolayer and bilayer of PS nanospheres on content of nanospheres in solution; (b–d) microphotographs of nanosphere coatings obtained at varying content of nanospheres in solution. The final series of experiments was aimed at determining the nature of the influence of spin-coating time on the coverage area of the substrate with mono- and bilayer of nanospheres. According to the obtained plot shown in Fig. 7a, when the rotation time is less than 10 s, a significant number of bilayer areas are formed, dem- onstrated in Fig. 7b. This is due to the fact that the bulk of the suspension still remains on the substrate, and the solvent does not have time to evaporate. Moreover, in addition to the bilayers, a short rotation time during the spin-coating process formed a thickening on the periphery of the substrate, which flowed over a large area of the substrate and reduced its working area. On the contrary, at spin-coating times longer than 10 s, as follows from the plot (Fig. 7a) and the microphotograph (Fig. 7d), the formation of voids was observed, because by this time, probably, too many nanospheres leave the substrate. Using the obtained dependences, the optimal spin-coating time was chosen to be 10 s (Fig. Results and discussionh 7c).h ( g ) The optimal technological parameters of the nanosphere lithography process for obtaining the close-packed hexagonal array of polystyrene nanospheres with a diameter of 300 nm were determined on the basis of analyz- ing the obtained experimental data (Table 1). To check the reproducibility of the results, five experiments with the selected optimal parameters were conducted, and the coverage area with mono- and bilayer was estimated for each (Table 2). As a result, the average coverage area of the substrate with monolayer of the nanospheres (diameter 300 nm) was 97.8%, with a small number of bilayer regions (0.5%). ( ) y g ( ) To estimate the accuracy characterizing the reproducibility of the results, the relative root-mean-square was calculated according to the formula (1) (1) ν = n i (xi −x)2 x2 · (n −1) · 100% (1) where xi is the monolayer coverage area of the substrate in each experiment, x is the average value of the mon- olayer coverage area of the substrate, and n is the number of experiments, equal to 5. Thus, the relative root- mean-square error was 1.37%, which can be considered quite acceptable result. q q p It is well known that self-assembled arrays are characterized by individual domains, or as they are called, grains, which can be differently oriented, but within which spheres are strictly ordered into a hexagonal lattice Scientific Reports \| (2023) 13:3350 \| https://doi.org/10.1038/s41598-023-29077-y www.nature.com/scientificreports/ Figure 5. (a) Plot of dependence of substrate coverage area by monolayer and bilayer of PS nanospheres on content of isopropyl in solution; (b–d) microphotographs of nanosphere coatings obtained at varying content of isopropyl in solution. Figure 5. (a) Plot of dependence of substrate coverage area by monolayer and bilayer of PS nanospheres on content of isopropyl in solution; (b–d) microphotographs of nanosphere coatings obtained at varying content of isopropyl in solution. with one orientation. In order to estimate the degree of ordering of the resulting coatings, a Fourier transform of SEM images covering different areas was performed. The corresponding 2D Fourier images of the arrays are shown in the insets in Fig. 8a–c. The maximum covered area, where a hexagonal ordered domain with one orientation was obtained, was found to be 1535 μm2, as confirmed by the peaks in the Fourier image located at the vertices of the regular hexagon (Fig. 8a). Results and discussionh However, despite the ordering, dislocations can also be present in one domain and are clearly visible in SEM images at high magnification (Fig. 8b,c). Using the HEXI software, the percentage of defective areas in each image was calculated. Close-packed nanospheres are marked with green circles and nanospheres entering dislocation areas are marked with red circles (Fig. 8d–f). The resulting percentage of dislocations was 22% in an area of 1535 µm2 (Fig. 8d), 24% in an area of 175 µm2 (Fig. 8e) and 25% in an area of 45 µm2 (Fig. 8f).h µ ( g ) The next step to obtain an array of silicon nanostructures of a certain geometry is the process of reducing the size of polystyrene nanospheres in inductively coupled oxygen plasma, to form an ordered, not close-packed array with gaps between nanospheres. For controlled reduction of nanosphere size in oxygen plasma, it is necessary to study physicochemical regularities of polystyrene etching process, as well as processes occurring in plasma during etching. In this regard, the nature of the influence of the main process technological parameters (high- frequency (HF) power, etching time, bias voltage on the substrate holder, pressure in the chamber, and oxygen flow rate) on the etching rate and the diameter of nanospheres was determined.h l g p The etching of polystyrene in oxygen plasma occurs by breaking the aromatic ring, formation of oxygen- containing functional groups on the surface, and subsequent formation of volatile products such as carbon monoxide and carbon dioxide40. Figures 9c–h show how the size of nanospheres etched at different HF power for 1 min changes. From the plot (Fig. 9a) it can be observed that the polystyrene etching rate increases mono- tonically from 83 to 175 nm/min when the applied power is increased from 250 to 500 W. Such nature of the dependence is related to the fact that with increasing absorbed power, the concentration and average energy of electrons in plasma grows, therefore, the intensity of inelastic collisions of electrons with oxygen molecules increases. As a result, it leads to more efficient formation of such active particles as radicals and ions which react with polystyrene41. This is confirmed by the increasing concentration of atomic oxygen in plasma when the applied power is increased (Fig. 9b). Scientific Reports \| (2023) 13:3350 \| https://doi.org/10.1038/s41598-023-29077-y www.nature.com/scientificreports/ Figure 6. Results and discussionh (a) Plot of dependence of substrate coverage area by monolayer and bilayer of PS nanospheres on content of propylene glycol in solution; (b–d) microphotographs of nanosphere coatings obtained at varying content of propylene glycol in solution. Figure 6. (a) Plot of dependence of substrate coverage area by monolayer and bilayer of PS nanospheres on content of propylene glycol in solution; (b–d) microphotographs of nanosphere coatings obtained at varying content of propylene glycol in solution. Next, the reduction in the size of the nanospheres at different etching times was studied, while the other technological parameters remained constant. As can be seen from the plot (Fig. 10a), the diameter of the nano- spheres decreases from 240 to 15 nm when the etching time is increased from 1 to 5 min, while the etching rate changes insignificantly. When the nanospheres are still large enough, during the first 2 min of etching, bridges with a length of about 50 nm are formed between them, thanks to which the nanospheres are well stabilized in their initial positions (Fig. 10b). The formation of such bridges is caused by the tendency of the nanospheres to reduce the surface energy42 With further increasing the etching time, these bridges, like the spheres, are etched in oxygen plasma and disappear (Fig. 10c). After that the nanospheres can start moving along the substrate surface, because their size by this time strongly decreases, therefore, the contact area with the substrate also decreases43. In addition, as can be seen from Fig. 10d–f, when the etching time reached 3 min, the nanospheres deformed, taking an irregular shape. In this regard, for further experiments the optimal etching time of 2 min was chosen, at which the nanospheres are still well stabilized in their places, are circular in shape and are ≈ 210 nm in diameter (Fig. 10c). The pressure in the reaction chamber determines both the energy distribution of electrons and ions and the number of chemical reactions occurring on the surface of the treated material. So, as the gas pressure increases, the energy of ions bombarding the surface decreases due to decreasing the mean free path of the particles. In addition, the average energy of electrons, which determines the rate of generation of active oxygen particles, also decreases44. As can be seen from the plot (Fig. 11a) and microphotographs (Fig. Results and discussionh 11c–g), the etching rate of polystyrene nanospheres decreased from 115 to 47 nm/min when the pressure in the chamber was increased from 0.4 to 1.25 Pa. However, the dependence of the concentration of oxygen atoms on pressure shown in Fig. 11b demonstrates only a slight increasing atomic oxygen with increasing pressure. Thus, we assume that the contribution of the physical component (i.e., ion bombardment) to the polystyrene etching process prevails in this case over the contribution of the chemical component (i.e., chemical reaction).i p ( ) In addition, decreasing the intensity of ion bombardment of the surface with increasing pressure is confirmed by the fact that at low pressure (< 1 Pa) and higher ion energy the nanospheres become irregularly shaped (Fig. 11c–e). The intensity of ion bombardment is primarily determined by the magnitude of the negative bias potential on the substrate, which also has a great influence on the etching rate and the morphology of the Scientific Reports \| (2023) 13:3350 \| https://doi.org/10.1038/s41598-023-29077-y www.nature.com/scientificreports/ Figure 7. (a) Plot of dependence of substrate coverage area by monolayer and bilayer of PS nanospheres on spin-coating time; (b–d) microphotographs of nanosphere coatings obtained at varying spin-coating time. Figure 7. (a) Plot of dependence of substrate coverage area by monolayer and bilayer of PS nanospheres on spin-coating time; (b–d) microphotographs of nanosphere coatings obtained at varying spin-coating time. Table 1. Values of technological parameters of control experiments. N, rpm T, s V1, μl V2, μl V3, μl 3300 10 80 40 40 Table 2. Coverage areas with mono- and bilayers in control experiments. No 1 2 3 4 5 Average Area of monolayer, % 98.3 96.4 99.2 96.3 98.7 97.8 Area of bilayer, % 0.5 0.1 0.9 0 0.9 0.5 Table 1. Values of technological parameters of control experiments. N, rpm T, s V1, μl V2, μl V3, μl 3300 10 80 40 40 Table 1. Values of technological parameters of control experiments. Table 2. Coverage areas with mono- and bilayers in control experiments. Table 2. Coverage areas with mono- and bilayers in control experiments. nanospheres45. As can be seen from the plot (Fig. 12a), a higher bias voltage leads to a more efficient bombard- ment of the material, therefore, a higher etching rate. Results and discussionh Namely, as the modulus of the bias voltage on the substrate holder increases from 12 to 100 V, the diameter of the plasma-treated nanospheres decreases from 207 to 14 nm (Fig. 12b–f).hill nanospheres45. As can be seen from the plot (Fig. 12a), a higher bias voltage leads to a more efficient bombard- ment of the material, therefore, a higher etching rate. Namely, as the modulus of the bias voltage on the substrate holder increases from 12 to 100 V, the diameter of the plasma-treated nanospheres decreases from 207 to 14 nm (Fig. 12b–f).hill g The final series of experiments was aimed at determining the nature of the influence of oxygen flow rate on the etching rate of polystyrene nanospheres. Despite the fact that the concentration of atomic oxygen increases by approximately two times when the oxygen flow rate is increased from 9 to 15 sccm (Fig. 13b), the polystyrene etching rate does not show significant changes and remains at an average level of 82 nm/min (Fig. 13a,c–g). Probably, in the selected range of technological parameters, the weak variation of the etching rate depending on the oxygen flow rate is related to the fact that there is a certain amount of oxygen sufficient to saturate the chemical etching process27. In other words, we assume that in our case the flow rate of 9 sccm is sufficient to saturate the polystyrene surface with functional groups, and a further slight increasing the flow rate to 15 sccm does not significantly affect the probability of interaction of oxygen atoms with carbon atoms in polystyrene. Scientific Reports \| (2023) 13:3350 \| https://doi.org/10.1038/s41598-023-29077-y www.nature.com/scientificreports/ Figure 8. Qualitative and quantitative analysis of the hexagonal lattice on different substrate areas (a–c) using Fourier transform and HEXI processing. Figure 8. Qualitative and quantitative analysis of the hexagonal lattice on different substrate areas (a–c) usin Fourier transform and HEXI processing. As a result, silicon nanostructures in the form of nanoneedles of various sizes were fabricated using the devel- oped nanosphere lithography technology. For this purpose, at the first stage, ordered close-packed nanosphere masks were formed using spin-coating technique. Then, by PCE in oxygen plasma, the sizes of nanospheres were reduced to different values (220, 175, and 115 nm), as shown in Fig. 14aI–III. Results and discussionh Finally, plasma-chemical etching of silicon on the formed mask was conducted in SF6/C4F8 gas mixture, resulting in arrays of nanoneedles with base diameters ranging from 70 to 125 nm, with needle diameters ranging from 10 nm (aspect ratio over 90) to 50 nm, and heights ranging from 170 to 1000 nm depending on the initial size of the nanospheres (Fig. 14bI–III). It should be noted that the dimensions of the nanostructures were estimated as average values from measure- ments of several needles.t After silicon etching, polystyrene residues still remained on the tops of the nanoneedles (Fig. 14cI–III), which were removed by treatment in oxygen plasma. The optimal time of plasma etching for complete removal of polystyrene nanospheres was controlled in real time using the method of optical emission spectroscopy by changes in the polystyrene-specific spectral lines. Figure 15 shows the plasma emission spectrum during the etching process. We can identify the oxygen lines (844 and 777 nm) and the line belonging to carbon or hydrogen, which have very close values of the emission wavelength (656.28 and 656.87 nm, respectively) and cannot be identified exactly. However, both carbon and hydrogen are formed as a result of polymer chain splitting and atom detachment by active oxygen particles in plasma, therefore, this line can be used to determine the end point of the polystyrene etching process. The time variation of the intensity of the H/C emission line can be clearly seen in the enlarged view in Fig. 15. It can be observed that as the oxygen plasma cleaning time increases, the intensity of the H/C line gradually decreases and approaches 0 when plasma etching reaches 30 min. This indicates the disappearance of reaction byproducts by this time, therefore, the removal of polystyrene nanospheres from the surface of silicon nanostructures (Fig. 14d).ht g Thus, after the formation of the nanosphere mask, the process of fabricating silicon nanoneedles, which includes reduction of nanosphere size in oxygen plasma, silicon etching and removal of polystyrene residues from the surface of nanostructures, can be conducted in a single technological cycle without additional stages of sample unloading to the atmosphere. In addition, it is possible to use the OES method as in situ control of the end point of polystyrene etching to determine the time of complete removal of polystyrene nanospheres from the sample surface. Conclusion h In the course of this study, the technology of spin-coating was developed for creating a monolayer close-packed coating on the substrate using nanospheres with a diameter of 300 nm. For this purpose, the influence of the main technological parameters of the spin-coating process on the solvent evaporation process in the colloidal suspension and, consequently, the self-assembly process of nanospheres on the substrate, was experimentally studied. It was found that at spin speeds less than 3300 rpm, spin-coating time less than 10 s, and contents of isopropyl and propylene glycol less than 83 and 33%, respectively, a significant number of double layers were formed. Increasing the listed parameters above these limiting values resulted not only in the disappearance of bilayers, but also in decreasing the total coverage area of the substrate. An opposite dependence was observed https://doi.org/10.1038/s41598-023-29077-y Scientific Reports \| (2023) 13:3350 \| www.nature.com/scientificreports/ Figure 9. (a) Plot of dependence of nanosphere diameter and etching rate on applied HF power; (b) plot of dependence of oxygen atom concentration on applied HF power; (c–h) microphotographs of nanosphere mask obtained at varying HF power Figure 9. (a) Plot of dependence of nanosphere diameter and etching rate on applied HF power; (b) plot of dependence of oxygen atom concentration on applied HF power; (c–h) microphotographs of nanosphere mask obtained at varying HF power. Figure 9. (a) Plot of dependence of nanosphere diameter and etching rate on applied HF power; (b) plot of dependence of oxygen atom concentration on applied HF power; (c–h) microphotographs of nanosphere mask obtained at varying HF power. for the content of nanospheres in solution, with the increase of which from 8 to 43% the substrate coverage area increased from 50 to 99.5%. However, similarly there was a limiting value of the content (33%), after which bilayers appeared in addition to the close-packed monolayer of nanospheres. Based on the obtained results, the optimal technological parameters of the nanosphere lithography process were selected, and a monolayer nanosphere coating was obtained with the coverage area of 97.8% on the working area of 76 mm Si substrate and process reproducibility of 98.6%. It should be noted that the selected parameters are suitable for spin-coating using 76 mm Si substrate (in a studied range of technological parameters), but can be extended and used to find conditions for other substrate sizes. Conclusion h In addition, in this work we showed that using etching of polystyrene nanospheres in inductively coupled oxy- gen plasma it is possible to easily control the size of nanospheres and distances between them, and, consequently, the size of silicon nanostructures etched through the nanosphere mask. For this purpose, the influence of PCE technological parameters (HF power, bias voltage on the substrate holder, pressure in the chamber, oxygen flow rate, and etching time) on the etching rate of polystyrene nanospheres and their diameter was studied. When HF power was increased from 250 to 500 W, the etching rate pf polystyrene increased from 83 to 175 nm/min, which was confirmed by the increase of the concentration of atomic oxygen in plasma, approximately, by 3 times. On the contrary, the etching rate decreased from 115 to 47 nm/min when the pressure was increased from 0.4 to 1.25 Pa at constant concentration of atomic oxygen. Therefore, it was concluded that ion bombardment made the main contribution to the etching process in this case. As a result, using the developed technology, silicon nanoneedles of various sizes with a maximum aspect ratio of more than 90 were created. Scientific Reports \| (2023) 13:3350 \| https://doi.org/10.1038/s41598-023-29077-y www.nature.com/scientificreports/ Figure 10. (a) Plot of dependence of nanosphere diameter and etching rate on etching time; (b–f) microphotographs of nanosphere mask obtained at varying etching time. Figure 10. (a) Plot of dependence of nanosphere diameter and etching rate on etching time; (b–f) microphotographs of nanosphere mask obtained at varying etching time. https://doi.org/10.1038/s41598-023-29077-y Scientific Reports \| (2023) 13:3350 \| www.nature.com/scientificreports/ Figure 11. (a) Plot of dependence of nanosphere diameter and etching rate on pressure in the chamber; (b) plot of dependence of oxygen atom concentration on pressure in the chamber; (c–g) microphotographs of nanosphere mask obtained at varying pressure in the chamber. Figure 11. (a) Plot of dependence of nanosphere diameter and etching rate on pressure in the chamber; (b) plot of dependence of oxygen atom concentration on pressure in the chamber; (c–g) microphotographs of h k b i d i i h h b Figure 11. (a) Plot of dependence of nanosphere diameter and etching rate on pressure in the chamber; (b) plot of dependence of oxygen atom concentration on pressure in the chamber; (c–g) microphotographs of nanosphere mask obtained at varying pressure in the chamber. https://doi.org/10.1038/s41598-023-29077-y Scientific Reports \| (2023) 13:3350 \| www.nature.com/scientificreports/ Figure 12. Data availabilityh The datasets generated during and/or analyzed during the current study cannot be shared at this time as the data also forms part of an ongoing study, but are available from the corresponding author on reasonable request. Received: 10 October 2022; Accepted: 30 January 2023 Received: 10 October 2022; Accepted: 30 January 2023 Received: 10 October 2022; Accepted: 30 January 2023 Conclusion h (a) Plot of dependence of nanosphere diameter and etching rate on bias voltage on the substrate holder; (b–f) microphotographs of nanosphere mask obtained at varying bias voltage. Figure 12. (a) Plot of dependence of nanosphere diameter and etching rate on bias voltage on the substrate holder; (b–f) microphotographs of nanosphere mask obtained at varying bias voltage. Figure 12. (a) Plot of dependence of nanosphere diameter and etching rate on bias voltage on the substrate holder; (b–f) microphotographs of nanosphere mask obtained at varying bias voltage. https://doi.org/10.1038/s41598-023-29077-y Scientific Reports \| (2023) 13:3350 \| www.nature.com/scientificreports/ Figure 13. (a) Plot of dependence of nanosphere diameter and etching rate on oxygen flow rate; (b) plot of dependence of oxygen atom concentration on oxygen flow rate; (c–g) microphotographs of nanosphere mask obtained at varying oxygen flow rate. Figure 13. (a) Plot of dependence of nanosphere diameter and etching rate on oxygen flow rate; (b) plot of dependence of oxygen atom concentration on oxygen flow rate; (c–g) microphotographs of nanosphere mask obtained at varying oxygen flow rate. https://doi.org/10.1038/s41598-023-29077-y Scientific Reports \| (2023) 13:3350 \| www.nature.com/scientificreports/ Figure 14. (a) Microphotographs of nanosphere masks with different size of nanospheres; (b,c) microphotographs of nanoneedle arrays after Si etching in SF6/C4F8 plasma; (d) microphotographs of nanoneedle arrays after removal of nanospheres in O2 plasma. Microphotographs taken at angle of 45°. Figure 14. (a) Microphotographs of nanosphere masks with different size of nanospheres; (b,c) microphotographs of nanoneedle arrays after Si etching in SF6/C4F8 plasma; (d) microphotographs of nanoneedle arrays after removal of nanospheres in O2 plasma. Microphotographs taken at angle of 45°. https://doi.org/10.1038/s41598-023-29077-y Scientific Reports \| (2023) 13:3350 \| www.nature.com/scientificreports/ Figure 15. Emission spectra recorded during polystyrene etching in oxygen plasma. Figure 15. Emission spectra recorded during polystyrene etching in oxygen plasma. References References 1. Mizuta, H. & Oda, S. Bottom-up approach to silicon nanoelectronics. Microelectron. J. 39, 171–176 (2008).i 1. Mizuta, H. & Oda, S. Bottom-up approach to silicon nanoelectronics. Microelectron. J. 39, 171–176 (2008). 2 Ravipati S Kuo C J Shieh J Chou C T & Ko F H Fabrication and enhanced field emission properties of novel silicon nan 2. Ravipati, S., Kuo, C. J., Shieh, J., Chou, C. T. & Ko, F. H. 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https://openalex.org/W3098240448	https://www.e3s-conferences.org/10.1051/e3sconf/202020207065/pdf	English	null	Entrepreneurship spirit to survive on culture and environmental development in Jepara, Indonesia	E3S web of conferences	2,020	cc-by	3,292	Keywords Carving; entrepreneur; business; cultural product; pray. Keywords Carving; entrepreneur; business; cultural product; pray. 1 Introduction The carving handicraft industry in Jepara experienced receding in its development. Various components of flux and reflux in the carving handicraft industry, took an important role. Both in the environment of craftsmen, entrepreneurs, informal institutions and local government institutions that have links with the industry. Especially after the presence of foreign investors (investors) in the 1980s from various countries, especially Korea, Japan, Netherlands and Singapore, a process of cultural shift took place [1]. E3S Web of Conferences 202, 07065 (2020) ICENIS 2020 E3S Web of Conferences 202, 07065 (2020) ICENIS 2020 https://doi.org/10.1051/e3sconf/202020207065 © 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/). Entrepreneurship spirit to survive on culture and environmental development in Jepara, Indonesia Sri Indrahti 1, Bintang Andini2 1Department of History, Faculty of Humanities, Diponegoro University, Semarang – Indonesia 2Master Program of Accounting, Faculty of Economics and Business, Diponegoro University, Semarang – Indonesia 1Department of History, Faculty of Humanities, Diponegoro University, Semarang – Indonesia 2Master Program of Accounting, Faculty of Economics and Business, Diponegoro University, Semarang – Indonesia Abstract. This paper seeks to portray the entrepreneurial spirit that originates from the values of local culture after touching and interacting with (new) foreign cultures. The entrepreneurial spirit of a Jepara carving industry entrepreneur is from the cultural products as their background. The livelihood system is one of the cultural elements that gave rise to an entrepreneurial spirit. The entrepreneurial spirit is generated from economic activities that come from livelihoods. Data collection is from observation and in-depth interview with carving entrepreneurs in Jepara, Indonesia. Entrepreneurial spirit of Jepara carving entrepreneurs are working as pray, working for profit and doing rituals for looking business fortune. The result is evident to understanding that work is part of pray. The entrepreneurial spirit of Jepara carving industry entrepreneurs is much influenced by local cultural values, especially those related to religious values. Enthusiasm of work is carried out by increasing the friendship in exchanging work information. An effort to strengthen the value of togetherness, entrepreneurs also often perform religious rituals. This is done as an effort to obtain fluency in the work as well as at the same time hoping for abundant profits. Corresponding author : indrahti@gmail.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/e3sconf/202020207065 E3S Web of Conferences 202, 07065 (2020) ICENIS 2020 E3S Web of Conferences 202, 07065 (2020) Fig 1. Carving handicraft in Jepara Fig 1. Carving handicraft in Jepara Culture as an expression of ideas and ideas, activities and cultural products, which is sourced from cultural elements, that carving is one of the livelihoods that absorbs almost 40% of the active workforce in Jepara. From the aspect of livelihood, family management which initially colored the carving handicraft business slowly adopted a more modern business management. The process of meeting these cultures, led to a culture of business management that is different from family business management and modern business management [2]. This acculturation process is what the writer wants to present through the results of research on handicraft industry players, as well as raising local values that are believed to have wisdom. How do these local values survive in the onslaught of new, more modern values. Will it be able to survive, in what form. This paper tries to present local values that can still be found in some traditions of carving practitioners. The role of entrepreneurs in a Jepara carving handicraft industry is, of course, related to the entrepreneurial spirit inherent in it based on local socio-cultural values. Even this condition becomes interesting when local values interact with cultural values brought by migrants (foreign entrepreneurs). So that it is possible to mix the two cultural values that give rise to a new culture or often called the acculturation process [3]. Of course it is new to see how the process of mixing, and the efforts of old cultural values to survive with local wisdom that is owned. Entrepreneurship is an entrepreneurial attitude and behaviour, someone who is innovative, anticipatory, initiative, risk-taking and profit-oriented [4]. In this way the entrepreneurial spirit is related to the spirit, attitude, behaviour and enthusiasm of a person in handling businesses or activities that lead to efforts to find, create, apply new ways of working, technology and production by increasing efficiency in order to provide better services and or gain the greater one. During the 1990s, the marketing of the carving industry was much enlivened by demand from abroad at quite high prices. 2 Data Collection Method In this study, data collection consisted of primary data and secondary data. Primary data collection methods are in-depth interviews (in depth interview) and participant observation. In this case, in-depth interviews were conducted with informants that included carving industry players consisting of elements of labor / workers, artisans, entrepreneurs (local and foreign), intermediary / liaison / broker / traders, and community leaders both formal and informal. This in depth interview was conducted to explore and discover the social and cultural values associated with economic activities in the carving industry in Jepara. The selection of informants is preceded by creating categories that can represent elements of the carving industry, especially those who begin to have capacity as entrepreneurs with indicators that the informants are workers in the carving handicraft industry and have the ability to market them by involving several workers under their coordination. Thus the informant has an understanding of production, marketing and leading a business. For participant observation conducted at the research location, namely: Jepara City, especially in the Mantingan Village as the embryo of carving art, Annual Village and Senenan which is a traditional market for carving handicrafts, Mlonggo Village which is an area of carving handicraft industry development. In participant observation, researchers carefully observe all activities related to the carving industry through direct observation and accompanying / joining together with informants who are carrying out their activities. The main target of observations other than carving handicraft industry products is to include activities carried out by groups of local entrepreneurs. Meanwhile secondary data was collected from written sources both archives, documents, newspapers, magazines, official reports from relevant agencies regarding a general description of the potential of the region and community activities related to the carving industry. Data from archives, documents, personal records, official reports from relevant agencies regarding events that have occurred related to the history of the development of the carving industry, can be constructed so as to obtain a clear picture of past collective experience. The data contained in documentary material was not only intended for historical research, but could also be used in research in the social sciences and culture. This seems inseparable from the understanding that society as a phenomenon has a temporal dimension. Social and cultural systems in society consist of pre-predicted interactions and continuity. * Corresponding author : indrahti@gmail.com Indigenous entrepreneurs who do not have overseas networks only rely on local marketing and have difficulty in developing their businesses [5]. It is precisely in these conditions it will be seen how the ability of the entrepreneurial spirit of indigenous entrepreneurs to deal with changes in market conditions that are very rapid development. According to Erman Suherman in his book Entrepreneurship Learning Design, every successful entrepreneur must be equipped with four main things which include ability, courage, determination and creativity. So that an entrepreneur is required to remain oriented to the tasks and results by maintaining the quality that has become the standard, has an optimistic spirit and is supported by the flexibility to adjust the existing development 2 E3S Web of Conferences 202, 07065 (2020) ICENIS 2020 https://doi.org/10.1051/e3sconf/202020207065 conditions. These characteristics will be explored from the socio-cultural values that exist in Jepara carving entrepreneurs and the religious values maintained by the local community. conditions. These characteristics will be explored from the socio-cultural values that exist in Jepara carving entrepreneurs and the religious values maintained by the local community. Even the community besides having capacity as a consultant can also be a producer at the same time. The community becomes an arena for events that affect each other's patterns of behaviour and human life [6]. In this study the data collected consisted of primary data and secondary data. Primary data collection methods are in-depth interviews (in-depth interviews) and participant observation. In this case, in-depth interviews were conducted with informants including carving industry players consisting of elements of labour/workers, artisans, entrepreneurs (local and foreign), broker/traders, and community leaders both formal and informal. This in-depth interview was conducted to explore and discover the social and cultural values associated with economic activities in the carving industry in Jepara. 2 Data Collection Method In addition to the social system is a description of the past of customs, cultural values and institutions, also has the potential to make predictions of the future. 3 3 https://doi.org/10.1051/e3sconf/202020207065 E3S Web of Conferences 202, 07065 (2020) ICENIS 2020 E3S Web of Conferences 202, 07065 (2020) 3 Result and Discussion From the results of the study can be categorized several things that are part of the entrepreneurial spirit of Jepara carving entrepreneurs, including: 3.2 Aim for profit Based on the results of the interview, Jepara carving industry players have diverse work orientations, namely: to meet the necessities of life (primary needs), continuing the family business, gaining new experiences, meeting the increasing demands for material needs (Interview with H. Ahmad Kosim and Rasyidi). Those who are engaged in carving handicraft industries with an orientation to meet the increasing material needs, want to be able to have their own business, not working for others. The spirit to continue working at an age that is already in the pension category has indeed become one of the attractions to foster work spirit. Besides that, the spirit of work or entrepreneurial spirit is also motivated by challenges that are always present in the work [7]. Fig 3. Continuing family business Fig 3. Continuing family business The measure of success has indeed shifted. In the past, success was seen from honesty and discipline, as well as being on time in fulfilling orders. When it can convince people, then when people order will dare to give an advance for the purchase of raw materials or raw materials. Now, the measure of success is more viewed in terms of material. This will gradually reduce the element of trust. As a result, to get a job must have their own capital, because there are no customers who provide advance payments. The size of success of entrepreneurs in Nigeria is determined by internal factors, external factors also play an important role, such as lack of financial capital, inadequate infrastructure facilities, competition from large companies, unfavorables government policies, scarcity of machinery and spare parts and lack of raw materials. Internal barriers such as incompetent planning, skills poor organization, and limited knowledge are also obstacles [8]. Halal work is work done by establishing good relationships with partners, meaning "not eating one's own friends" (Interview with H. Umar Dhani). If you get a lot of luck, the fortune must be shared with colleagues, for example by dividing the work. In this way the fortune that is obtained even though it is reduced is halal (Interview with H. Ahmad Mukhlisin). The perception of ways that can be associated with the enjoyment and comfort of work is also the main thing in building construction work [9]. 3.1 Work as part of pray As is known that the majority of carving handicraft industry players in Jepara are Muslims. Therefore Islamic religious values are one source of work philosophy for them. Islamic values provide a very basic philosophy of work, work as pray. This philosophy of work as pray implies that work is not merely a matter of looking for material income, but also has a spiritual dimension. This will certainly affect the ways how people work. Work that has no spiritual dimension will easily plunge people into practices that justify the means and practices of exploitation when they employ others. Conversely, if work is considered to have a spiritual and religious dimension, then the work is not only responsible to humans but also to God. Thus honesty becomes the main element in doing work. Fig 2. Jepara carving craft business Fig 2. Jepara carving craft business The real philosophy of visiting can be used as a basis for establishing cooperation in the world of Jepara carving craft business. One informant stated that honesty and discipline are work philosophies or basic guidelines in working, with the hope that their business will run smoothly. In addition, people must work hard so that their business can be more developed and developed (Interview with H.M. Sarimin). There is a tendency that if the employer feels to lead, often the employee's respect for him will decrease. The form of relations between employers and workers is a relationship of mutual need (Interview with Iman Qusri). Among the entrepreneurs created friendships, especially if in the beginning they were friends. Meanwhile, the form of the relationship between the entrepreneur and the craftsman is not a relationship that is dependent. This is caused by the craftsmen cannot be bound by only one entrepreneur. They generally also deposit or supply goods to more than one entrepreneur. 4 4 E3S Web of Conferences 202, 07065 (2020) ICENIS 2020 https://doi.org/10.1051/e3sconf/202020207065 3.2 Aim for profit Thus the carving industry business also has a strong social dimension which is currently also facing challenges, because of the demands of a consumeristic lifestyle that encourage people to think of themselves by collecting as much wealth as possible without caring about others. The business that raises challenges also becomes one of the driving forces in the business. New demands provide opportunities for workers and employers to create creations [10]. The family dimension must also be strengthened to build a good spirit of national entrepreneurship. Corrective steps from cultural aspects must also be taken [11]. 5 5 https://doi.org/10.1051/e3sconf/202020207065 E3S Web of Conferences 202, 07065 (2020) ICENIS 2020 Fig 4. Wood craft waste Wood craft waste can be treated in four ways. They are reduction, reuse, recovery and disposal [12]. Reduction is try to reduce the volume, concentration, toxicity and hazard level of waste that spreads to the environment preventively at pollutant source. Reuse is reusing waste that can still be used as material. Recovery is repair damaged good or waste for reuse. Disposal is safe disposal of waste to the environment. Fig 4. Wood craft waste Fig 4. Wood craft waste Wood craft waste can be treated in four ways. They are reduction, reuse, recovery and disposal [12]. Reduction is try to reduce the volume, concentration, toxicity and hazard level of waste that spreads to the environment preventively at pollutant source. Reuse is reusing waste that can still be used as material. Recovery is repair damaged good or waste for reuse. Disposal is safe disposal of waste to the environment. 3.3 Rituals for looking business fortune Jepara carving industry players make the values of Islam as a spirit of work. According to them work is worship. At work, as Muslims, they are guided by the proposition that reads: "Work as if you will live forever and worship you as if you want to die tomorrow, and scatter all of you on earth to seek good fortune" Interview with Junoto). The proposition can be interpreted that a person must work to live a life in the world, without neglecting worship for matters in the hereafter. Thus there is a balance between work and worship. Although they are busy working to find a fortune to meet the needs of the world, one must not forget pray. Fortune is something that comes from God. Fortune is not only in the form of material, but can be in the form of health. Barokah is fortune that can be used for good (Interview with Suakhmadi). Although few in number but can be used to make ends meet, because it is obtained by lawful means and based on honesty. In other words, blessing that blessing is fortune obtained has more value, meaning that it can be useful or has a certain value, for example, bring peace. To get a blessing fortune, the Jepara carving industry performers perform selametan, tahlilan, manakiban, and pilgrimage. (Interview with Adib Faiz). These activities are also carried out for soul balance. 6 E3S Web of Conferences 202, 07065 (2020) ICENIS 2020 https://doi.org/10.1051/e3sconf/202020207065 Fig 5. Spirit of work Fig 5. Spirit of work 4 Conclusions The entrepreneurial spirit of Jepara carving industry entrepreneurs is much influenced by local cultural values, especially those related to religious values. This is evident from the understanding that work is part of worship that originates in Islam. Given that work is part of worship, in doing work must be based on honesty and not mutually harmful. For this reason, the enthusiasm of work is carried out by increasing the friendship in exchanging work information. 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Low, Crafted culture: governmental sculpting of modern Singapore and effects on business environment, Journal of Organizational Change Management, Vol. 11 No. 6, pp. 530-553, (1998) 12. Ed Suttie, Wood Waste Management – UK Update, Enviromental Optimisation of Wood Protection, pp. 1-10, (2004) Informants List 1. Name : H. M. Sarimin Age : 43 years old Address : Tahunan, Jepara 2. Name : Iman Qusri Age : 49 years old Address : Tahunan, Jepara 3. Name : H. Ahmad Khosim Age : 45 years old Address : Tahunan, Jepara 4. Name : Rasyidi Age : 34 years old Address : Tahunan, Jepara 5. Name : H. Umar Dhani Age : 43 years old Address : Tahunan, Jepara 6. Name : H. Ahmad Mukhlisin Age : 39 years old Address : Tahunan, Jepara 7. Name : Suakhmadi Age : 34 years old Address : Jepara 8. Name : Junoto Age : 45 years old Address : Tahunan, Jepara 9. Name : Adib Faiz Age : 35 years old Address : Tahunan, Jepara 8 8
https://openalex.org/W1851930522	https://europepmc.org/articles/pmc4645612?pdf=render	English	null	Hierarchal Autophagic Divergence of Hematopoietic System	Journal of biological chemistry/The Journal of biological chemistry	2,015	cc-by	11,281	Hierarchal Autophagic Divergence of Hematopoietic System* Recei ed for p blication March 5 2015 and in re ised form J l 22 2015 Published JBC Papers in Press August 5 2015 DOI 101074/jbcM115650028 Yan Cao, Suping Zhang, Na Yuan, Jian Wang, Xin Li, Fei Xu, Weiwei Lin, Lin Song, Yixuan Fang, Zhijian Wang, Zhen Wang, Han Zhang, Yi Zhang, Wenli Zhao, Shaoyan Hu, Xueguang Zhang, and Jianrong Wang1 From the Hematology Center of Cyrus Tang Medical Institute, Jiangsu Institute of Hematology, Jiangsu Key Laboratory for Stem Cell Research, Collaborative Innovation Center of Hematology, Affiliated Children’s Hospital, Soochow University School of Medicine, Suzhou 215123, China Background: Autophagy is required in hematopoiesis and protects against leukemogenesis. Results: When ATG7-dependent canonical autophagy is impaired, ATG7-independent alternative autophagy engages in mye- loid cells but not in hematopoietic stem cells. Conclusion: The integrity of hematopoietic stem cells is jeopardized by a lack of alternative autophagy. Significance: Learning autophagy organization in hematopoietic system is crucial for understanding hematopoietic stem cell transformation. Background: Autophagy is required in hematopoiesis and protects against leukemogenesis. Results: When ATG7-dependent canonical autophagy is impaired, ATG7-independent alternative autophagy engages in mye- loid cells but not in hematopoietic stem cells. l h f h ll d d b l k f l h g p gy q p p g g Results: When ATG7-dependent canonical autophagy is impaired, ATG7-independent alternative autophagy engages in mye- loid cells but not in hematopoietic stem cells. Conclusion: The integrity of hematopoietic stem cells is jeopardized by a lack of alternative autophagy. Significance: Learning autophagy organization in hematopoietic system is crucial for understanding hematopoietic stem cell transformation. Autophagy is integral to hematopoiesis and protects against leukemogenesis. However, the fundamentals of the required molecular machinery have yet to be fully explored. Using condi- tional mouse models to create strategic defects in the hemato- poietic hierarchy, we have shown that recovery capacities in stem cells and somatic cells differ if autophagy is impaired or flawed. An in vivo Atg7 deletion in hematopoietic stem cells completely ablates the autophagic response, leading to irrevers- ible and ultimately lethal hematopoiesis. However, while no adverse phenotype is manifested in vivo by Atg7-deficient mye- loid cells, they maintain active autophagy that is sensitive to brefeldin A, an inhibitor targeting Golgi-derived membranes destined for autophagosome formation in alternative auto- phagy. Removing Rab9, a key regulatory protein, in alternative autophagy, disables autophagy altogether in Atg7-deficient macrophages. 2 The abbreviations used are: HSC, hematopoietic stem cells; MACS, magnet- ic-activated cell sorting; LC, light chain; ROS, reactive oxygen species; pIpC, polyinosine-polycytosine; BFA, brefeldin A; HSPC, hematopoietic stem and progenitor cells. * This work was supported by grants from the National Natural Science Foun- dation of China (No.31071258, No.81272336, No.31271526, and No.31201073), The Ministry of Science and Technology of China (No. 2011CB512101), The Department of Science and Technology of Jiangsu Province of China (No. BK20130333), and a project funded by the Priority AcademicProgramDevelopmentofJiangsuHigherEducationInstitutions. The authors declare no conflicts of interest. Hierarchal Autophagic Divergence of Hematopoietic System* Recei ed for p blication March 5 2015 and in re ised form J l 22 2015 Published JBC Papers in Press August 5 2015 DOI 101074/jbcM115650028 Functional analysis indicates that ATG7-depen- dent canonical autophagy is physiologically active in both hema- topoietic stem cells and in terminally differentiated hematopoi- etic cells; however, only terminally differentiated cells such as macrophages are rescued by alternative autophagy if canonical autophagy is ineffective. Thus, it appears that hematopoietic stem cells rely solely on ATG7-dependent canonical autophagy, whereas terminally differentiated or somatic cells are capable of alternative autophagy in the event that ATG7-mediated autophagy is dysfunctional. These findings offer new insight into the transformational trajectory of hematopoietic stem cells, which in our view renders the autophagic machinery in stem cells more vulnerable to disruption. Hematopoietic stem cells (HSC),2 armed with the capacity to self-renew and generate somatic/differentiated progenies, are essential for normal hematopoiesis and are largely compelled to transform by imbalances in hematopoietic homeostasis. Autophagy is a conserved catabolic mechanism that protects cells by delivering potentially toxic macromolecular aggregates (e.g. proteins, lipids, and glycans) and damaged or superfluous organelles to lysosomes for degradation (1–6). Various stimuli, such as starvation, endoplasmic reticular stress, DNA damage, and reactive oxygen species, may trigger autophagy. Although studied extensively in somatic cells, our understanding of autophagy in stem cells is very limited. Deletion of the autophagy gene Beclin1 leads to early embryonic lethality (7). Recent research has implicated autophagy in hemostatic con- trol and maintenance of the capacity for self-renewal in stem cells. Autophagy is up-regulated during early differentiation of mouse and human embryonic stem cells (8, 9); is known to regulate maintenance, self-renewal, and differentiation of human mesenchymal stem cells (10, 11); and participates in somatic reprogramming (12, 13) and regulating stem-cell status as well (14). In short, autophagy is required for maintenance of HSCs (15–17). Deletion of essential autophagy genes Atg7 or Fip200 in mouse HSCs leads to defective self-renewal and dys- regulated myeloproliferation (15, 17). In addition, recent stud- ies of ours have shown that ATG7-dependent autophagy regu- lates cell cycles of HSCs and progenitor cells (18), promotes megakaryopoiesis, megakaryocyte differentiation, and throm- bopoiesis (19), and regulates hematopoiesis largely via direct targeting Notch (20). ATG7-dependent autophagy, or canonical autophagy, is characterized by lipidation and processing of microtubule-as- sociated protein light chain 3 (LC3) to form LC3-II, an essential step in autophagosme structuring (2). Previous investigations have documented an ATG5/ATG7-independent alternative autophagic mechanism in mouse embryonic fibroblasts, regu- VOLUME 290•NUMBER 38•SEPTEMBER 18, 2015 1 To whom correspondence should be addressed: Hematology Center of Cyrus Tang Medical Institute, Soochow University School of Medicine, 199 Ren’ai Road, Suzhou 215123, China. Tel/Fax: 86-512-65880877; E-mail: jrwang@suda.edu.cn. ssmark ssmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 38, pp. 23050–23063, September 18, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 38, pp. 23050–23063, September 18, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. ierarchal Autophagic Divergence of Hematopoietic System* eived for publication,March 5, 2015, and in revised form, July 22, 2015 Published, JBC Papers in Press,August 5, 2015, DOI 10.1074/jbc.M115.650028 n Cao, Suping Zhang, Na Yuan, Jian Wang, Xin Li, Fei Xu, Weiwei Lin, Lin Song, Yixuan Fang, Zhijian Wang, en Wang, Han Zhang, Yi Zhang, Wenli Zhao, Shaoyan Hu, Xueguang Zhang, and Jianrong Wang1 om the Hematology Center of Cyrus Tang Medical Institute, Jiangsu Institute of Hematology, Jiangsu Key Laboratory for Stem ll Research, Collaborative Innovation Center of Hematology, Affiliated Children’s Hospital, Soochow University School of dicine, Suzhou 215123, China Background: Autophagy is required in hematopoiesis and protects against leukemogenesis. Results: When ATG7-dependent canonical autophagy is impaired, ATG7-independent alternative autophagy engages in mye- loid cells but not in hematopoietic stem cells. Conclusion: The integrity of hematopoietic stem cells is jeopardized by a lack of alternative autophagy. Significance: Learning autophagy organization in hematopoietic system is crucial for understanding hematopoietic stem cell transformation. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 38, pp. 23050 23063, September 18, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Hierarchal Autophagic Divergence of Hematopoietic System* Received for publication,March 5, 2015, and in revised form, July 22, 2015 Published, JBC Papers in Press,August 5, 2015, DOI 10.1074/jbc.M115.650028 Yan Cao, Suping Zhang, Na Yuan, Jian Wang, Xin Li, Fei Xu, Weiwei Lin, Lin Song, Yixuan Fang, Zhijian Wang, Zhen Wang, Han Zhang, Yi Zhang, Wenli Zhao, Shaoyan Hu, Xueguang Zhang, and Jianrong Wang1 From the Hematology Center of Cyrus Tang Medical Institute, Jiangsu Institute of Hematology, Jiangsu Key Laboratory for Stem Cell Research, Collaborative Innovation Center of Hematology, Affiliated Children’s Hospital, Soochow University School of Medicine, Suzhou 215123, China Background: Autophagy is required in hematopoiesis and protects against leukemogenesis. Results: When ATG7-dependent canonical autophagy is impaired, ATG7-independent alternative autophagy engages in mye- loid cells but not in hematopoietic stem cells. Conclusion: The integrity of hematopoietic stem cells is jeopardized by a lack of alternative autophagy. ssmark Significance: Learning autophagy organization in hematopoietic system is crucial for understanding hematopoietic stem cell transformation. Stem Cell Autophagy Is More Vulnerable p q HSCs reside in niche locations and behave differently than differentiated blood cells that are actively exposed to a variety of intra- and extracellular stimuli. Despite a rapidly growing inter- est in autophagy, the potential divergence in the autophagic profiles of stem cells and somatic/differentiated cells is still fun- damentally unknown in mammalian systems. Through the use of conditional mouse models harboring autophagy-essential gene deletions in the hematopoietic hierarchy, we show that two distinct mechanisms of autophagy are operant. HSCs rely solely on canonical autophagy, which is ATG7-dependent and non-recoverable if impaired, whereas disruption of canonical autophagy in myeloid cells triggers an alternative compensa- tory pathway, thereby maintaining cellular viability and function. Confocal Microscopy Analysis of GFP-LC3 Fluorescence Puncta—Macrophages grown on coverslips were incubated with CD11b antibody after removing uncombined antibody using PBS. Cells were fixed in 4% formaldehyde for 30 min at room temperature prior to cell permeabilization with 0.1% Tri- ton X-100 (4 °C, 10 min). The cells were washed in PBS three times for 5 min, incubated on 0.1 g DAPI for 9 min, and then rinsed with PBS. Fluorescence signals were analyzed using con- focal microscope. Experimental Procedures Animals—Atg7f/f mice (kindly from Dr. Komatsu, Japan) (23) were crossed to Vav-Cre mice (Jackson Lab) to obtain Atg7f/f; Vav-Cre and Atgf/;Vav-Cre mice. Atg7f/f mice was crossed to Lyz-Cre mice (Jackson Lab) to obtain Atg7f/f;Lyz-Cre. Atg7f/f; Lyz-Cre mice was further crossed to GFP-LC3 transgenic mice (Jackson Lab) to obtain Atg7f/f;Lyz-Cre;GFP-LC3 mice. Atg7f/f mice was crossed to Mx1-Cre mice ((Jackson Lab) to obtain Atg7f/f;Mx1-Cre mice. Genotyping was performed on tail genomic DNA. Male and female mice were used equally in all experiments, and littermates were always used as controls. Each group contains at least 6 mice. All experiments with animals are complied with the institutional protocols on animal welfares and approved by the Ethics Committee of Soochow University. Reagents and Antibodies—CD11b-APC(553312), Ly-6G and Ly-6C-APC, Ter119-FITC, CD71-PE were from BD Biosci- ences; F4/80-PE(12–4801) was from eBioscience; Ly-6C-FITC was from Biolegend; AnnexinV-FITC PI Apoptosis Kit was from Biouniquer; M-CSF was from Sigma; Anti-Atg7, anti-Be- clin1, anti-PI3 Kinase Class III, and anti-GAPDH were from Cell Signaling Technology; anti-LC3 was from Medical & Bio- logical Laboratories. Animals—Atg7f/f mice (kindly from Dr. Komatsu, Japan) (23) were crossed to Vav-Cre mice (Jackson Lab) to obtain Atg7f/f; Vav-Cre and Atgf/;Vav-Cre mice. Atg7f/f mice was crossed to Lyz-Cre mice (Jackson Lab) to obtain Atg7f/f;Lyz-Cre. Atg7f/f; Lyz-Cre mice was further crossed to GFP-LC3 transgenic mice (Jackson Lab) to obtain Atg7f/f;Lyz-Cre;GFP-LC3 mice. Atg7f/f mice was crossed to Mx1-Cre mice ((Jackson Lab) to obtain Atg7f/f;Mx1-Cre mice. Genotyping was performed on tail genomic DNA. Male and female mice were used equally in all experiments, and littermates were always used as controls. Each group contains at least 6 mice. All experiments with animals are complied with the institutional protocols on animal welfares and approved by the Ethics Committee of Soochow University. Transmission Electron Microscopy of Autophagy in Mac- rophages—Macrophages were isolated from mice (each treat- ment sample requires 20 mice of each animal model) and the cell pellets from the macrophage pool were fixed in 3% glutar- aldehyde solution in 0.1 M phosphate buffer at 4 °C. After 2 h of fixation in osmium tetroxide (3%), the macrophages were then dehydrated in graded acetone, and embedded in Araldite (Fluka, Buchs, Switzerland). Ultrathin sections were prepared using a diamond knife, collected on copper grids (G 300 Cu), contrasted using both lead citrate and uranyl acetate, and then examined with a transmission electron microscope (JEM- 1010, JEOL, Japan). 23050 JOURNAL OF BIOLOGICAL CHEMISTRY 23050 JOURNAL OF BIOLOGICAL CHEMISTRY Stem Cell Autophagy Is More Vulnerable lated by proteins such as RAB9, Unc-51-like kinase 1 (ULK1), and Beclin1. Unlike canonical autophagy, autophagosomes are generated in a RAB9-dependent manner by the fusion of isola- tion membranes with vesicles of trans-Golgi and late endo- somal derivation (20, 21). ATG3-independent autophagy, which resembles the ATG7-deletion phenotype, has also been described (21, 22). Although canonical autophagy has been amply and intensively studied, and non-canonical or alternative autophagy similarly has been well documented, the particulars of these mechanisms in differing mammalian systems and the biological significance of their functional heterogeneity remain open to question. lated by proteins such as RAB9, Unc-51-like kinase 1 (ULK1), and Beclin1. Unlike canonical autophagy, autophagosomes are generated in a RAB9-dependent manner by the fusion of isola- tion membranes with vesicles of trans-Golgi and late endo- somal derivation (20, 21). ATG3-independent autophagy, which resembles the ATG7-deletion phenotype, has also been described (21, 22). Although canonical autophagy has been amply and intensively studied, and non-canonical or alternative autophagy similarly has been well documented, the particulars of these mechanisms in differing mammalian systems and the biological significance of their functional heterogeneity remain open to question. Blood Routine Examination—20 l of mouse peripheral blood was added into 500 l CPK-303A (37 °C). Blood routine examination was performed using sysmex KX-21N. p g y Forced in Vivo or ex Vivo Monocyte-Macrophage Differ- entiation—For forced in vivo monocyte-macrophage differen- tiation, mice were treated with thioglycolate. Basically, the thio- glycolate-activated macrophages were isolated from Atg7f/f; Lyz-Cre mice or wild-type mice by peritoneal lavage 1–4 days after intraperitoneal injection of 1 ml of 3% sterile thiogly- colate medium. The cells from thioglycolate-induced perito- nitis were costained with anti-CD11b and anti-F4/80, and F4/80CD11b populations were sorted by FACS. The thiogly- colate-induced macrophages were then incubated, respec- tively, with Baf-A1 (10 nM), BFA (0.1 ng/ml), and cells were cultured in RPMI 1640 with 10% FBS or RPMI 1640 without amino acids and serum for 1.5 h (for starvation). Myeloid cells were finally measured with flow cytometry using specific bio- markers. For forced ex vivo monocyte-macrophage differentia- tion, monocytes were first isolated from mice by FACS or MACS (Mouse Monocyte Enrichment Kit, Stem Cell), and then cultured in 10% FBS RPMI 1640 with or without M-CSF (25 ng/ml) for 72 h. BFA (0.1 ng/ml) or Baf-A1 (10 nM) was added into culture medium after cultured for 48 h. Differentiation was accessed using flow antibodies indicated. Results Atg7-gene Deletion Confers Differing Phenotypes in HSCs and Terminally Differentiated Hematopoietic Cells—Autophagy in HSCs and in differentiated hematopoietic cells was assessed through conditional mouse models by deleting a critical gene (Atg7) in the hematopoietic hierarchy (Fig. 1A). The Atg7 gene encodes an E1-like enzyme that functions in the two ubiquitin- like conjugation systems essential for autophagosome forma- tion (24). Whole-body homozygous Atg7 knock-out is lethal in mice neonates (23). In our mouse model, Atg7 deletion was selective, limited to the hematopoietic system, and was con- firmed by semi-quantitative real-time polymerase chain reac- tion (RT-PCR). The Vav gene encodes a guanine nucleotide exchange factor in HSCs (25, 26), and its promoter effectively directs exogenous gene expression in HSCs. In Atg7f/f;Vav-Cre mice, Atg7 deletion by Vav promoter-directed Cre expression occurs at stem-cell level. This leads to complete floxing of Atg7 throughout the hematopoietic system (Fig. 1B), given that all descendant hematopoietic cells originate from HSCs (27). In contrast, lysozyme M (Lyz) expression gradually increases dur- ing myeloid differentiation, peaking in mature granulocytes and macrophages (28, 29). In Atg7f/f;Lyz-Cre mice, Atg7 dele- tion is limited to myeloid-cell lineage, including granulocytes, monocytes, and mature macrophages, whereas early hemato- poietic cells, including stem cells and progenitors, retain intact Atg7 alleles (Fig. 1C) (30, 31). In Vitro Phagocytosis Assay—Bone marrow CD11b cells were combined with reconstituted Escherichia coli that expressed EGFP and incubated at a ratio of 10:1 (E. coli:macro- phages) at 37 °C. After 2 and 4 h, macrophage phagocytosis was analyzed by flow cytometry. Blood Smear—Blood smears are made by placing a drop of blood on one end of a slide, and using a spreader slide to dis- perse the blood over the slide’s length. The slide is left to air dry, after which the blood is fixed to the slide by immersing it briefly in methanol and performed on blood smears stained with Giemsa stain. After staining, the monolayer is viewed under a microscope using magnification up to 400. Individual cells are examined, and their morphology is characterized and recorded. Measurement of Reactive Oxygen Species—DCFH-DA dis- solved in serum-free medium was added at 10 M (final con- centration). Cells were resuspended in serum-free medium containing DCFH-DA and incubated at 37 °C 15 min, then ana- lyzed by FACS-Calibur Determination of ROS production. Cells were incubated with CM-H 2DCFDA (Molecular Probes) in the dark for 15 min at 37 °C. FIGURE 1. Differential deletion of Atg7 in stem cells and terminally differentiated cells of hematopoietic system reveals different phenotypes. A, scheme for deferential deletion of atg7 in mouse hematopoietic hierarchy. B, identification of Atg7f/f;Vav-Cre mice. Atg7 gene is deleted in bone marrow LSK hematopoietic stem and progenitor cells. C, identification of Atg7f/f;Lyz-Cre mice. Atg7 gene is deleted in the myeloid cells including granulocytes, monocytes, and macrophages, but their upstream cells (bone marrow lineage negative and positive cells) maintain atg7 transcript. The designated types of cells from the mouse models were directly sorted from bone marrow cells by FACS, and the presence of Atg7 transcript was determined by RT-PCR. D, Atg7 deletion in hematopoietic stem cells, not myeloid cells, caused mouse death, but atg7 deletion in myeloid cells did not affect mouse death under nutrient-rich or starvation conditions. For starvation, mice were provided with water but without feed. E, blood routine examination of Atg7f/f;Vav-Cre mice and Atg7f/f;Lyz-Cre mice. F, flow cytometric analysis of the percentage of bone marrow myeloid cells (CD11b) and granulocyte (Ly-6G and Ly-6C) in Atg7f/f;Vav-Cre and wild-type mice. G, flow cytometric analysis of bone marrow and peripheral blood myeloid cells (CD11b) and granulocyte (Ly-6G and Ly-6C) in Atg7f/f;Lyz- Cre and wild-type mice. H, apoptosis of myeloid cells from the mouse models was assessed using Annexin V/PI staining on flow cytometer. I, flow cytometric analysis of ROS levels in Lin cells of Atg7f/f;Vav-Cre mice or myeloid cells of Atg7f/f;Lyz-Cre mice. Data are representative or statistical results of three experiments. n 6. , p 0.05; , p 0.01. Stem Cell Autophagy Is More Vulnerable min at 4 °C. The protein was detected by immunoblotting. Equal amounts of protein were loaded on a 12% SDS-polyacryl- amide gel electrophoresis (SDS-PAGE) and transferred to poly- vinylidene difluoride membrane (Millipore). Anti-Atg7, anti- LC3 polyclonal antibodies were revealed using an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling) and detected by an enhanced chemilumines- cence kit (Pierce). In conjunction, blots were probed with anti- GAPDH antibody (Cell Signaling) to confirm equal loading of protein. Statistical Analysis—The significance of differences was determined by Student’s t test or one-way ANOVA followed by Tukey test. p 0.05 was considered statistically significant. min at 4 °C. The protein was detected by immunoblotting. Equal amounts of protein were loaded on a 12% SDS-polyacryl- amide gel electrophoresis (SDS-PAGE) and transferred to poly- vinylidene difluoride membrane (Millipore). Anti-Atg7, anti- LC3 polyclonal antibodies were revealed using an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling) and detected by an enhanced chemilumines- cence kit (Pierce). In conjunction, blots were probed with anti- GAPDH antibody (Cell Signaling) to confirm equal loading of protein. Experimental Procedures Reagents and Antibodies—CD11b-APC(553312), Ly-6G and Ly-6C-APC, Ter119-FITC, CD71-PE were from BD Biosci- ences; F4/80-PE(12–4801) was from eBioscience; Ly-6C-FITC was from Biolegend; AnnexinV-FITC PI Apoptosis Kit was from Biouniquer; M-CSF was from Sigma; Anti-Atg7, anti-Be- clin1, anti-PI3 Kinase Class III, and anti-GAPDH were from Cell Signaling Technology; anti-LC3 was from Medical & Bio- logical Laboratories. AnnexinV/PI Apoptosis Assays—Cells were costained with FITC-annexin V and propidium iodide (PI) according to the manufacturer’s protocol and analyzed with a flow analyzer. Mouse Primary Monocytes Isolation from Bone Marrow— Bone marrow cells were isolated by removing leg bones from muscles. Cells were detected by flow analysis. In addition, pure mouse primary monocytes were sorted against anti-Ly6C and anti-F4/80. Flow Cytometry—Flow cytometry experiments were per- formed with BD fluorescence-activated cell sorting (FACS) Calibur or BD FACS Aria III, followed by magnetic-activated cell sorting (MACS). Real-time PCR—Total RNA was extracted with TRIzol re- agent according to the manufacturer’s guidelines (Invitrogen), and reverse transcription was performed with Thermo Scien- tific Revert Aid First Strand cDNA Synthesis Kit on an ABI 7500 instrument. Samples were run in triplets. Western Blotting Analysis—Cells were cultured and lysed in lysis buffer (Cell Signaling) containing protease inhibitor mix- ture and PhosSTOP phosphatase inhibitor mixture (Roche) on ice for 30 min. Crude lysates were obtained by centrifugation centrifuged at 13,000 g for 20 min and heated at 95 °C for 5 SEPTEMBER 18, 2015•VOLUME 290•NUMBER 38 23051 JOURNAL OF BIOLOGICAL CHEMISTRY Stem Cell Autophagy Is More Vulnerable VOLUME 290•NUMBER 38•SEPTEMBER 18, 2015 23052 JOURNAL OF BIOLOGICAL CHEMISTRY Results After washing, cells were ana- lyzed by flow cytometry using a FACS Calibur (BD Biosciences). Data were analyzed by using the CellQuest program (BD Biosciences). Phenotypic analysis revealed that the Atg7f/f;Vav-Cre mice developed progressive anemia and splenomegaly, survived for a mean of only 12 weeks, and were sterile (data not shown), con- sistent with previous reports (16, 17). However, survival rates of wild-type and Atg7f/f:Lyz-Cre mice did not differ under nutri- ent-rich or starvation conditions (Fig. 1D), suggesting that autophagy in macrophages of Atg7f/f;Lyz-Cre mice remain functional and respond to starvation. As shown by blood smears and cell counts, Atg7f/f;Vav-Cre mice developed wors- ening anemia, whereas Atg7f/f;Lyz-Cre mice did not differ from wild-type in this regard (Fig. 1E). Lentivirus-mediated RNA Interference—Lentiviral vectors to silence Rab9 together with negative control vector (shRNA- NC) were purchased from GenePharma Co, Ltd (Shanghai, China). The sequences to silence Rab9 were shRNA: 5-GGA- GGCAGTTCGAAGAATTCTT-3. Virus was produced by co-transfection of 293T cells with shRab9 or shRNA-NC and packaging plasmids. For viral infection, viral supernatant were filtered, collected and used to infect macrophages. Monocytes were cultured in M-CSF medium for 48 h, and lentivirus was then added into culture medium. After 48 h, GFP macroph- ages were sorted for further Q-PCR and indicated treatment. The Q-PCR primers used were mouse Rab9, the forward primer was 5-TACCGGGGTTCTGACTGTTG-3, and the reverse primer was 3-GGCTCTTTCACATCTGCGTAATA-5. We also observed that autophagic defects in HSCs of the Atg7f/f;Vav-Cre mouse model culminated in irreversible failure of hematopoiesis, again in agreement with previous findings (16, 17), whereas the ATG7-mediated autophagy defects in Atg7f/f;Lyz-Cre mice did not result in peripheral blood changes relative to wild-type mice (data not shown). To validate these peripheral blood findings, bone marrow samples were analyzed SEPTEMBER 18, 2015•VOLUME 290•NUMBER 38 23053 JOURNAL OF BIOLOGICAL CHEMISTRY 23053 SEPTEMBER 18, 2015•VOLUME 290•NUMBER 38 Stem Cell Autophagy Is More Vulnerable 2A), with near-complete floxing of the Atg7 gene in these cells (Fig. 2B). To determine if any lingering ATG7 protein in macrophages of Atg7f/f;Lyz-Cre mice was capable of sustaining canonical autophagy, or if biallelic deletion of the Atg7 gene in macro- phages of Atg7f/f;Lyz-Cre mice was sufficient to disrupt this process, comparative analysis of monoallelic Atg7 deletion in HSCs of another conditional Atg7 knock-out mouse model (Atg7f/;Vav-Cre) was performed. Although 50% of ATG7 protein remained in bone marrow lineage-negative cells of Atg7f/;Vav-Cre mice (Fig. 2C), monoallelic Atg7 deletion in this mouse model resulted in hematopoietic stem-cell exhaus- tion, signaled by higher percentages of LSKCD34 cells and LK cells and subsequent reduction in colony-forming ability (Fig. 2D). Hence, HSCs of heterozygous Atg7f/;Vav-Cre mice proved relatively sensitive to disrupted ATG7-dependent autophagy, even with partial loss of ATG7 protein. By all accounts, biallelic Atg7 deletion in myeloid cells of Atg7f/f;Lyz-Cre mice should disrupt ATG7-dependent autophagy, having lost the vast majority of ATG7 protein (Fig. 2B). To our surprise, flow cytometry revealed that percent- ages of monocytes (Ly6CF4/80), mature macrophages (Ly6CF4/80), and progenitors at monocyte-macrophage stage of differentiation (Ly6CF4/80) in bone marrow of Atg7f/f;Lyz-Cre and wild-type mice (Fig. 2E) did not differ. Thus, an ATG7-dependent autophagic defect in Atg7f/f;Lyz- Cre mice did not compromise in vivo physiologic monocyte- macrophage differentiation. Given the above outcomes, we then examined myeloid cells in Atg7f/f;Lyz-Cre mice, checking their responsiveness to extra- cellular stimuli. Phagocytic activity in bone marrow or perito- neal macrophages of Atg7f/f;Lyz-Cre and wild-type mice was investigated by ex vivo addition of GFP-expressing E. coli to macrophage cultures. Uptake of GFP-expressing E. coli by mye- loid cells was comparable for both strains (data not shown). This supported our prior observation that survival of Atg7f/f; Lyz-Cre mice harboring an Atg7 deletion in myeloid cells is not compromised under starvation conditions (Fig. 1D). p g To determine if ATG7-dependent autophagy is actually functional during physiologic monocyte-macrophage differen- tiation, a forced in vivo differentiation assay was done by treat- ing wild-type and Atg7f/f;Lyz-Cre mice with thioglycolate, an inducer of monocytic differentiation (34). Mice were sacrificed on Days 1 and 3 post-treatment, and thioglycolate-exposed peritoneal cells were collected for flow analysis. In wild-type mice, the percentage of Ly6CF4/80 macrophages increased from Day 1 to Day 3, whereas the percentage of Ly6CF4/80 monocytes decreased, reflecting in vivo physiologic monocyte- macrophage differentiation in response to the induction agent. FIGURE 2. Atg7 deletion in myeloid cells cripples the artificial but not physiological induction of monocyte-macrophage differentiation. A, sorting for myeloid cells at the monocyte-macrophage differentiation stage (Ly6CF4/80). Bone marrow cells from Atg7f/f;Lyz-Cre mice and wild-type mice were sorted against the markers with FACS, shown by representative sorting plots (left). The purity of the sorted cells from these two mouse models is shown (right). B, examination of the efficiency for atg7 gene floxing in macrophages of the mouse models as indicated, and the majority of atg7 protein is absent due to the floxing of atg7 gene. To obtain sufficient macrophages for analysis, macrophages were sorted with FACS after M-CSF induction of mononuclear cells isolated with MACS from bone marrow cells. Shown is a representative Western blotting result (left) and quantified data (right). C, examination of the efficiency for atg7 gene floxing in HSPCs of Atg7f/f;Vav-Cre mice and Atg7f/;Vav-Cre mice by Western analysis (left), and their protein levels were quantified (right). Monoallelic deletionofatg7onlypartiallyreducedatg7proteinlevelbutbiallelicdeletioncausedcompleteabsenceofatg7proteininHSPCs.D,monoallelicdeletionofthe atg7 gene in Atg7f/;Vav-Cre mice caused hematopoietic stem cell exhaustion. Percentage of LSKCD34 and LK cells were measured by flow cytometry (left and middle panels). Colony-forming ability of LSK cells from wild-type mice and Atg7f/;Vav-Cre mice were determined, and the result was quantified (right panel).E,flowcytometricanalysisofthepercentageofmonocytes(Ly-6CF4/80)andmacrophages(Ly-6CF4/80)frombonemarrowcellsofAtg7f/f;Lyz-Cre mice and wild-type mice. F, examination of artificial induced monocyte-macrophage differentiation by thioglycolate in Atg7f/f;Lyz-Cre mice and wild-type mice. Differentiation was measured by flow cytometry on days 1 and 3 after induction. G, morphological changes associated with or without M-CSF (25 ng/ml)-induced monocyte-macrophage differentiation.H–J, analysis of differentiation and apoptosis of the atg7-deleted monocytes. Primary monocytes from Atg7f/f;Lyz-Cre and wild type mice were sorted against the indicated markers and incubated with or without M-CSF (25 ng/ml) for 72 h, the differentiation and apoptosis levels of monocytes were measured by flow cytometry. Data are representatives or statistical results of three experiments. n 6. , p 0.05; *, p 0.01. Stem Cell Autophagy Is More Vulnerable In Atg7f/f;Lyz-Cre mice, the percentage of Ly6CF4/80 macrophages or Ly6CF4/80 monocytes surprisingly showed no obvious change (Fig. 2F), clearly representing a failure of artificial induction in this setting and underscoring the in vivo ATG7-dependent Autophagic Defect in Myeloid Cells Cripples Artificial but Not Physiologic Induction of Mono- cyte-Macrophage Differentiation—Focusing on canonical autophagy in myeloid cells, the effect of an ATG7-dependent autophagic defect on terminal differentiation of monocytes was examined. Fluorescence-activated cell sorting at monocyte- macrophage stage of differentiation (Ly6CF4/80) in bone FIGURE 2. Atg7 deletion in myeloid cells cripples the artificial but not physiological induction of monocyte-macrophage differentiation. A, sorting for myeloid cells at the monocyte-macrophage differentiation stage (Ly6CF4/80). Bone marrow cells from Atg7f/f;Lyz-Cre mice and wild-type mice were sorted against the markers with FACS, shown by representative sorting plots (left). The purity of the sorted cells from these two mouse models is shown (right). B, examination of the efficiency for atg7 gene floxing in macrophages of the mouse models as indicated, and the majority of atg7 protein is absent due to the floxing of atg7 gene. To obtain sufficient macrophages for analysis, macrophages were sorted with FACS after M-CSF induction of mononuclear cells isolated with MACS from bone marrow cells. Shown is a representative Western blotting result (left) and quantified data (right). C, examination of the efficiency for atg7 gene floxing in HSPCs of Atg7f/f;Vav-Cre mice and Atg7f/;Vav-Cre mice by Western analysis (left), and their protein levels were quantified (right). Monoallelic deletionofatg7onlypartiallyreducedatg7proteinlevelbutbiallelicdeletioncausedcompleteabsenceofatg7proteininHSPCs.D,monoallelicdeletionofthe atg7 gene in Atg7f/;Vav-Cre mice caused hematopoietic stem cell exhaustion. Percentage of LSKCD34 and LK cells were measured by flow cytometry (left and middle panels). Colony-forming ability of LSK cells from wild-type mice and Atg7f/;Vav-Cre mice were determined, and the result was quantified (right panel).E,flowcytometricanalysisofthepercentageofmonocytes(Ly-6CF4/80)andmacrophages(Ly-6CF4/80)frombonemarrowcellsofAtg7f/f;Lyz-Cre mice and wild-type mice. F, examination of artificial induced monocyte-macrophage differentiation by thioglycolate in Atg7f/f;Lyz-Cre mice and wild-type mice. Differentiation was measured by flow cytometry on days 1 and 3 after induction. G, morphological changes associated with or without M-CSF (25 ng/ml)-induced monocyte-macrophage differentiation.H–J, analysis of differentiation and apoptosis of the atg7-deleted monocytes. Primary monocytes from Atg7f/f;Lyz-Cre and wild type mice were sorted against the indicated markers and incubated with or without M-CSF (25 ng/ml) for 72 h, the differentiation and apoptosis levels of monocytes were measured by flow cytometry. Data are representatives or statistical results of three experiments. n 6. , p 0.05; *, p 0.01. Stem Cell Autophagy Is More Vulnerable Stem Cell Autophagy Is More Vulnerable ex vivo monocyte-macrophage differentiation (33). Not unex- pectedly, loss of autophagy in myeloid cells of the Atg7f/f;Lyz- Cre mouse model would thus have profound consequences in terms of in vivo terminal differentiation. To explain why in vivo Atg7 deletion in myeloid cells did not culminate in dys- functional terminal differentiation, we examined the poten- tial influence of this deletion on intrinsic myeloid cell func- tion in Atg7f/f;Lyz-Cre mice. To this end, we compared myeloid-cell apoptotic response in Atg7f/f;Vav-Cre and Atg7f/f;Lyz-Cre strains, using wild-type mice as controls. Bone marrow and peripheral blood cells collected from each subset were stained, using anti-CD11b antibody and PI/An- nexin V. Although the ratio of apoptotic CD11b-positive cells (monocytes/macrophages) in wild-type and Atg7f/f; Lyz-Cre mice did not differ (data not shown), Atg7 deletion in HSCs of Atg7f/f;Vav-Cre mice clearly boosted apoptosis (Fig. 1H). Previous studies have shown that the absence of autophagy leads to mitochondrial accumulation of reactive oxygen species (ROS) (34, 35). We thus measured ROS level in the CD11b-positive cells of wild-type and Atg7-deficient mice. Again, Atg7f/f;Lyz-Cre and wild-type mice did not dif- fer in ROS clearance (data not shown), suggesting that the capacity for ROS clearance in myeloid cells is not under- mined by an ATG7-dependent autophagic defect. In con- trast, Atg7 deletion in HSCs from Atg7f/f;Vav-Cre mice ablated ROS clearance, as evident by accumulation of ROS (Fig. 1I). marrow reached at least 95% of respective myeloid-cell totals for both Atg7f/f:Lyz-Cre and wild-type mice (Fig. 2A), with near-complete floxing of the Atg7 gene in these cells (Fig. 2B). To determine if any lingering ATG7 protein in macrophages of Atg7f/f;Lyz-Cre mice was capable of sustaining canonical autophagy, or if biallelic deletion of the Atg7 gene in macro- phages of Atg7f/f;Lyz-Cre mice was sufficient to disrupt this process, comparative analysis of monoallelic Atg7 deletion in HSCs of another conditional Atg7 knock-out mouse model (Atg7f/;Vav-Cre) was performed. Although 50% of ATG7 protein remained in bone marrow lineage-negative cells of Atg7f/;Vav-Cre mice (Fig. 2C), monoallelic Atg7 deletion in this mouse model resulted in hematopoietic stem-cell exhaus- tion, signaled by higher percentages of LSKCD34 cells and LK cells and subsequent reduction in colony-forming ability (Fig. 2D). Hence, HSCs of heterozygous Atg7f/;Vav-Cre mice proved relatively sensitive to disrupted ATG7-dependent autophagy, even with partial loss of ATG7 protein. marrow reached at least 95% of respective myeloid-cell totals for both Atg7f/f:Lyz-Cre and wild-type mice (Fig. Stem Cell Autophagy Is More Vulnerable by flow cytometry for specific markers, namely CD11b (mono- cytes/macrophages) or Ly6G/C (granulocytes). Subsequently, Atg7f/f:Vav-Cre mice displayed marked myeloproliferation (Fig. 1F), but there were no signs of altered differentiation in Atg7f/f;Lyz-Cre mice (Fig. 1G). The genetic and phenotypic data imply that Atg7 deletion in HSCs induces irreversible impairment in hematopoiesis and eventual animal death; how- ever, this outcome does not occur in myeloid cells with this deletion. We have reported that autophagy is required for terminal differentiation of myeloid cells in vitro (31, 32) and for mega- karyocytic differentiation in vivo (19) Another recent study involving exposure to an inducer of monocytic differentiation has demonstrated that induction of autophagy is necessary for 23054 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290•NUMBER 38•SEPTEMBER 18, 2015 VOLUME 290•NUMBER 38•SEPTEMBER 18, 2015 VOLUME 290•NUMBER 38•SEPTEMBER 18, 2015 23054 JOURNAL OF BIOLOGICAL CHEMISTRY 23054 JOURNAL OF BIOLOGICAL CHEMISTRY Stem Cell Autophagy Is More Vulnerable physiologic role of ATG7-dependent autophagy in myeloid cells. In turn, this indicated that Atg7 deletion in myeloid cells of Atg7f/f;Lyz-Cre mice had the intended effect. Although per- centages of monocytes and macrophages in Atg7f/f;Lyz-Cre and wild-type mice did not differ under physiologic conditions, ter- minal differentiation patterns were not identical with artificial stimulation. Subsequent to thioglycolate treatment, mice with defective ATG7-dependent autophagy had much lower mono- cyte counts (Ly6CF4/80) and much larger macrophage pop- ulations (Ly6CF4/80) than wild-type mice on Day 1 of treat- ment; and because these levels held steady through Day 3 (Fig. 2F), it is likely that autophagy was sustained though an alternate pathway, ensuring homeostasis despite the terminal differenti- ation failure imposed by Atg7 deletion. autophagy is needed for artificial induction of monocyte- macrophage differentiation in vivo and ex vivo. Autophagy Is Maintained in Myeloid Cells Not HSCs Despite Impaired or Dysfunctional ATG7-dependent Mechanism— Biallelic Atg7 deletion in HSCs of Atg7f/f;Vav-Cre homologous mice resulted in loss of Atg7 in all descendant cells of the hema- topoietic hierarchy (Figs. 1B and 2C), leading to a complete lack of LC3 lipidation, total absence of LC3-II (Fig. 3A), general fail- ure of hematopoiesis, and animal demise (Fig. 1, D and C). However, HSCs of Atg7f/;Vav-Cre heterozygous mice with monoallelic Atg7 deletion are still capable of active LC3 lipida- tion (Fig. 3A), despite an overriding functional ablation (Fig. 2D. Thus, even subtotal Atg7 gene deletions impair HSC func- tion. Similarly, macrophages of Atg7f/;Vav-Cre heterozygous mice with monoallelic Atg7 deletion displayed severely impaired canonical autophagic flux in response to starvation or inhibition by bafilomycin A1 (Baf-A1), which targets the deg- radation stage of ATG7-dependent autophagy (Fig. 3B). Nota- bly, constitutive conversion of LC3-II from LC3-I was sustained at high levels in Atg7/ macrophages of the heterozygous mouse model, even without activation of autophagy via starva- tion (Fig. 3B). Consequently, LC3 conversion is a reasonable index of ATG7-dependent (canonical) autophagy, rather than a bona fide index of ATG7-independent (noncanonical) autophagy. Similarly, crippled canonical autophagic flux was evident in macrophages of Atg7f/f;Lyz-Cre homologous mice with biallelic Atg7 gene deletions; and Atg7 floxing was fairly efficient in these mice, as indicated by Western blot analysis (Fig. 3C). To further test for sustained differentiation of monocytes with ATG7-dependent autophagic defects, we performed an ex vivo monocyte-macrophage differentiation assay. Stem Cell Autophagy Is More Vulnerable Ly6CF4/ 80 monocytes in bone marrow cellular isolates mice were enriched by flow sorting and then selectively treated with macrophage colony-stimulating factor (M-CSF) to induce monocyte-macrophage differentiation. Morphology of cells in Atg7f/f;Lyz-Cre and wild-type mice did not differ. Untreated monocytes from both wild-type and Atg7-deficient mice were rounded and gradually died, whereas monocytes cultured with M-CSF differentiated into clusters of macrophages (Fig. 2G). As evident by the percentage of Ly6CF4/80 cells, differentiation in Atg7-deficient myeloid cells, was partially crippled, with or without M-CSF treatment (Fig. 2H). In response, Atg7 deletion in myeloid cells blocked monocyte-macrophage differentia- tion, resulting in diminished expression of the CD11b (mono- cyte/macrophage) marker (Fig. 2I) and a higher percentage of apoptotic cells (Fig. 2J). These findings are consistent with those of an earlier study, reporting apoptotic cell death in cir- culating monocytes that fail to differentiate (34). Likewise, they confirm that ATG7-dependent autophagy is essential for ex vivo induction of monocyte-macrophage differentiation and again illustrate the ATG7-dependent autophagic defect in mye- loid cells of Atg7f/f;Lyz-Cre mice. For visual examination of in vivo autophagic response in Atg7-deficient myeloid cells, we created an Atg7f/f;Lyz-Cre; GFP-LC3 mouse model from Atg7f/f;Lyz-Cre conditional mice and GFP-LC3 transgenic mice. Driven by CAG, a ubiquitous promoter, the latter express GFP-LC3 in a host of cell types, including myeloid cells (36). Confocal microscopic results showed punctate dispersion of GFP-LC3 in Atg7/ wild-type macrophages of GFP-LC3 transgenic mice, which was not pres- ent in Atg7/ macrophages of Atg7f/f;Lyz-Cre;GFP-LC3 mice under starvation conditions. The puncta observed in wild-type macrophages were due to inhibition of autophagosome-lyso- some fusion, with Baf-A1 accumulating in autophagosomes. However, Baf-A1 did not accumulate in GFP-LC3 puncta of Collectively, above results provide substantial evidence that an Atg7 deletion in HSCs readily inflicts a functional abnormity. On the other hand, knock-out of Atg7 in myeloid cells does not compromise cell function, although ATG7-dependent FIGURE 3. Macrophages but not hematopoietic stem and progenitor cells maintain autophagic response when atg7 is deleted. A, effect of monoallelic or biallelic deletion of atg7 gene in stem and progenitor cells on autophagy response. Western analysis on HSPCs from homogenous Atg7f/f;Vav-Cre mice, Atg7f/;Vav-Creheterozygousmiceandwild-typemiceshowthatbiallelicdeletionofagt7geneinHSPCscausedcompletelossofLC3-IIconversionfromLC3-I, but monoallelic deletion of atg7 gene maintained the LC3 lipidation and processing. B, effect of monoallelic deletion of atg7 gene directed by Vav promoter in macrophages on autophagy response. Stem Cell Autophagy Is More Vulnerable SEPTEMBER 18, 2015•VOLUME 290•NUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 23055 SEPTEMBER 18, 2015•VOLUME 290•NUMBER 38 Stem Cell Autophagy Is More Vulnerable 6 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290•NUMBER 38•SEPTEMBER 18, 20 VOLUME 290•NUMBER 38•SEPTEMBER 18, 2015 23056 JOURNAL OF BIOLOGICAL CHEMISTRY Stem Cell Autophagy Is More Vulnerable Western analysis on macrophages from heterozygous Atg7f/;Vav-Cre mice shows that monoallelic deletion of agt7 gene caused loss of canonical autophagic flux response, albeit constitutive LC3-II conversion from LC3-I maintained. For starvation, serum was deprived for 90 min. For inhibition on atg7-dependent canonical autophagy, bafilomycin A1 of 10 nM was used. The upper panel is a representative Western blotting result, and the lower panel is quantified results from three independent experiments. C, effect of biallelic deletion of atg7 gene directed by Lyz promoter in macrophages on autophagy response. The primary macrophages were lysed after being plated in the culture plate for 2 h under starvation or with/without Baf-A1 (10 nM) treatment and were then immunoblotted with antibodies against Atg7 and LC3 (upper panel). Autophagic flux was quantified (lower panel). Shown is a representative result of three independent experiments. GAPDH served as a loading control in experiments A, B, and C. D, confocal microscopic analysis of GFP-LC3 localization in the macrophages of Atg7f/f;Lyz-Cre;GFP-LC3 mice and GFP-LC3 transgenic mice. The GFP-LC3 transgenic mice served as a control. Representative images are shown for non-starvation (left panel) and for starvation 1.5 h (middle panel) with or without 10 nM of Baf-A1 (right panel). The nucleus was stained with DAPI (blue), CD11b (red) is a marker for macrophages. GFP-LC3 (green) is expressed in all tissue cells including macrophages of the GFP-LC3 transgenic mice. E, macrophages acid phosphatase activity (AcPA) in wild-type and Atg7f/f;Lyz-Cre mice was measured by spectrophotometry. F, transmission electron microscopic analysis of macrophage autophagy in response to starvation treated with or without bafilomycin A1 (Baf, 10 nM), a specific inhibitor on atg7-dependent canonical autophagy. Macrophages were isolated from wild-type mice or Atg7f/f;Lyz-Cre mice and followed by starvation for 3 h. Autopha- gosomes and autolysosomes are indicated with arrows. Data are representatives or statistical results of three experiments. n6. , p 0.05; *, p 0.01. SEPTEMBER 18, 2015•VOLUME 290•NUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 23057 SEPTEMBER 18, 2015•VOLUME 290•NUMBER 38 Stem Cell Autophagy Is More Vulnerable Stem Cell Autophagy Is More Vulnerable Atg7-deficient macrophages (Fig. 3D). These confocal data fur- ther confirm that Atg7 deletion in myeloid cells impairs the ATG7-dependent canonical autophagic machinery. Cre mice bearing monoallelic (rather than biallelic) Atg7 dele- tion (Fig. 4C), indicating differing capacities for compensatory autophagy in somatic cell types. Cre mice bearing monoallelic (rather than biallelic) Atg7 dele- tion (Fig. 4C), indicating differing capacities for compensatory autophagy in somatic cell types. p gy yp To further assess autophagic regulatory genes in other Atg7- deficient somatic/differentiated cells, we examined their expression levels in a Mx1 promoter-directed Atg7 conditional knock-out mouse model (Atg7f/f;Mx1-Cre). At 4 weeks of age, these mice were treated with polyinosine-polycytosine (pIpC) three times every other day to induce deletion of Atg7 alleles. In hepatic cells, such treatment did result in up-regulation of Ulk1, Beclin1, and Vps34 genes (Fig. 4D). Consistent with the above transcriptional data, protein levels of PI3KC3, Beclin1, and ULK1 in Atg7-deficient macrophages of Atg7f/f;Lyz-Cre mice were all significantly up-regulated (Fig. 4E). The Rab9 gene, a critical positive regulator of alternative autophagy, appeared to be up-regulated upon starvation in Atg7-deficient macrophages of Atg7f/f;Lyz-Cre mice (Fig. 4F). To evaluate whether autophagy is dispensable in macro- phages, or if a compensatory autophagic pathway is activated once ATG7-dependent canonical autophagy is impaired in myeloid cells in vivo, we first examined the functional status of lysosomes in Atg7-deficient myeloid cells. Intact lysosomes are essential for cellular degradation of any autophagic cargo. Spe- cifically, the activity of acid phosphatase (a lysosomal marker enzyme) was screened, disclosing that Atg7/ and wild-type macrophages did not differ significantly. It was therefore appar- ent that defective ATG7-dependent autophagy had no impact on the lysosomal system in myeloid cells (Fig. 3E). These data provide evidence that a compensatory pathway for autophagy, relying instead on lysosomal degradation, may be activated in Atg7-deficient macrophages. Finally, transmission electron microscopy (TEM) was engaged to further determine if an autophagic response actually occurs in Atg7-deficient macrophages. We subsequently found that Baf-A1 (a selective inhibitor of canonical autophagy) could suppress formation of autolysosomes induced by starvation in wild-type macrophages. However, Atg7-deficient macrophages did not respond similarly. Baf-A1 failed to suppress starvation- induced autolysosome formation in these cells (Fig. 3F). This TEM data again imply that a compensatory pathway of autophagy may be prompted by dysfunctional ATG7-depen- dent autophagy in macrophages. Stem Cell Autophagy Is More Vulnerable A brief look at altered expression of several proteins essential for canonical autophagy was done in the context of Atg7 dele- tion. In myeloid cells, Atg7 deletion resulted in significant reductions of key proteins (ATG4A, ATG4B, ATG4C, ATG5, and LC3) in canonical autophagy, but in Atg7-deficient lineage- negative cells, expression of these proteins was largely unchanged (Fig. 4, G and H). Atg7-deficient macrophages may augment expression of proteins for alternative autophagy by reducing levels of proteins specific for canonical autophagy. In doing so, the metabolic burden of an Atg7-deficient functional cell is lowered, encouraging its survival under stress. Atg7 Deletion in Macrophages Raises Levels of Key Proteins Regulating Alternative Autophagy—A previous study has dem- onstrated that in mouse embryonic fibroblast cells, ULK1, Beclin1, VPS34, and RAB9 proteins are involved in an alterna- tive autophagic response that is independent of ATG5/ATG7 (21). Embryonic fibroblasts are differentiated somatic cells that retain limited capacity for further differentiation. To ascertain whether a similar compensatory autophagic mechanism exists in Atg7-deficient myeloid cells, we examined above gene expression levels by real-time PCR, revealing that Vps34, Ulk1, and Beclin1 were transcriptionally up-regulated in Atg7-defi- cient macrophages of Atg7f/f;Lyz-Cre mice (Fig. 4A, left panel); but this up-regulation was not observed in HSCs and progeni- tors (LSK and LK cells) of Atg7f/f;Lyz-Cre mice (Fig. 4A, middle and right panels). Given the Lyz promoter specificity, restrict- ing Cre floxing of Atg7 to myeloid lineage (Fig. 1C), Atg7 genes are intact in these upstream cells. Furthermore, in the Atg7f/f; Vav-Cre mouse model, where Atg7 deletion starts at HSC level (Fig. 1A), all Atg7/ LSK, Atg7/ LK, and Atg7/ Lin cells failed to upregulate above autophagic regulatory genes, and Ulk1 expression was in fact down-regulated (Fig. 4B). However, these genes were up-regulated in splenic cells of Atg7f/f;Vav- Collectively, above data suggest that somatic/differentiated cells, including myeloid cells, splenic cells, and hepatic cells, but excluding HSCs, are capable of up-regulating the proteins needed to drive alternative autophagy, in the event that ATG7- dependent canonical autophagy is impaired. Brefeldin A Treatment or Rab9 Knockdown Specifically Inhibits Alternative Autophagic Response in Atg7-deficient Myeloid Cells—To confirm that a compensatory mechanism of autophagy is operant in Atg7-deficient macrophages, we exam- ined the autophagic response to starvation in mice exposed to selective inhibitors of canonical or alternative autophagic path- ways. FIGURE 4. Atg7 deletion leads to an elevated level of key regulatory proteins for alternative autophagy. A–D, RT-PCR determination of transcriptional expression of Vps34, ULK1 and Beclin1 genes in peripheral macrophages (A, left), bone marrow LSK cells (A, middle), and bone marrow LK cells of Atg7f/f;Lyz-Cre mice (A, right); bone marrow LSK cells (B, left), bone marrow LK cells (B, middle), and bone marrow Lin cells of Atg7f/f;Vav-Cre mice (B, right); spleen cells of Atg7f/f;Vav-Cre mice (C); liver cells of pIpC induced Atg7f/f;Mx-Cre mice (D). Wild-type mice were used as a control. E, Western blotting of PI3KC3, Beclin1, and ULK1 of macrophages from wild-type and Atg7f/f;Lyz-Cre mice. Shown is a representative result. F, Western analysis of Rab9 of the macrophages from Atg7f/f;Lyz-Cre mice and wild-type mice. Representative Western blotting result (left) and quantified data (right) were shown. GAPDH served as a loading control. G and H, quantatative PCR was used to measure the expression of Atg4A, Atg4B, Atg4C, Atg5, and LC3 in myeloid cells (CD11b) or hematopoietic Lin negative cells from Atg7f/f;Vav-Cre mice. Relative expression level is normalized to GAPDH. Data are representative or statistical results of three experiments. n 6. , p 0.05; *, p 0.01. Stem Cell Autophagy Is More Vulnerable VOLUME 290•NUMBER 38•SEPTEMBER 18, 2015 VOLUME 290•NUMBER 38•SEPTEMBER 18, 2015 23058 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290•NUMBER 38•SEPTEMBER 18, 2015 Stem Cell Autophagy Is More Vulnerable Baf-A1 alters autophagic flux in wild-type macrophages under starvation, whereas brefeldin A (BFA), an inhibitor tar- geting an alternative autophagic process (21), has little effect on the dynamics of LC3-II accumulation in macrophages of wild-type mice (Fig. 5A, left panel). Thus, BFA does not inhibit the canonical autophagy pathway in wild-type mye- loid cells. However, even in monoallelic Atg7-deficient macrophages of Atg7f/;Vav-Cre mice, Baf-A1 no longer altered the canonical autophagic flux, nor did BFA (Fig. 5A, middle panel). Examination of peripheral mononuclear cells (PMNCs), as differentiated blood cells in Atg7f/f;Lyz-Cre 23059 SEPTEMBER 18, 2015•VOLUME 290•NUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 23059 SEPTEMBER 18, 2015•VOLUME 290•NUMBER 38 Stem Cell Autophagy Is More Vulnerable Stem Cell Autophagy Is More Vulnerable mice, disclosed severely abnormal autophagic flux; and LC3-II accumulation in these cells was unchanged by BFA treatment (Fig. 5A, right panel). Hence, non-inhibition of canonical autophagy by BFA was confirmed, and unlike wild-type coun- terparts, BFA, not Baf-A1, noticeably downregulated RAB9 protein, a key regulator of alternative autophagy, in starved Atg7-deficient macrophages of Atg7f/f;Lyz-Cre mice (Fig. 5B). These data further suggest that Atg7-deficient macrophages possess an alternative autophagic mechanism. cient macrophages. As anticipated, BFA did not inhibit starva- tion-induced autolysosome formation in wild-type macro- phages, where ATG7-dependent canonical autophagy is functional. However, BFA effectively suppressed the starva- tion-induced autophagic response in Atg7-deficient macro- phages of Atg7f/f;Lyz-Cre mice (Fig. 5F). It is therefore quite apparent that Atg7-deficient macrophages use an alternative mechanism of autophagy if ATG7-dependent canonical autophagy is impaired or dysfunctional. p p g Autophagy is essential for ex vivo monocyte-macrophage dif- ferentiation (33). To demonstrate that macrophages use two autophagic processes, relying on the alternative autophagic response if the canonical pathway is impaired, we examined M-CSF-induced monocyte-macrophage differentiation in the presence of selective inhibitors targeting either canonical or alternative autophagy. Monocytes of Atg7f/f;Lyz-Cre and wild- type mice were sorted and cultured with M-CSF to induce dif- ferentiation. After 48 h of culture, Baf-A1 was first added, and the cells were harvested at 72 h for microscopic assessment and flow cytometry. M-CSF effectively induced macrophage differ- entiation ex vivo in wild-type monocytes but was inhibited by Baf-A1, a canonical autophagy inhibitor. Interestingly, M-CSF- induced differentiation of Atg7-deleted monocytes only reached the same level to M-CSF-induced wild-type monocyte differentiation when its canonical autophagy was inhibited with Baf-A1 (Fig. 5C), again indicating Atg7 deletion in myeloid cells of the Atg7f/f;Lyz-Cre mice impairs canonical autophagy. In contrast, BFA did not inhibit M-CSF-induced monocyte- macrophage differentiation in wild-type monocytes, but in Atg7-deficient monocytes, M-CSF-induced monocyte-macro- phage differentiation showed significant inhibition (Fig. 5D). Once more, the alternative autophagic response that is pre- dominant in Atg7-deleted monocytes was thus illustrated. RAB9 is a critical regulatory protein and a specific require- ment in ATG7-independent alternative autophagy, as opposed to ATG7-dependent canonical autophagy (21). Failure of alter- native autophagy is then expected with depletion of RAB9. Indeed, knockdown of Rab9 by lentiviral infection of a DNA construct targeting Rab9 crippled the autophagic response, sig- naled by loss of autolysosomes and accrual of swollen mito- chondria in Atg7-deficient macrophages (Fig. 5G). Stem Cell Autophagy Is More Vulnerable This was not manifested in wild-type macrophages and confirms that alter- native autophagy is triggered if canonical autophagy in macro- phages is damaged. FIGURE 5. Brefeldin A treatment or knockdown of Rab9 inhibits alternative autophagic response in atg7-deleted macrophages. Examination of canonical autophagic flux in response to starvation (1.5 h) with/without bafilomycin A1 (Baf-A1) or brefeldin A (BFA, 0.1 ng/ml, a specific inhibitor on atg7-independent autophagy) in macrophages of wild-type mice (left), Atg7f/;Vav-Cre heterozygous mice (middle), and in peripheral mononuclear cells (PMNCs) of Atg7f/f;Lyz-Cre homozygous mice (right) by Western analysis. Representative Western blotting results (upper panels) and statistical data of three independent experiments (lower panels) are shown. B, BFA but not Baf-A1 downregulated Rab9 level of macrophages from Atg7f/f;Lyz-Cre mice in response to autophagy stimuli (starvation 1.5 h). Macrophages from wild-type mice served as a control. A representative Western blotting result (left) and quantative data (right) are shown. C, atg7-deleted macrophages of Atg7f/f;Lyz-Cre mice showed an inhibition on M-CSF induced monocyte-macrophage differentiation to the samedegreeasBaf-A1inhibitionontheM-CSFinducedmonocyte-macrophagedifferentiationofwild-typemice.D,BFAbutnotBaf-A1inhibitsM-CSFinduced monocyte-macrophage differentiation in atg7-deleted myeloid cells of Atg7f/f;Lyz-Cre mice. Representative colony-forming results (right) and statistical data (left) are shown. E, BFA inhibition on monocyte differentiation increases apoptosis of atg7-deleted monocytes of Atg7f/f;Lyz-Cre mice. F, transmission electron microscopic analysis of macrophage autophagy in response to starvation treated with or without Brefeldin A (0.1 ng/ml). Macrophages were isolated from wild-type mice or Atg7f/f;Lyz-Cre mice and followed by starvation for 90 min. Autophagosomes and autolysosomes are shown in the representative TEM images. G, knockdown of Rab9 crippled autophagy response in atg7-deleted, but not wild-type macrophages. Red arrows: autophagosmes or autolysosomes; black arrows: damaged mitochondria. Data are representative or statistical results of three experiments. n 6. , p 0.05; **, p 0.01. Stem Cell Autophagy Is More Vulnerable Stem Cell Autophagy Is More Vulnerable 23060 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290•NUMBER 38•SEPTEMBER 18, 2015 23060 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290•NUMBER 38•SEPTEMBER 18, 2015 23060 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290•NUMBER 38•SEPTEMBER 18, 2015 Stem Cell Autophagy Is More Vulnerable functional autophagy in instances of Atg7 mutation or deletion. In our hands, partial loss (50%) of ATG7 protein through monoallelic Atg7 deletion produced dysfunctional autophagic flux, given that LC3-II did not accumulate in Atg7/ macro- phages of Atg7f/;Vav-Cre mice upon exposure to Baf-A1 (Fig. 3B). This suggests that ATG7-dependent autophagy in Atg7/ macrophages of Atg7f/f;Lyz-Cre mice should be disabled if a majority of ATG7 protein is absent. autophagy becomes dysfunctional. Nevertheless, it should be noted that starvation-induced autophagy in Atg7-deficient macrophages of Atg7f/f;Lyz-Cre mice is not as robust as that seen in wild-type macrophages. Hence, canonical autophagy is likely the mainstay, whereas alternative autophagy is complementary. Collectively, the experiments conducted here indicate that the Atg7f/f;Lyz-Cre mouse model is valid for assessing biomo- lecular dynamics in response to loss of ATG7-dependent canonical autophagy in macrophages. A recent study with the same Atg7f/f;Lyz-Cre mouse model has shown that when infected with Mycobacterium tuberculosis or M. tuberculosis var. bovis, macrophages lacking Atg7 may accumulate p62, thereby inducing expression of Nrf2 target genes that encode scavenger receptors, MARCO and MSR1, for an increase in phagocytosis (41). Our observations and those of others indi- cate that macrophages lacking Atg7 actually mount a varied response to differing infectious pathogens. While indispensable in HSCs, macrophages are capable of instigating a com- pensatory autophagic mechanism when ATG7-dependent autophagy is impaired. Although autophagosome accumulation corresponds with LC3 lipidation, autophagic degradation is not guaranteed, unless LC3-II further accrues upon inhibiting autophagic deg- radation. Therefore, autophagy may still be defective, even if LC3 lipidation is maintained (39, 40). In this study, autophagic flux analysis suggested that residual ATG7 protein and LC3 lipidation/processing in Atg7/ macrophages of Atg7f/;Vav- CremiceareinsufficienttomaintainafunctionalATG7-depen- dent autophagic response. In other words, a major reduction in the AGT7 protein within myeloid cells of Atg7f/f;Lyz-Cre ho- mologous mice should suffice to impair ATG7-dependent autophagy in these differentiated cells. Because ATG7-dependent autophagy impairment in mye- loid cells scarcely displaced functional defect, nor lost autophagy response, one might think that functional somatic/ differentiated cells may compensate in some way, resuming obligatory ATG7-dependent canonical autophagy. Surpris- ingly, M-CSF-induced differentiation in Atg7-deficient mono- cytes of Atg7f/f;Lyz-Cre mice only reached the same level as wild-type monocytic counterparts when canonical autophagy was inhibited by Baf-A1 (Fig. 5C), thus supporting our con- tention that Atg7 deletion in myeloid cells impairs canonical autophagy. Discussion ATG7 mediates ubiquitin-like protein systems where ATG8 and ATG12 are conjugated with phosphatidylethanolamine and ATG5, respectively, and ATG16 interacts with ATG5.37 ATG7 is thus considered critical for autophagic machinery (23, 24). However, other research has indicated that autophagy in mouse embryonic fibroblasts functions independently of ATG5/ATG7 (21). Our data here suggest that biallelic Atg7 deletion leads to irreversible loss of autophagy in HSCs that rely solely on ATG7-dependent canonical autophagy, whereas the same Atg7 deletion in myeloid cells triggers an alternative autophagic pathway. Atg7 deletion in monocytes of Atg7f/f;Lyz-Cre mice increases apoptosis. Consequently, ATG7-dependent autophagy is needed for apoptotic balance in macrophages, as well as for normal progression of cellular differentiation. Forced differen- tiation with M-CSF inhibited apoptosis in both wild-type and Agt7-deficient monocytes. However, BFA treatment increased apoptosis only in Atg7-deficient monocytes (Fig. 5E), reinforc- ing our contention that compensatory autophagy is primarily operant in Atg7-deficient monocytes, equating with previously described alternative autophagy (21, 37). Theoretically, Atg7/ myeloid cells of Atg7f/f;Vav-Cre ho- mologous mice would be optimal for studying ATG7 depen- dence in differentiated hematopoietic cells. The floxing of Atg7 in HSCs is truly complete, so all descendant cells, including myeloid lines, should lack Atg7 alleles. Unfortunately, multilin- eage differentiation in such HSCs is simply not feasible, so Atg7/ myeloid cells cannot be generated for analysis, West- ern blotting in particular. Examining the monoallelic Atg7/ macrophages of Atg7f/; Vav-Cre mice shows that constitutive conversion of LC3-II from LC3-I remains at high levels in the heterozygous mouse model, even without starvation-induced autophagy (Fig. 3B). This suggests that LC3 conversion is not a bona fide indicator of In a further attempt to reconcile compensatory and alterna- tive autophagy (21), transmission electron microscopy was used to examine the autophagic response to BFA in Atg7-defi- SEPTEMBER 18, 2015•VOLUME 290•NUMBER 38 23061 SEPTEMBER 18, 2015•VOLUME 290•NUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 23061 Stem Cell Autophagy Is More Vulnerable designed the experiment, Y. C. did most of the experiments; S. Z., N. Y., X. L., W. L., Z. W., F. X., L. S., Z. W., Y. F., H. Z., Y. Z., W. Z., S. H., and X. Z. conducted experiments. J. W. wrote the paper, and all authors read and approved the paper. Knockdown of Rab9, removing a key regulatory protein required in alternative autophagy, disrupted alternative autophagy in Atg7-deficient macrophages but not in wild-type macrophages (Fig. 5G), attesting to our impression that alter- native autophagy in macrophages is active only if canonical Stem Cell Autophagy Is More Vulnerable Unlike Baf-A1, which inhibits endoplasmic re- ticulum-derived autophagosomes essential for canonical autophagy (38), BFA inhibits Golgi-derived membranes, lead- ing to inhibition of alternative autophagy (21). BFA inhibited differentiation in M-CSF-stimulated Atg7-deficient monocytes of Atg7f/f;Lyz-Cre mice, but comparably treated monocytes of wild-type mice were unaffected (Fig. 5D), reinforcing the con- cept that an alternative autophagic pathway is operant in Atg7- deficient myeloid cells of Atg7f/f;Lyz-Cre mice to compensate for functional loss of canonical autophagy. Our results further demonstrate that in myeloid cells, splenic cells, and hepatic cells, albeit not stem cells, Atg7 deletion results in up-regulation of PI3K, Beclin1, Ulk1, and Rab9 (Fig. 4), and BFA down-regulates Rab9 (Fig. 5B), all of which are required for alternative autophagy (21, 37). Such divergence within the hematopoietic system may be applicable to all types of cellular hierarchies in mammalian tissues. Hence, unlike stem cells that rely solely on AGT7-dependent canonical autophagy, somatic/differentiated/functional cells have at least two autophagic options. Stem cells are largely dominant figures with low rates of metabolism, generating the least intracellular stress and residing where extracellular stress is minimized (42). Thus, only one autophagic pathway may suf- fice in this context. In the absence of autophagy, mitochondrial ROS accumulate (34, 43), ultimately leading to malignant trans- formation from cumulative DNA damage. Somatic/differenti- ated cells are exposed to various extracellular and environmen- tal stresses through their respective functions and thus require more innate protection. Such divergence of autophagy within the hematopoietic hierarchy enables functional blood cells to deal with stress more efficiently and effectively. Our findings indicate that autophagy is more vulnerable in stem and progen- itor cells, and if damaged, is only recoverable in somatic/differ- entiated cells. These revelations expand our understanding of malignant transformation, which is more apt to involve cells of lesser differentiation. 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https://openalex.org/W3166828429	https://pure.uva.nl/ws/files/67230527/socsci_10_00200_v2.pdf	English	null	Learning from, through and about Differences: A Multiple Case Study on Schools as Practice Grounds for Citizenship	Social sciences	2,021	cc-by	13,603	UvA-DARE (Digital Academic Repository) Learning from, through and about Differences: A Multiple Case Study on Schools as Practice Grounds for Citizenship Rinnooy Kan, W.F.; März, V.; Volman, M.; Dijkstra, A.B. DOI 10.3390/socsci10060200 Publication date 2021 Document Version Final published version Published in Social Sciences License CC BY Link to publication Citation for published version (APA): Rinnooy Kan, W. F., März, V., Volman, M., & Dijkstra, A. B. (2021). Learning from, through and about Differences: A Multiple Case Study on Schools as Practice Grounds for Citizenship. Social Sciences, 10(6), Article 200. https://doi.org/10.3390/socsci10060200 UvA-DARE (Digital Academic Repository) Citation: Rinnooy Kan, Willemijn F., Virginie März, Monique Volman, and Anne Bert Dijkstra. 2021. Learning from, through and about Differences: A Multiple Case Study on Schools as Practice Grounds for Citizenship. Social Sciences 10: 200. https:// doi.org/10.3390/socsci10060200 Keywords: citizenship education; diversity; differentiation; school as practice ground Academic Editors: Liyuan Liu, Steven Donbavand, Bryony Hoskins, Jan Germen Janmaat and Dimokritos Kavadias Learning from, through and about Differences: A Multiple Case Study on Schools as Practice Grounds for Citizenship Rinnooy Kan 1,* , Virginie März 2 , Monique Volman 1 and Anne Bert Dijkstra 1 Willemijn F. Rinnooy Kan 1,* , Virginie März 2 , Monique Volman 1 and Anne Bert Dij Willemijn F. Rinnooy Kan 1 Willemijn F. Rinnooy Kan 1 Educational Sciences, Faculty of Social and Behavioural Sciences, University of Amsterdam, 1012 Amsterdam, The Netherlands; m.l.l.volman@uva.nl (M.V.); a.b.dijkstra@uva.nl (A.B.D.) 2 Institute for the Analysis of Change in Contemporary and Historical Societies, Faculty of Psychology and Educational Sciences, Université Catholique de Louvain, 1348 Louvain-la-Neuve, Belgium; virginie.marz@uclouvain.be * Correspondence: W.F.RinnooyKan@uva.nl Abstract: Learning to relate to others that differ from you is one of the central aims of citizenship education. Schools can be understood as practice grounds for citizenship, where students’ citizenship is not only inﬂuenced by the formal curriculum, but also by their experiences in the context of teacher–student and student–student relations. In this article we therefore investigate how the practice of dealing with difference is enacted in schools. Data were collected through an exploratory multiple case study in four secondary schools, combining interviews and focus groups. Despite the differences between the schools in terms of population and location, in all schools the reﬂection on the enactment of ‘dealing with differences’ was limited in scope and depth. ‘Being different’ was understood primarily in terms of individual characteristics. Furthermore, in all schools there was limited reﬂection on being different in relation to teachers and the broader community. Finally, relevant differences for citizenship were conﬁned to the category of ‘ethnic and cultural diversity’. This article calls for preparing teachers to consider a broader array of differences to practice dealing with differences with their students and to support students in reﬂecting on the societal implications of being different from each other. $€ £ ¥ social sciences $€ £ ¥ social sciences social sciences Learning from, through and about Differences: A Multiple Case Study on Schools as Practice Grounds for Citizenship Citation for published version (APA): Rinnooy Kan, W. F., März, V., Volman, M., & Dijkstra, A. B. (2021). Learning from, through and about Differences: A Multiple Case Study on Schools as Practice Grounds for Citizenship. Social Sciences, 10(6), Article 200. https://doi.org/10.3390/socsci10060200 Citation for published version (APA): Rinnooy Kan, W. F., März, V., Volman, M., & Dijkstra, A. B. (2021). Learning from, through and about Differences: A Multiple Case Study on Schools as Practice Grounds for Citizenship. Social Sciences, 10(6), Article 200. https://doi.org/10.3390/socsci10060200 General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Disclaimer/Complaints regulations UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:24 Oct 2024 1. Introduction Increased participation in education, migration, emancipation have made schools more diverse in their composition and have created new challenges for teachers in the recent decades. The reality of classrooms that are becoming increasingly diverse, e.g., academically and socio-culturally, is an invitation to researchers and practitioners alike to reﬂect on the most desirable way to deal with these changing teaching and learning environments. As a result, differences between students have been studied from an array of perspectives, often related to individuals’ academic capacities, with the goal of increasing educational equality and ensuring equity of access to high-quality learning (e.g., Lindsay 2007; Steenbergen-Hu et al. 2016; Thijs and Verkuyten 2014; Tomlinson et al. 2003). Teachers are well aware of the importance of addressing differences between students; however, many teachers experience attending to those differences as a challenge (Gable et al. 2000; Subban 2006; Tomlinson 2001). Received: 27 February 2021 Accepted: 21 May 2021 Published: 29 May 2021 Received: 27 February 2021 Accepted: 21 May 2021 Published: 29 May 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). ( ) Citizenship education as an educational priority and topic has emerged explicitly in the context of increased societal diversity, especially in terms of their ethnic and cultural composition. One of its main goals is preparing students to function in societies with fellow citizens who differ from them (Banks 2007). During recent decades, an increased focus on the promotion of compulsory citizenship education programs all over the world has been particularly evident (European Commission/EACEA/Eurydice 2017). In educational research, citizenship of adolescents has been conceptualized in different ways (e.g., Banks https://www.mdpi.com/journal/socsci Soc. Sci. 2021, 10, 200. https://doi.org/10.3390/socsci10060200 Soc. Sci. 2021, 10, 200 2 of 18 2004; Rychen and Salganik 2003). In this study, following Lawy and Biesta (2006), we approach it as a continuing developmental practice concerned with negotiating the rights and responsibilities that are tied to the membership of the various communities to which people belong. Through this deﬁnition of citizenship, schools can be understood as practice grounds for citizenship (Veugelers 2011). 1. Introduction Students not only learn about their rights and responsibilities in schools, but practice them through social interactions within the school: using their voice in a student council, solving conﬂicts, and debating sensitive issues (Flanagan 2013). Learning to deal with differences is at play in all of these examples. Thus, when we understand schools to be practice grounds for citizenship, the differences that students encounter within the classroom and the school as well as the way teachers deal with these differences matter as relevant experiences (Gurin et al. 2004). In this exploratory multiple case study, our main research question is: how is the practice of dealing with differences enacted in schools? Through our analysis, we aim to make transparent what the abstract notion of learning to ‘deal with differences’—as a key element of the ambitions of citizenship education—may actually entail within schools. Consequently, we provide insight into the functioning of schools as practice grounds for citizenship, where citizenship learning takes place beyond the formal citizenship curriculum. This approach allows for new insight for researchers and practitioners, in how we can best proﬁt from the school environment to practice (for) citizenship and in particular, to practice (for) the crucial social skill of dealing with differences. 2.1. Dealing with Differences: Categorizing and Group Hierarchy The ability to categorize ourselves and others into social groups helps us to make sense of the complexity of the social world (Jenkins 2000). There are many differences that we can use to categorize ourselves and others, for example: gender, socio-economic background, ethnic and cultural background, religion, and socio-emotional development. Schools are typically the place where at least some of these differences are consciously encountered for the ﬁrst time in children’s lives (Flanagan 2013). Additionally, even when these differences are not reﬂected in the characteristics of the school population itself, they can be introduced and reﬂected upon by teachers. Children learn to categorize groups of people through a combination of similarities they observe themselves and labelling of groups by authority ﬁgures, for example, their teachers. According to the social identity approach (Tajfel 1978), our positive expectations of in-group members are connected to the human motivation to have a positive self-concept, and as a consequence to believe that ‘our’ group is a good group. This is not harmful in itself. Young children already categorize and attribute certain positive characteristics to fellow in-group members; however, their social categorization is not automatically negative for the out-group. It simply separates in-group from out-group (Liberman et al. 2017). One of the main ways information about social categories is transmitted is through language, for example, through the use of generic language in which groups are ad- dressed instead of individuals (i.e., girls always like . . . or Moroccan kids prefer . . . ). Such use of language reinforces group distinctions and an essentialist understanding of groups (Liberman et al. 2017). If children learn that members of a category share important essences beyond what is observable, an essentialist understanding of groups may develop (Patterson and Bigler 2006). Meanwhile, in society, not all groups are equal, and some groups have a relatively low status in comparison to the “dominant or most valued group or groups in society” (Nesdale and Flesser 2001, p. 506). This perceived hierarchy of groups adds a layer of complexity to dealing with differences, as it suggests that practicing dealing with differences is not the same exercise for those who do and those who do not belong to a dominant group. Soc. Sci. 2021, 10, 200 3 of 18 2.2. 2.1. Dealing with Differences: Categorizing and Group Hierarchy The School as a Practice Ground for Dealing with Differences School is one of the social environments in which children practice dealing with differences. Practicing dealing with differences can be understood as part of citizenship education. This is in line with a developmental deﬁnition of citizenship (Lawy and Bi- esta 2006), in which schools are understood to be more than places where adolescents formally learn about their future as citizens. Students also informally develop their agency as citizens on a daily basis within the school, through social interactions with other stu- dents and teachers (Lawy and Biesta 2006; Torney-Purta and Amadeo 2011). Within the school students learn what being part of a community means, what the consequences and implications of their membership in that community actually are, and how they can best deal with the differences they encounter between themselves and others (Flanagan 2003). Many opportunities for citizenship learning that present themselves within schools are unintentional, not consciously linked with citizenship education (Kahne and Sporte 2009; Kissling 2018). The practice of citizenship happens in relation to others: their fellow students, their teachers, and other members of the professional community (Veugelers 2011). Consequently, insight into the functioning and characteristics of these relations is crucial for understanding how dealing with differences is enacted within the school as a practice ground. p g There are different ways in which practicing dealing with differences can take place within the context of the school. One of the ways proposed in the literature is learning to take perspective; positioning oneself in the other’s shoes. Research has indicated that the mere presence of others in schools (other in terms of race, social class, gender, for example) is not sufﬁcient to support perspective taking; speciﬁc conditions are necessary, for example, equality of status, and normalization and negotiation of conﬂict (Gurin et al. 2004; Zirkel and Cantor 2004). Educators act as ‘proximate authority ﬁgures’ who play a signiﬁcant exemplary role when it comes to experiencing and practicing dealing with differences. In terms of perspective taking, they are the ones that can convey ‘equality of status’ and model ‘the normalization and negotiation of conﬂict’. Teachers, intentionally and unintentionally, set the example of how students are supposed to deal with others that differ from them. However, the way educators deal with differences also gives students an impression of how authority will deal with people ‘like them’ (Flanagan 2013). 2.1. Dealing with Differences: Categorizing and Group Hierarchy This role as proxy for political authority, is illustrated by a recent study by Bruch and Soss (2018) on different expressions of school authority and their relation to students’ civic attitudes. A stricter and more punitive expression of authority resulted in lower trust in government and less political engagement for already-disadvantaged students. p g g y g What complicates the enactment of dealing with differences in schools is that for many teachers, their position as proximate authority ﬁgure is combined with membership in a dominant group. For members of a dominant group, categorizing can be seen as an innocent act, while for those who perceive themselves as different from a dominant group, being categorized may be experienced as being ‘othered’ or having their ‘being different’ conﬁrming their lower status (Borrero et al. 2012). As such for students dealing with differences can also be learning to deal with being different oneself, as difference is inherently relational. Diversifying the teacher population, which has been on the educa- tional agenda for several decades, can help students in several ways in this process. Firstly, a more diverse teacher population could provide mentors and role models for minority students. Furthermore, a more diverse teacher population can provide insight into ‘being different’ from a dominant group and help students reﬂect on what that means in society (Cherng and Halpin 2016; Villegas et al. 2012). 2.4. The Present Study Our conceptual framework tries to elucidate the complex socializing processes that underly dealing with differences. The school environment, as one of the arenas where these processes take place, provides different opportunities besides curricular content for students to learn how to deal with differences: on the one hand through the opportunities to practice dealing with differences themselves and on the other hand through the modelling of their teachers, as authority ﬁgures. When we combine both these aspects, the question remains: how is the practice of dealing with differences enacted in schools as practice grounds for citizenship? To answer this research question, an exploratory multiple case study was conducted (Yin 2003). This approach allowed us to compare schools and to gain an in-depth under- standing of how schools differ in their teachers’ practices related to dealing with differences and students’ experiences with dealing with difference. 2.3. Different Citizenship Approaches for Different Students To complicate matters further, in light of the school as a practice ground for citizenship, it is also relevant to point at a pattern that has been revealed in the research: the lines along which society is stratiﬁed, particularly educational level and socio-economic status, Soc. Sci. 2021, 10, 200 4 of 18 are mirrored in how citizenship education itself is interpreted for different groups (see, e.g., Nieuwelink et al. 2019; Ten Dam and Volman 2003). Research has shown that not all students have equal access to citizenship learning opportunities. A different type of citizenship may be promoted in the classroom, depending on the educational level of the students. For example, within pre-vocational education, adaptation was found to be the main theme, while pre-university students were more often offered a critical perspective (Ten Dam and Volman 2003). In line with that ﬁnding, a recent study showed that pre-vocational students are given fewer opportunities to practice their citizenship in terms of taking part in discussions or debates (Nieuwelink et al. 2019). Yet another study showed that educators in lower socio-economic status school communities had a more local understanding of citizenship, whereas in a school community with a higher socio-economic status, an international or global perspective on citizenship was more common (Goren and Yemini 2017; Sincer et al. 2019; Wood 2015). Finally, Kahne and Middaugh (2008) used the concept of the ‘civic opportunity gap’ as a way to convey that different students have different opportunities to develop their citizenship competences. Their study, analyzing a Californian sample (2737 students) from the IEA civic education study, showed that academically successful, white and higher socioeconomic status students were presented with more classroom-based civic learning opportunities. As such, while learning to deal with difference in a socially just way is an important goal of citizenship education, research indicates that the way citizenship education itself is enacted in schools often reproduces societies’ inequalities. 2.5. Dutch Context The study was conducted in four Dutch secondary schools. Schools in the Netherlands have a statutory obligation to promote citizenship, yet schools have a great deal of freedom in fulﬁlling this obligation (Bron and Thijs 2011). What citizenship implies in this context is very broadly deﬁned in terms of ‘promoting active citizenship and social integration’ and ‘learning to participate in a pluriform democratic society’ (Dutch Foundation for Curriculum Development[Stichting Leerplan Ontwikkeling] 2018). This combination of statutory obligation and school autonomy has led to wide variation in school policies and educational practices concerning citizenship education (Dijkstra et al. 2020). In more general terms ‘learning to deal with differences’ is one of the ﬁfty-eight central aims of secondary education. It is conceptualized in terms of different ‘worldviews’ and ‘lifestyles’ and understanding the importance for society to learn to deal ‘respectfully with diversity’.1 3.2. Data Collection Data collection took place from September 2014 until February 2015, and included a combination of different data sources: document analysis, observations, and semi- structured interviews. A total of 24 interviews (with 16 staff members and 24 students) were conducted by two interviewers, one of whom was the ﬁrst author. More speciﬁcally, two semi-structured interviews were conducted with each school principal, or the team leader who assumed the role of principal (n = 4). Additionally, one interview was conducted with the building service workers (n = 4), civics teachers (n = 4), and mentors (n = 4), as well as one focus group session with six pre-vocational students per school (14- to 15-year- olds; n = 24). These students were selected by their mentors, based on our instruction of choosing students that represented the diversity present in the classroom. The mentors we interviewed were the mentors of the students we included, all were responsible for one class of students and all of them were also teachers in the school. To prepare the interviews, we analyzed the school plan, focusing speciﬁcally on in- formation related to citizenship education in general and about dealing with differences in particular. Additionally, we spent a full day in the school and conducted an initial ex- ploratory interview with the school leader. We observed the course of events during breaks and attended at least one class were all students from the focus group were present, as well as being there at the start and end of the school day. We used all of these observations as input for our interviews, but did not include the observations themselves in the data analysis. The interviews with the teachers and building service workers, as well as the ﬁrst interviews with the school principals, had the following structure: we started with some introductory questions regarding the school vision and practices related to citizenship education and the functioning of the school as community. Next, and in line with the conceptual framework, we focused on understanding how differences were dealt with within the school. 3.1. Sample For this study, we selected four schools that differed in terms of student population, tracks offered by the schools, the school’s vision (mainstream versus speciﬁc pedagogical philosophies), degree of urbanization in the school’s location (urban versus rural), and Soc. Sci. 2021, 10, 200 5 of 18 5 of 18 school size (small versus large). This maximum variation sampling (Maxwell 2004) was intended to allow for large variation in practices related to dealing with differences. The selection of schools with different track combinations (one versus several tracks) led to variations in academic and sociocultural characteristics and the status of student groups, due to the considerable differences in tracks. All four schools offered pre-vocational education, while two of them also offered pre-university education, allowing us to compare how the schools approached the issue of the handling of the related differences (Table 1). Table 1. Overview of the cases. Cases Location Student Population2 Tracks3 Waldorf (School 1) Urban 800 (approx. 90% native Dutch) Pre-vocational (theory) to pre-university (3 tracks) Rural (School 2) Rural 300 (approx. 95% native Dutch) Pre-vocational (job-oriented) (1 track) Urban Pre-Vocational (School 3) Urban 300 (approx. 5% native Dutch) Pre-vocational (theory) (1 track) Mixed Urban (School 4) Urban 1600 (approx. 30% native Dutch) Pre-vocational (job-oriented) to pre-university (4 tracks) 3.2. Data Collection More speciﬁcally, questions concerned the participants’ vision with regard to differences (e.g., In your school plan you mention the diversity of the student population: how would you deﬁne diversity?), practices related to differences (e.g., What differences between students are relevant in your practice as a teacher?), and perceived student experience with differences (e.g., Do you see clear groups in your student popu- lation? Along what lines do groups exist?). We included the building service workers to gain an inside perspective on the functioning of the school beyond what happened inside the classrooms. In these interviews we did not focus on citizenship education, but we did touch upon all the other themes. The second interview with the principals was usually the last interview in the school which we used to clarify and ask for additional information. Soc. Sci. 2021, 10, 200 6 of 18 Students were interviewed during a focus group session. The focus group session started with an open-ended conversation regarding the relations between students at the school and how differences were addressed by teachers (15 min). Next, we presented three short ﬁlms to the students (≈2 min each) on groups within the student population, bullying and student teacher relations to further open up conversations about dealing with differences at school. After the ﬁrst ﬁlm, we asked for example: Are there groups in the student population? How do you experience these groups? What are characteristics of these groups? g p Thus, in the interviews with different members of the school community, both practices and student experiences were addressed; however, the emphasis differed between the structured interviews and the focus group sessions. Through the interviews and focus groups we tried to reconstruct as complete as possible an image of how actors within the school experience and practice dealing with differences. 3.3. Data Analysis All interviews were audio-taped, transcribed verbatim, and interpretatively coded (Miles and Huberman 1994). The data analysis was performed through an iterative process of reading and re-reading of the data, by selecting and coding (data reduction), and by displaying the data in within-case and cross-case matrices (Miles and Huberman 1994). For the coding (in Atlas.ti), we used both ‘a priori’ and ‘in vivo’ codes (Bogdan and Biklen 1997). The ‘a priori’ codes were inspired by our conceptual framework and the ‘in vivo’ codes were inductive, data-driven codes that arose during analysis, either as a new main code or as a speciﬁcation of a priori codes in subcodes (see Table 2). The coding and selection of quotes was carried out by the ﬁrst author. The second and third author provided feedback on the coding and interpretation of the selected quotes. Table 2. 3.3. Data Analysis Coding scheme.4 Codes Deﬁnition Subcodes Type of difference Statements referring to different categories of differences Religion Gender Cognitive Cultural background Mother tongue (native Dutch/other) Parents’ Socio-Economic Status Socio-emotional development Hometown Evaluation of difference Statements describing difference as being something positive/negative/irrelevant Difference_positive (as an asset) Difference_negative (as a problem) Difference_blind (as irrelevant) Source of difference Statements concerning the context and relation in which difference is understood Within School—student/student or teacher/student Outside school—local or national School policy for dealing with differences Statements describing school policy and practices related to dealing with differences Differentiation (early tracking) Diversifying teacher population Diversity in classroom composition Teacher practices for dealing with differences Statements describing teacher practices related to dealing with differences Discussing being different in the class Teacher as role model/example Teacher as representing authority Essentializing language Difference understood in a hierarchical way Difference understood as an individual characteristic Difference understood as a relational characteristic Goal of practices/policies Statements describing the goal of practices related to dealing with difference Interpersonal (as opportunity for students to learn from each other) Intrapersonal (as opportunity to learn for the student who is ‘different’) Citizenship (as an opportunity for citizenship learning) Deﬁnition Religion Gender Cognitive Cultural background Mother tongue (native Dutch/other) Parents’ Socio-Economic Status Socio-emotional development Hometown Difference_positive (as an asset) Difference_negative (as a problem) Difference_blind (as irrelevant) Within School—student/student or teacher/student Outside school—local or national Differentiation (early tracking) Diversifying teacher population Diversity in classroom composition Discussing being different in the class Teacher as role model/example Teacher as representing authority Essentializing language Difference understood in a hierarchical way Difference understood as an individual characteristic Difference understood as a relational characteristic Interpersonal (as opportunity for students to learn from each other) Intrapersonal (as opportunity to learn for the student who is ‘different’) Citizenship (as an opportunity for citizenship learning) Statements referring to different categories of differences Evaluation of difference Source of difference School policy for dealing with differences Soc. Sci. 2021, 10, 200 7 of 18 7 of 18 The second step of the data analysis consisted of two phases. In the ﬁrst phase—the within-case analysis—the individual school was taken as our unit of analysis. Transcript fragments with the same codes were grouped and categorized. This resulted in an individ- ual, structured case report for each school, encompassing all of the results of the analysis as well as illustrative interview fragments. 4. Results Our results show that the way in which most differences were addressed in the schools limited students’ citizenship learning opportunities, although schools differed in the extent to which this happened. Four underlying themes emerged from our analysis. First, differences between students were mainly understood in terms of individual characteristics. Second, the differences between teachers and students were rarely understood to be relevant in terms of students’ citizenship learning. Third, the characteristics of the school’s student population were taken as a reference point for differences. There was little reﬂection on how being different from others in society outside of the school might be relevant in terms of citizenship learning. Finally, at all schools, ethnic and cultural differences between students were the only differences that were consciously considered as relevant for practicing dealing with differences as part of citizenship learning, yet the understanding of these types of differences was superﬁcial at all schools. 3.3. Data Analysis The structured case reports described school policy and teacher practices related to dealing with differences as well as the way in which dealing with difference was understood in connection to citizenship education in each school. The structural analogy across the different case reports enabled us to organize the data in a more manageable form, and facilitated comparisons between schools in the second phase. In this phase—the cross-case analysis—we looked for systematic differences, similarities, patterns and processes across the four schools. By comparing the different schools four main themes emerged that were relevant across all four schools: difference as an individual characteristic, the difference between teachers and students, a school internal understanding of difference and a focus on ethnic and cultural diversity in light of citizenship education. Participation in the study was entirely voluntary. During data collection, the partic- ipants were given information about the procedure of data collection and analysis. We explained why we recorded the interviews as well as what would happen with the data (i.e., recording, transcribing, and publishing). At each data collection occasion, we asked them if they had any questions and reminded them that they were free to withdraw at any given point (which none of the participants did). In the process of data collection and data analysis, conﬁdentiality and anonymity were guaranteed. The interview data were anonymized and the results of this study were reported without compromising the identities of the respondents. 4.1. Differences as Individual Characteristics Students differed in many ways from each other, for example, in terms of educational level, gender, socio-economic background, and socio-emotional development. These differences were mostly addressed by teachers in an individualized way; educational level, background and so forth were understood as individual characteristics, and in some cases as an obstacle for individual students that teachers could possibly help them overcome. Regarding differences in educational level, early tracking separated students from the start of their secondary school careers. School 1 was an example of that early separation; students were assigned to different tracks in their ﬁrst year and thus to different classrooms. Although early tracking is foremost a system feature in the Netherlands, in the ﬁrst two or three years of secondary school (depending on the tracks), mixing of students is possible. School 1 used to mix students from different tracks, but for several reasons the principal decided to switch to separating students along educational tracks from the very beginning. Both Schools 2 and 3 served only pre-vocational students. However, these students did differ in other ways, for example, in terms of gender. Gender was an interesting Soc. Sci. 2021, 10, 200 8 of 18 8 of 18 characteristic at School 2, which is pre-vocational, because the preparation for a speciﬁc ﬁeld provided the basis for different classes, and these ﬁelds are very gendered. More speciﬁcally, after the ﬁrst two years of secondary school there was an all-boys class for technical jobs and an all-girls class for care-related jobs. This resulted in limited interaction between these groups. As students were separated along the lines of their different characteristics, interactions and possibilities for learning from each other and directly experiencing a different perspective were limited. p p School 4 was an exception; at this school, differences between students was a key theme, was deﬁned in a very broad sense, and was characterized by a relational approach: “Therefore, learning about differences is not only learning about cognitive differences but also about different capabilities, different cultures, gender, ethnicity, religion, language” (principal, School 4). At this school, students across different educational levels shared classrooms as long as possible, tracking was delayed, which is quite uncommon in the Netherlands, and the population itself was mixed in terms of origin, SES, cultural back- ground, gender, and religion. 4.1. Differences as Individual Characteristics The presence of diverse others was the norm, and classes were explicitly put together in a way that would ensure a diverse population as possible in each classroom in terms of student background and characteristics. Learning to deal with differences was understood to happen automatically when students encountered differences. Besides the diverse classrooms, other conditions for perspective-taking were present. Classroom practices aimed at dealing with differences mostly concerned creating an open climate, for example, by starting and ending each class in a circle to share: “In the circle we are all equal, there is no one to hide behind”, as one of the teachers put it. This open climate was used not only for contact, but also to solve conﬂicts, as one of the mentors explained. Differences in educational level were also sometimes used within the classroom. One of the teachers remarked that it was a wonderful exercise for pre-university level students to explain a subject to a pre-vocational level student, as according to him, “it helps them to really understand the material”. As illustrated by this quote, School 4 was the only school in which differences between students were understood as an explicit asset in their relationships. p At the same time, at three (Schools 1, 2 and 4) of the four schools, teachers reﬂected on the quality of their care for individual students’ needs in terms of socio-emotional wellbeing. For example, at School 2 the principal took pride in the school for having a well-developed system of mentors and care coordinators to support individual students in their speciﬁc needs related to their socio-emotional development. Additionally, at School 1, the principal described the continual process of trying to ﬁgure out the different needs of individual students and how they could best be supported as part of the process of “becoming who you are”. When differences between students are only understood in terms of how best to meet the individual needs that might be associated with those differences, the consequence for the school as a practice ground is that students are not invited to actively practice dealing with differences themselves. They are treated in a way that addresses their individual way of ‘being different’ but they are rarely invited to deal with differences between themselves and others, and to reﬂect on the meaning and impact of differing from each other. 4.1. Differences as Individual Characteristics At all schools a myriad of opportunities to practice dealing with differences, for example through the experience of different perspectives, thus remained underused. School 3) However, even at this school, beyond aiming at a diverse teacher population, there was no vision as to what this diversity should lead to, or what practices within the school should contribute to bringing about the desired effect. However, there seemed to be the implicit idea at this school that teachers can act as role models. Additionally, indeed, at all schools, students looked for similarities with their teachers. The teachers who were understood to be part of their in-group were treated differently by students. At School 3, one of the teachers, who wore a headscarf, talked about being conﬁded in by students looking for advice on how to interpret some of the religious laws. At School 4, the building service worker described a similar dynamic as he explained that after students realized that he had a Surinamese background (which was not obvious at ﬁrst sight), this helped him to interact with many of them in a friendly and interested manner. Additionally, while similarities between members of the professional community seemed to be an invitation to connect, differences seemed to invite the exact opposite. For example, at School 3, a teacher who differed from most of her students in terms of ethnic background and educational level talked about how shocked she was by the level of mistrust the students had in her intentions, accusing her of “wanting them to fail”. That effect of detecting differences seemed to work both ways. Differences between teachers and students were seldom used as an invitation to collective reﬂection. For example, one of the civics teachers (School 4) shared his frustration regarding the garbage on the streets after a bike ride through the neighborhood where most students live, and remarked: “Additionally, then one must reach the conclusion that not everyone has the same intrinsic motivation for citizenship as you and I”. In terms of citizenship learning, this illustrates the perceived difference in starting point between teacher and student, but it did not invite the teacher to use this observation to reﬂect on it together with his students: is this about motivation? What do the students think? What could be reason for the trash on the streets? The same teacher talked about having to lower his expectations with some of his students, and learning to accept that when a student has two or more homes the chances are a bit better for things to become lost. 4.2. Different from Your Teacher and it would, of cours ff f f y y f f be idiotic if in a big city in which there are different cultures, and this is true for th 4.2. Different from Your Teacher Students did not just differ from each other; in general, they also shared few charac- teristics with their teachers. This was especially true in pre-vocational classrooms, where teachers all completed a higher education program, something the majority of their stu- dents probably never will do. Teachers in our sample were also predominantly white. At School 1 (the Waldorf school), however, students and teachers were similar on many counts: in background, in ideology, and in socio-economic status. In general, the teacher population was hardly ever understood as a signiﬁcant part of the school population Soc. Sci. 2021, 10, 200 9 of 18 9 of 18 when differences were reﬂected upon, and consequently the differences between teachers and students were ignored as an opportunity for reﬂection and citizenship learning for students. At School 3, however, the analysis showed that diversity in the teacher population was considered important; the ambition was to have a teacher population that reﬂected the surrounding environment within which the school was located. That is a considered choice [to have a diverse teacher population], also in view of our environment. But now it is no longer a considered choice, it is more the normal state of That is a considered choice [to have a diverse teacher population], also in view of our environment. But now it is no longer a considered choice, it is more the normal state of affairs. Because half of this city, 50-50 obviously, consists of . . . and it would, of course, be idiotic if in a big city in which there are different cultures, and this is true for the Netherlands as a whole, that all your teachers are still native Dutch only. (team leader, School 3) That is a considered choice [to have a diverse teacher population], also in view of our environment. But now it is no longer a considered choice, it is more the normal state of affairs. Because half of this city, 50-50 obviously, consists of . . . and it would, of course, be idiotic if in a big city in which there are different cultures, and this is true for the Netherlands as a whole, that all your teachers are still native Dutch only. (team leader, School 3) g affairs. Because half of this city, 50-50 obviously, consists of . . . 4.3. Being Different: Within and Outside of the School 4.3. Being Different: Within and Outside of the School Learning to deal with differences is also learning to deal with being different. The student population within the school seemed to serve as the only reference point for difference. We rarely encountered a broader reﬂection on what being part of the speciﬁc population the school serves implies in the context of the broader society. p p p y At School 2, ‘the city’ was far away, not only geographically. “These students do not move far beyond the tractor”, the civics teacher remarked. He was very focused on giving the students pride in their local identity, for example, by sometimes using the local dialect. Other teachers seemed less occupied with the question of identity; they were more concerned with the students obtaining their diplomas and keeping the students in school. Citizenship at this school was understood in a local sense: “Contributing to the local community” and “Making the world around you a bit better” (civics teacher 1, School 2). y g y The other civics teacher the rural school, School 2, explicitly aimed at making these students’ world larger: “They must understand that the world is bigger than their small village” (team leader and civics teacher, School 2). This example illustrates the complexity of ﬁnding ‘the right way’ to deal with the perceived distance between the student population and the dominant group. At this school, two teachers used different approaches: the ﬁrst civics teacher stimulated local pride, strengthening the sense of community within the school, while the other civics teacher tried to move beyond the local, trying to make the ‘normal’ a bit less narrow by encouraging students to take a broader perspective. At School 3, there was the same tendency to using the students’ background as a reference point for what is ‘normal’. Teachers dealt with the fact that many of their students differed from the dominant group in a similar fashion: they connected this ‘being different’ to characteristics of the local community. Most of the students had an Islamic background; at this school, that was just “normal”. This is illustrated by the fact that the school emptied out during Islamic holidays and teachers did something fun with the few non-Islamic students. School 3) This observation resulted in a changed, more tolerant attitude towards students, but did not actively include the students, for example, through a discussion on the broader implications of divorce or the effects of multiple caregivers on children. p g In terms of the school as a practice ground, this illustrated the importance of teacher reﬂexivity on their position in relation to their students as well as to the dominant group. This is a complex skill that most of them probably were not trained for as part as their teacher education. Besides the difference in position within the school, possibly the fact that teachers are representatives of the dominant group plays a role here: they may have unconsciously considered themselves ‘the norm’ and their students as ‘different’. However, teachers’ lack of reﬂection on their position diminished the opportunity for students to learn about the signiﬁcance of hierarchies of groups in society, as well as about their own relative position and its implication. At the same time, it became clear that the students experienced relationships with their teachers, both positively and negatively, as relevant and as having an inﬂuence on their learning to deal with differences. Soc. Sci. 2021, 10, 200 10 of 18 10 of 18 (focus group students, School 1) The openness of the within-school relations was cherished by teachers, and the dis- tance from the outside world was accepted as part of the ambition to keep the shared ideological roots intact. The effect was comparable to that at Schools 2 and 3; the practice ground for citizenship remained narrow. g p Teachers and school leaders at all three of these schools tended to use the characteristics of their student population as a reference point for difference, possibly as a means to make students feel included and connected to the school. What the examples illustrate is that to gain greater insight into how dealing with differences is enacted within schools, an important aspect to consider is the presence and effect of using the within-school student population as a reference point for what is ‘normal’. While teachers might have focused on what was shared from the perspective of community, a relatively homogenous environment complicated learning to deal with differences. Despite the positive motivation behind it, this process limits the opportunities of students to practice dealing with differences and dealing with being different. It also limits the scope of their understanding of their relative position in the larger society, a missed opportunity in terms of citizenship learning. At School 4, the mere presence of a diverse student population and the fact that this diversity was understood and used as an asset created a different starting point for a shared norm. Here, diversity was normalized, instead of relative homogeneity. 4.4. Diversity, Not Difference While principals and teachers ignored many types of difference as opportunities to practice dealing with differences, they all discussed and reﬂected upon ethnic and cultural differences, usually under the header of ‘diversity’. This type of difference was also the only type of difference that was explicitly connected to citizenship education at all schools. At School 1, the Waldorf school, ethnic and cultural diversity was almost entirely absent within the school population. The principal described the homogenizing effect of attending the school on an already homogenous population: “students seem to get more and more alike during their time here”. There was a clear wish for increased diversity, but little reﬂection on how minority students could be made to feel welcome. To compensate for the lack of this type of diversity a ‘World Day’ was organized every year, where “people with different cultural backgrounds, for example Surinamese, Hindu and Indonesian” were invited to talk about ‘their culture’. In terms of the school as a practice ground one might say that this conﬁrms ‘other’ cultures as being far removed from the daily lives of students, and exoticized them rather than creating opportunities to practice taking a different perspective. p p Citizenship education at this school was focused on ‘becoming who you are’; sup- porting self-expression was the main goal, in this context. Meanwhile, “unworldly”, disconnected to the reality of the ‘real world’, was what some students in the school called themselves: However, I do feel that there are few people from different backgrounds at our school, and people thus more easily shy away from other cultures and such. And that is not a good thing, in my opinion, because the whole world is a mix, like, and it is also good to get to know other cultures and not be afraid of them. ( . . . ) And later you are simply in the real world and there will be other cultures all around you and I do feel that you should learn about this, like, earlier. (student, School 1) However, I do feel that there are few people from different backgrounds at our school, and people thus more easily shy away from other cultures and such. And that is not a good thing, in my opinion, because the whole world is a mix, like, and it is also good to get to know other cultures and not be afraid of them. 4.3. Being Different: Within and Outside of the School Citizenship was also understood in a local way at this school; the national level was explicitly reduced to that of the city where the school was situated: “When we are talking about the Netherlands, we are actually mainly referring to [city]” (school leader, School 3). In terms of the school as a practice ground, this implied that students mostly practiced for the world they already know, a world that is nearby. p y y y School 1, the Waldorf school, illustrated the costs, in terms of citizenship learning opportunities related to dealing with differences, of a school community where ideological characteristics are shared by students and teachers. Despite having a completely different population than the other three schools, it illustrated the presence of the implicit reference category of the dominant group in a different way. Furthermore, what was different about the population at this school was that it did not reﬂect the diversity of the urban context it was located within, whereas the other three schools mirrored characteristics of the members of the surrounding community. At this school, the perceived connection of the school community to the dominant, white, group was frowned upon. Throughout the professional community frustration was expressed at being reduced to working at a ‘white school’, a term used in the Netherlands to describe a school that has a predominantly native Dutch population. The association diminished the importance of what they themselves understood to be their shared identity; for them it was the ideology they shared, not their skin color. The consequence of the exclusive character of the school, a relatively homogeneous population, was negatively discussed within the professional community, but there was little reﬂection on the possible role that the shared ideology could play in maintaining that exclusive character. However, students did consciously experience its ‘othering’ implications and did experience how the rules worked differently for people ‘like them’. Student A: There was this boy ( . . . ), who has a foreign background, and who is not your stereotypical ‘Waldorf’ child but, like, he was a student here, and he was such a pain in his class, but when he did something it was regarded as ten times more serious than for someone else. If a real ‘Waldorf’ child did that, it was judged upon in a very mild way, 11 of 18 11 of 18 Soc. Sci. 4.3. Being Different: Within and Outside of the School 2021, 10, 200 and if he did something like that, the reaction immediately was “If this goes on, you will be suspended”. So, actually, I often think that if you are not completely what they want you to be, they may still, well, how shall I put it . . . and if he did something like that, the reaction immediately was “If this goes on, you will be suspended”. So, actually, I often think that if you are not completely what they want you to be, they may still, well, how shall I put it . . . Student C: begin to hate you. Student C: begin to hate you. Student A: Yes, it may be that this school will start to hate you. 4.4. Diversity, Not Difference ( . . . ) And later you are simply in the real world and there will be other cultures all around you and I do feel that you should learn about this, like, earlier. (student, School 1) While some students qualiﬁed their being ‘unworldly’ as somewhat problematic, they did not consider it their school’s task to help them become more worldly. The principal Soc. Sci. 2021, 10, 200 12 of 18 12 of 18 realized that this inward look did contribute to the strong sense of community in the school. However, at the same time, it limited students’ exposure to the outside world, which is different not only in its greater diversity, but also, for example, in having stricter rules. realized that this inward look did contribute to the strong sense of community in the school. However, at the same time, it limited students’ exposure to the outside world, which is different not only in its greater diversity, but also, for example, in having stricter rules. At School 2, the rural school, ethnic and cultural diversity were also almost entirely absent within the school population, but the principal, who also taught civics, stressed that this type of diversity was nevertheless an important theme in the school, because students were often very stigmatizing when they talked about immigrants. The principal struggled with creating an awareness that the world is larger than the small villages the students come from, and also felt that the lack of ethnic and cultural diversity within the school was a limiting factor in terms of citizenship education. During his civics classes, he used the signiﬁcance of the local community to help students reﬂect on the experience of refugees, by discussing what had happened in the area during World War II, when many of the ancestors of the students had to ﬂee themselves. Even though the school population provided little opportunity to practice dealing with ethnic and cultural diversity, this was an attempt to at least nurture compassion and perspective-taking in light of encountering differences. The two schools with a population that was not predominantly native Dutch dealt with this fact in very different ways. At School 4, ethnic and cultural diversity was celebrated, as an important cornerstone of the school’s identity. Diversity was mostly approached as an opportunity to learn from each other, and the school’s responsibility to be an emancipating institution was emphasized. 4.4. Diversity, Not Difference However, at this school, teachers also categorized students by their ethnic or cultural background: “All the Surinamese kids . . . ”, “These kids have music in them, they are so different from the Turkish and Moroccan kids . . . ”. This way of speaking could reinforce essentialist notions of cultural identity and inﬂuenced what students learnt about being ethnically and culturally different from the native Dutch population. Students themselves said they appreciated the fact that their school was so diverse, because other kids could teach you about “their culture, for example what they eat”, reﬂecting a superﬁcial understanding of the implications of ethnic and cultural differences. The student population at School 3 was rather homogeneous non-native Dutch, mainly of Moroccan and Turkish descent, and the great majority of students shared an Islamic background. In terms of the school as a practice ground for citizenship, there was a strong focus on adaptation and rule obedience; for example, speaking any other language than Dutch was strictly forbidden, even in the hallways. The fact that the majority of the students in this school belonged to a minority in society, ethnically different from the dominant group, was not reﬂected upon or used. One of the teachers noted that students from a minority group within the school, such as those with a Hindu or atheist background, were sometimes hesitant when she asked them to speak their mind on what they believe: “They are afraid the others will ﬁnd them strange” (civics teacher, School 3). Schools 3 and 4 illustrate that the mere presence of an ethnically diverse or minoritized student population did not automatically result in more nuanced reﬂection on what being different implies or what being part of a minority implies. However, at the two other schools with a more homogeneous native Dutch population, the assumption did seem to be that a more ethnically diverse population would lead to more nuanced reﬂection. To conclude, at all schools, despite their differences in population, the understanding of ethnic and cultural differences, usually labelled as ‘diversity’, was quite superﬁcial, mentioning only surface elements. This superﬁcial understanding was visible in the schools’ practices and student experiences. This was the case even at schools that had ethnically and culturally diverse populations. 4.4. Diversity, Not Difference In terms of the school as a practice ground, despite the importance attached to this type of difference, students seemed to learn little about its complex implications. 5. Discussion This study explored how the practice of dealing with differences is enacted in schools as practice grounds for citizenship. Our main ﬁnding is that the reﬂection on the enactment Soc. Sci. 2021, 10, 200 13 of 18 of dealing with differences was limited in scope and depth within the schools that were studied. However, schools did differ in the extent to which and the way this happened. The differences between schools were related to four underlying themes. The ﬁrst theme speciﬁcally concerns the individual, instead of relational, understand- ing of difference. Differences between students were mainly understood in terms of individual characteristics. Differences were addressed in a way that focused on improving students’ individual learning trajectories instead of being seen as an opportunity to practice dealing with differences in relation with other students. This is in line with what other authors have pointed out concerning citizenship education in general, where a lack of ‘good citizenship’ has also been framed in individual terms, as an individual characteristic and problem to be solved through education instead of as a contexualized and relational practice (Biesta and Lawy 2006). p y The second theme was the lack of reﬂection on the difference between students and teachers and, as a consequence, the missed opportunities to practice with dealing with these differences. Our study illustrated that the enactment of dealing with differences that supports citizenship learning opportunities for students asks for speciﬁc competencies of teachers. Teachers at all four schools showed little reﬂexivity on their own position- ality as (predominantly) members of the white, native Dutch majority (Martin and Van Gunten 2002). As such, they did not make use of the opportunities to address the soci- etal implications of differences and model possible alternatives and as such contribute to the citizenship learning of their students. Furthermore, while the few minority teachers showed interest in their role as possible role models, other teachers reﬂected hardly at all on their position as representing authority outside of their classrooms (Flanagan 2003). Other research conﬁrms that there is work to be done in this realm in terms of teacher training. To promote a more relational and contextualized understanding of difference in particular, Goodman (2011) proposes that the only way to do justice to difference is to move beyond ‘accepting, appreciating and understanding cultural differences’ (p. 3) and to seek social justice by addressing issues of ‘privilege and power’(p. 4). 5. Discussion To do this, teachers must be taught to reﬂect on their own position of power as a starting point to help their students in this complex process (Paul-Binyamin and Haj-Yehia 2019). Additionally, a more inclusive approach to relevant differences instead of the narrow focus on ethnic and cultural difference could also contribute to better practices, as advantage and disadvantage, power and privilege, are at play in the context of many differences (e.g., differences in gender, ability, age and sexual identity; Knowles and Clark 2018). g y g y Thirdly, deﬁning difference was primarily done using the characteristics of the school’s student population as a reference point. The broader societal context was hardly under- stood as a reference point for difference. As such, there was little reﬂection on how being different from others in society outside of the school might be relevant in terms of citi- zenship learning. The inward focus on the school and the corresponding sense of school community were (unconsciously) prioritized over the introduction of the outside world and its complex relation with difference (Sincer 2021). As such, citizenship learning was primarily focused on a world that students already knew, a world nearby. It is clear that doing justice to diversity while supporting a sense of unity is a complicated task. As a solution, Shields (2000) proposed the notion of schools as ‘communities of difference’, in which both a sense of (school) community and the value of diversity are combined. This is an approach to community where what is shared is built upon reﬂection on how best to deal with differences, in all their nuances, within the school. This can be understood as an invitation to move beyond the narrow focus on ethnic and cultural differences and to embrace a broader understanding of relevant differences, including but not limited to educational level, gender identity, religious background, socio-economic status and socio-emotional development as possible differences that are relevant in students’ daily lives in the larger society. In terms of the school as a practice ground for citizenship, this approach could enable an enactment of dealing with differences that supports a much Soc. Sci. 2021, 10, 200 14 of 18 14 of 18 broader array of citizenship learning opportunities than we encountered in the schools that were part of this study. broader array of citizenship learning opportunities than we encountered in the schools that were part of this study. 5. Discussion p y Finally, our analysis shows that ‘ethnicity and/or culture as difference’ is understood as the most pressing difference within schools in the context of citizenship education. This was especially true in the schools that lacked this type of difference within the student population. Despite the relevance attached to it for all schools, differences within this category were treated in a superﬁcial way, for example, with a focus on tangible differences such as food preference or music, and essentializing language was often used to describe these differences. This limited nuanced reﬂection on the meaning and implications of ethnic and cultural diversity, and the way differences in this category interact with other differences. The presence of diverse others within the school community in this category was not sufﬁcient to avoid these pitfalls, although within schools that lacked this type of difference that seemed to be the idea: their population not their practices limited the opportunity to learn to deal with ethnic and cultural diversity (Zirkel and Cantor 2004). This seems to hold true not only for citizenship education, but within the discourse on citizenship in general (Bauböck and Rundell 1998; Bloemraad et al. 2008), which is not surprising as citizenship education and citizenship learning are connected to broader societal understandings of citizenship (e.g., Banks and Diem 2008). Here, we also recognize the tension between a desire for unity and the reality of diversity. The focus on ‘a shared culture’, deﬁned along the lines of the culture of the mainstream or dominant groups in society, as the cornerstone of how citizenship in general is understood is an illustration of the ﬁrst aspect (Alba and Duyvendak 2019). Meanwhile, the multicultural or even ‘super-diverse’ character of most Western nations has become a given. The dual forces of a narrow understanding of citizenship in general and the desire by an increasingly diverse population to feel included and acknowledged as citizens, create a complex playing ﬁeld for educators in the realm of citizenship education; they are expected to prepare their students for a world in which both these forces are present. As Ben-Porath (2012) described it using her framework of ‘citizenship as shared fate’: Schools in democratic societies are charged with responding to the multiplicity of afﬁli- ations, preferences, ideologies, languages, values, and memberships. 5. Discussion They are expected to celebrate the diversity of the student body, but also to minimize it by developing civic capacity and a host of shared dimensions, including language(s), civic knowledge, academic competency, and patriotic sentiments. (p. 328) The response from the perspective of citizenship education to this increasingly pluri- form context differs. Some authors have proposed a more global approach to citizenship education (e.g., Veugelers 2011), one that focuses on shared human rights (e.g., Alderson 2016) or one that speciﬁcally centers on cultural rights (e.g., Banks 2004). What these approaches share is a critique of the lack of reﬂection on hierarchy and power between different groups in society in the context of citizenship and citizenship education, a relevant critique in light of the results of this study. 6. Conclusions Learning to deal with differences is one of the main themes in the context of schools as practice grounds for citizenship. Despite limitations such as early tracking and school segregation, schools can make use of a broad array of opportunities for students to learn from, through and about differences, their implications and different ways to deal with them. In sum, this study showed that the way dealing with differences was enacted limited the citizenship learning opportunities of students. A broader understanding of relevant differences in schools, reﬂection on the fact that difference is inherently relational, as it is a characteristic of a relationship, as well as more reﬂection on the societal hierarchy related to differences by teachers would all contribute to increasing the quantity and quality of these opportunities. pp Practicing dealing with differences can be a conscious as well as an unconscious pro- cess. In this study, we have primarily relied on interviews to gain insight into how dealing Soc. Sci. 2021, 10, 200 15 of 18 15 of 18 with differences was enacted. This method allowed us to compare four different schools. However, to gain insight into the subtler processes by which dealing with differences is modeled by teachers, for example, an ethnographic approach might be a more appropriate method. Furthermore, schools are not islands; dealing with differences is also practiced by teachers and students outside of the school and their practices and understandings are thereby also inﬂuenced by peers, the media, and the community of which they are a part. In this study, we focused exclusively on the school. For future research, it would be interesting to ﬁnd out how students perceive the weight of the school’s inﬂuence in comparison to the inﬂuence of their peers, family, and media. Finally, we touched upon similar topics in all interviews. However, in some interviews we spoke more elaborately about some of the topics than in others. This was especially true for the focus group sessions with the students, in which some topics were difﬁcult to discuss in some of the groups, and probing did not always result in a deeper conversation. In scientiﬁc research, more insight is needed into (different groups of) students’ experiences with how differences are dealt with within their schools, in order to understand the effect of different approaches. 6. Conclusions Another important question for future studies is how different ways of dealing with differences affect civic and democratic attitudes and behaviors of students outside the classroom. The results of this study underline the importance of teacher competencies for dealing with differences in the context of schools as practice grounds for citizenship, and serve as an invitation to teacher educators to reﬂect on what practices will best serve the de- velopment of these competencies. This starts with the afﬁrmation that how dealing with differences is enacted by teachers matters for the content and quality of the citizenship learning opportunities of their students. Further research on effective teacher training in this domain is necessary, especially because what is effective might differ for different teachers and different (groups of) students, as well as within different national contexts (Leeman and Reid 2006; Wubbels et al. 2006; Seeberg 2003). Furthermore, for future re- search, it would be interesting to see if the notion of ‘schools as communities of difference’ in practice, if embraced by the professional community at a school, indeed contributes to a greater scope and depth of citizenship learning opportunities for students in the realm of dealing with differences. This interpretation of community might provide a crucial starting point to address the tension present within citizenship, and citizenship education, between craving unity and addressing diversity in a socially just way. Author Contributions: Conceptualization, W.F.R.K., V.M., M.V. and A.B.D.; methodology, W.F.R.K., V.M. and M.V.; software, W.F.R.K.; validation, W.F.R.K., V.M. and M.V.; formal analysis, W.F.R.K.; investigation, W.F.R.K.; resources and data curation, W.F.R.K.; writing—original draft preparation, W.F.R.K.; writing—review and editing, V.M. and M.V.; supervision, V.M., M.V. and A.B.D.; project administration, A.B.D.; funding acquisition, A.B.D. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Dutch Organization for Scientiﬁc Research (NOW): Grant number 411-12-035. Institutional Review Board Statement: Ethical review and approval were waived for this study, due to the fact that at our faculty during the period of data collection (in 2016) obtaining approval from an ethical review board was not yet common practice. In the Netherlands only in 2018 a general and shared ‘Code of Ethics for Research in the Social and Behavioral Sciences involving Human Participants’ was published and signed by the deans of all Dutch Social Sciences faculties. 6. Conclusions We have however adhered to common ethical guidelines such as consent of participants, the possibility to withdraw at any moment and entirely voluntary participation. Informed Consent Statement: Informed consent was obtained from the subjects involved in the study. Informed Consent Statement: Informed consent was obtained from the subjects involved in the study. D t A il bilit St t t N t li bl Informed Consent Statement: Informed consent was obtained from the subjects involved in the study. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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Available online: https://wetten.overheid.nl/BWBR0019945/2012-12-01 (accessed on 20 February 2021). ) 2 The percentage of native Dutch students is based on an estimation by school principals. ) 2 The percentage of native Dutch students is based on an estimation by school principals. 3 After completing primary school (age 11/12), students are placed in one of the three tracks within the Dutch school system: pre-vocational education (vmbo), general secondary education (havo) and pre-university education (vwo). Pre-vocational schools have job-oriented tracks (vmbo-b/k) and theory-oriented tracks (vmbo-tl). 3 After completing primary school (age 11/12), students are placed in one of the three tracks within the Dutch school system: pre-vocational education (vmbo), general secondary education (havo) and pre-university education (vwo). 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https://openalex.org/W2027002541	https://www.mdpi.com/1999-4915/4/11/3227/pdf?version=1431415106	English	null	Safety and Immunogenicity of a Plant-Produced Recombinant Hemagglutinin-Based Influenza Vaccine (HAI-05) Derived from A/Indonesia/05/2005 (H5N1) Influenza Virus: A Phase 1 Randomized, Double-Blind, Placebo-Controlled, Dose-Escalation Study in Healthy Adults	Viruses	2,012	cc-by	8,218	Viruses 2012, 4, 3227-3244; doi:10.3390/v4113227 viruses ISSN 1999-4915 www.mdpi.com/journal/viruses Article Safety and Immunogenicity of a Plant-Produced Recombinant Hemagglutinin-Based Influenza Vaccine (HAI-05) Derived from A/Indonesia/05/2005 (H5N1) Influenza Virus: A Phase 1 Randomized, Double-Blind, Placebo-Controlled, Dose-Escalation Study in Healthy Adults Jessica A. Chichester, R. Mark Jones, Brian J. Green, Mark Stow, Fudu Miao, George Moonsammy, Stephen J. Streatfield and Vidadi Yusibov * Fraunhofer USA, Center for Molecular Biotechnology, Newark, DE 19711, USA; E-Mails: jchichester@fraunhofer-cmb.org (J.A.C.); mjones@fraunhofer-cmb.org (R.M.J.); bgreen@fraunhofer-cmb.org (B.J.G.); mstow@fraunhofer-cmb.org (M.S.); fmiao@fraunhofer-cmb.org (F.M.); gmoonsammy@fraunhofer-cmb.org (G.M.); sstreatfield@fraunhofer-cmb.org (S.J.S.); vyusibov@fraunhofer-cmb.org (V.Y.) * Author to whom correspondence should be addressed; E-Mail: vyusibov@fraunhofer-cmb.org; Tel.: +1-302-369-3766; Fax: +1-302-369-8955. Received: 25 October 2012; in revised form: 15 November 2012 / Accepted: 16 November 2012 / Published: 19 November 2012 OPEN ACCESS Viruses 2012, 4, 3227-3244; doi:10.3390/v4113227 viruses ISSN 1999-4915 www.mdpi.com/journal/viruses OPEN ACCESS Safety and Immunogenicity of a Plant-Produced Recombinant Hemagglutinin-Based Influenza Vaccine (HAI-05) Derived from A/Indonesia/05/2005 (H5N1) Influenza Virus: A Phase 1 Randomized, Double-Blind, Placebo-Controlled, Dose-Escalation Study in Healthy Adults Jessica A. Chichester, R. Mark Jones, Brian J. Green, Mark Stow, Fudu Miao, George Moonsammy, Stephen J. Streatfield and Vidadi Yusibov * Viruses 2012, 4 The immune response elicited by the HAI-05 vaccine was variable with respect to both hemagglutination-inhibition and virus microneutralization antibody titers, with the highest responses observed in the 90 µg unadjuvanted group. Keywords: influenza A; H5N1; recombinant vaccine; hemagglutinin; plant-produced vaccine Keywords: influenza A; H5N1; recombinant vaccine; hemagglutinin; plant-produced vaccine 1. Introduction Infections with influenza viruses cause a serious respiratory disease, due to which three to five million cases of severe illness are recorded worldwide annually, with 250,000 to 500,000 deaths [3]. In addition to seasonal influenza outbreaks, occasional influenza pandemics can arise at any time when influenza A virus containing a novel hemagglutinin (HA) subtype is introduced and effectively transmitted among humans. In 1997, a highly pathogenic avian influenza strain of subtype H5N1 caused widespread disease in poultry in Hong Kong. The virus spread rapidly from 2004 onwards and has now caused serious poultry disease outbreaks in several Asian countries, Europe and Africa. More than 500 individuals have been infected with H5N1 viruses with a mortality rate of 50–60%. Fortunately, almost all reported human cases have had close contact with infected birds, with very rare instances of presumed human to human transmission. Nevertheless, considerable concern for human health remains, not only because of the severity of human cases, but also because the virus could undergo adaptive mutation or re-assortment and obtain the ability to spread efficiently among humans [4‒6]. Should these events occur, the H5N1 and/or other novel viruses could have the potential to cause a pandemic exhibiting a very aggressive clinical progression with devastating health and economic consequences globally. Such a scenario underscores the urgent need for rapid and robust influenza vaccine production methods to better prevent and mitigate the effects of pandemics in the future. The potential of plants as a platform for the production of subunit vaccines and therapeutic proteins has been recently demonstrated [7]. We have developed an approach for transient expression of target proteins in healthy Nicotiana benthamiana using a Tobacco mosaic virus-based ‘launch’ vector system [8] and demonstrated its utility by producing vaccine antigens derived from Bacillus anthracis [9], Yersinia pestis [10], and seasonal and pandemic strains of influenza virus [1,11,12]. Recently, we have developed a recombinant HA-based vaccine, HAI-05, for the prevention of disease caused by the novel A/Indonesia/05/2005 (H5N1) strain of influenza A virus, and produced this target in N. benthamiana at pilot plant scale under current Good Manufacturing Practice (cGMP) guidelines [2]. Pre-clinical studies have demonstrated immunogenicity of HAI-05 in mice, rabbits and ferrets [2] and prompted further investigation of this vaccine candidate in humans. Jessica A. Chichester, R. Mark Jones, Brian J. Green, Mark Stow, Fudu Miao, George Moonsammy, Stephen J. Streatfield and Vidadi Yusibov * Fraunhofer USA, Center for Molecular Biotechnology, Newark, DE 19711, USA; E-Mails: jchichester@fraunhofer-cmb.org (J.A.C.); mjones@fraunhofer-cmb.org (R.M.J.); bgreen@fraunhofer-cmb.org (B.J.G.); mstow@fraunhofer-cmb.org (M.S.); fmiao@fraunhofer-cmb.org (F.M.); gmoonsammy@fraunhofer-cmb.org (G.M.); sstreatfield@fraunhofer-cmb.org (S.J.S.); vyusibov@fraunhofer-cmb.org (V.Y.) * Author to whom correspondence should be addressed; E-Mail: vyusibov@fraunhofer-cmb.org; Tel.: +1-302-369-3766; Fax: +1-302-369-8955. Received: 25 October 2012; in revised form: 15 November 2012 / Accepted: 16 November 2012 / Published: 19 November 2012 Received: 25 October 2012; in revised form: 15 November 2012 / Accepted: 16 November 2012 / Published: 19 November 2012 Abstract: Recently, we have reported [1,2] on a subunit influenza vaccine candidate based on the recombinant hemagglutinin protein from the A/Indonesia/05/2005 (H5N1) strain of influenza virus, produced it using ‘launch vector’-based transient expression technology in Nicotiana benthamiana, and demonstrated its immunogenicity in pre-clinical studies. Here, we present the results of a first-in-human, Phase 1 randomized, double-blind, placebo-controlled study designed to investigate safety, reactogenicity and immunogenicity of three escalating dose levels of this vaccine, HAI-05, (15, 45 and 90 µg) adjuvanted with Alhydrogel® (0.75 mg aluminum per dose) and the 90 µg dose level without Alhydrogel®. Vaccine was administered intramuscularly in two injections three weeks apart to healthy adults of 18–49 years of age. At all dose levels the vaccine was generally safe and well tolerated, with no reported serious adverse events or dose-limiting toxicities. Mild local and systemic reactions were observed in all vaccine dose groups and the placebo group and their occurrence was not dose related. The incidence rates were higher in the groups receiving vaccine with Alhydrogel®. 3228 Viruses 2012, 4 Viruses 2012, 4 2. Results and Discussion This first-in-human, Phase 1 randomized, double-blind, placebo-controlled clinical trial was conducted to evaluate safety, reactogenicity and immunogenicity of two doses of the HAI-05 vaccine delivered at three escalating dose levels—15, 45 and 90 µg adjuvanted with Alhydrogel® and 90 µg without Alhydrogel®—in healthy adults 18–49 years of age. Viruses 2012, 4 Viruses 2012, 4 Viruses 2012, 4 3229 discontinuation: lost to follow-up. b: The “Per-protocol” population included all subjects who received two doses of vaccine and had a complete set of serum specimens for antibody determination. : The “Intent-to-treat” population included all subjects who received one dose of vaccine. p y c: Reason for discontinuation: lost to follow-up. set of serum specimens for antibody determination. c: Reason for discontinuation: lost to follow-up. p p j rotocol” population included all subjects who received two doses of vaccine and had a complete m specimens for antibody determination 1. Introduction Here, we report the results of a Phase 1 study conducted to determine safety, reactogenicity and immunogenicity of three escalating dose levels of HAI-05 (15, 45 and 90 µg) adjuvanted with Alhydrogel® and the 90 µg dose level without Alhydrogel®, delivered intramuscularly to healthy adults of 18–49 years of age. p y c: Reason for discontinuation: lost to follow-up. 2.1. Study Population Of the 100 subjects who were randomized and treated, all received both scheduled vaccinations (Table 1). The mean age of participants was 30–32 years for the active dose groups and approximately 34 years for the placebo group. The proportion of female subjects was higher in the active dose groups (55–72%), whereas equal numbers of female and male subjects were enrolled in the placebo group. Most subjects in all groups were white (65–85% in the active dose groups and 86% in the placebo group) (Table 2). Table 1. Subject disposition. Adjuvanted HAI-05 Unadjuvanted HAI-05 Placebo 15 µg n (%) 45 µg n (%) 90 µg n (%) 90 µg n (%) n (%) All subjects treated 18 (100) 20 (100) 20 (100) 20 (100) 22 (100) Intent-to-treat population a 18 (100) 20 (100) 20 (100) 20 (100) 22 (100) Per-protocol population b 18 (100) 20 (100) 20 (100) 20 (100) 22 (100) Completed the study 16 (88.9) 20 (100) 19 (95.0) 20 (100) 21 (95.5) Discontinued from the study c 2 (11.1) 0 1 (5.0) 0 1 (4.5) Table 1. Subject disposition. Table 1. Subject disposition. b: The “Per-protocol” population included all subjects who received two doses of vaccine and ha set of serum specimens for antibody determination. c: Reason for discontinuation: lost to follow-up. c: Reason for discontinuation: lost to follow-up. Viruses 2012, 4 3230 Table 2. Demographic characteristics. 2.2. Safety and Reactogenicity Adjuvanted HAI-05 Unadjuvanted HAI-05 Placebo 15 µg N = 18 45 µg N = 20 90 µg N = 20 90 µg N = 20 N = 22 Female, n (%) 13 (72.2) 11 (55.0) 12 (60.0) 12 (60.0) 11 (50.0) Age (years) n 18 20 20 20 22 Mean 31.8 31.1 32.0 30.1 34.1 SD 8.3 9.2 9.6 7.8 9.5 Median (Min, Max) 30.0 (18, 46) 30.5 (18, 49) 29.5 (19, 47) 28.5 (18, 42) 32.0 (21, 49) Race, n (%) White 15 (83.3) 17 (85.0) 13 (65.0) 14 (70.0) 19 (86.4) Black or African American 3 (16.7) 3 (15.0) 7 (35.0) 6 (30.0) 2 (9.1) Asian 0 0 0 0 1 (4.5) 2.2. Safety and Reactogenicity 2.2. Safety and Reactogenicity The HAI-05 vaccine was safe and well tolerated in all groups. No deaths, serious adverse events (SAEs), clinically significant laboratory abnormalities, or AEs of special interest were reported during the trial. No subject withdrew from the study due to an AE (Table 3). Treatment-related AEs that occurred after any vaccination (Table 3) included myalgia in the 15 µg adjuvanted dose group, injection site pain, pruritus, urticaria, back pain, headache and diarrhea in the 90 µg adjuvanted dose group, and pruritus in the 90 µg unadjuvanted dose group. Table 3. Incidence of AEs after any vaccination. a: Number (%) of subjects reporting symptom at any post-vaccination assessment. Each subject was counted only once. AE category, n (%) a Adjuvanted HAI-05 Unadjuvanted HAI-05 Placebo 15 µg N = 18 45 µg N = 20 90 µg N = 20 90 µg N = 20 N = 22 Any AEs 9 (50) 8 (40) 13 (65) 10 (50) 11 (50) Treatment-related AEs 2 (11.1) 0 4 (20) 1 (5) 0 Serious AEs 0 0 0 0 0 AEs leading to discontinuation from the study 0 0 0 0 0 AEs leading to death 0 0 0 0 0 Table 3. Incidence of AEs after any vaccination. The incidence of solicited local injection site reactions occurring within 7 days following either vaccination was similar across different adjuvanted HAI-05 dose groups, but was greater than in the unadjuvanted 90 µg dose group and in the placebo group. The most frequently reported reaction was pain at the injection site, with a relatively high incidence rate in the 90 µg adjuvanted dose group after Viruses 2012, 4 Viruses 2012, 4 3231 the first dose (80%), which was mild and self-limited. Swelling was infrequent, and no redness was reported in any of the groups following either dose of the vaccine or placebo (Table 4). The incidence of solicited systemic events occurring within 7 days following either vaccination was similar across different active dose groups and in general was similar to the placebo group. The most frequently reported systemic event was headache, which occurred in all dose groups, but most often in the 90 µg adjuvanted dose group following both the first (35%) and second (30%) vaccination (Table 5). Table 4. Incidence of solicited local injection site reactions after the first and second vaccination. a: Number (%) of subjects reporting symptom. Each subject was counted only once. a: Number (%) of subjects reporting symptom. Each subject was counted only once. b ( ) j b: Between Days 1 and 8. a: Number (%) of subjects reporting symptom. Each subject was counted only once. b: Between Days 1 and 8 : Between Days 1 and 8. c: Between Days 22 and 29. 2.3. Immunogenicity Mean geometric mean titers (GMTs) of the serum hemagglutination-inhibition (HAI) antibodies for the three adjuvanted dose groups were the same as the mean GMT for the placebo group on Days 1 and 22, and only slightly increased by Day 43. In contrast, the mean GMT for the unadjuvanted 90 µg dose group increased 6.4-fold from Day 1 through 43 (Table 6). Furthermore, seroconversion (10%) after the first vaccination (Day 22) was observed only in the unadjuvanted 90 µg dose group, and after the second vaccination (Day 43) only in the adjuvanted and unadjuvanted 90 µg dose groups (5% and 10%, respectively) (Table 6). The same results were obtained for the proportions of subjects who achieved HAI titers ≥40 (Table 6). Similar immunogenicity was observed for the adjuvanted dose groups based on mean GMTs of the virus microneutralization (MN) antibodies. In contrast to the adjuvanted dose groups, the mean GMT increased 16-fold from baseline in the unadjuvanted 90 µg dose group after the first vaccination (Days 22) and remained at the same level after the second vaccination (Days 43) (Table 7). At the cut-off titer of ≥20, the unadjuvanted 90 µg dose group was the only active treatment group in which seroconvertion was observed (10%) after the first vaccination (Day 22). However, after the second vaccination (Day 43), some subjects in all of the active dose groups seroconverted in the range from 5.6% to 15% (Table 7). In the post-hoc analysis using a MN antibody cut-off titer of ≥10, 40% of subjects who received the adjuvanted 45 µg vaccine dose seroconverted on Day 43, followed by only 20% of subjects who received the adjuvanted 90 µg dose (Table 7). Also, at the cut-off titer of ≥10 the response rates for the unadjuvanted and adjuvanted 90 µg doses were 15% and 0% on Day 22 and 15% and 20% on Day 43, respectively, further indicating that the presence of Alhydrogel® had no effect on HAI-05 vaccine immunogenicity (Table 7). Surprisingly, one subject (4.5%) in the placebo group (Table 7) who was seronegative at baseline (the titer of 5.0 on Day 1) was also determined as seroconverted after the first vaccination (the titer of 20 on Day 22). The titer on Day 22 was just at the borderline of seropositivity (≥20) and subsequently declined to 7.07 after the second vaccination (Day 43). Viruses 2012, 4 3232 Nearly all AEs were mild to moderate in intensity. The only notable unsolicited AE, localized rash, occurred in one female subject in the 90 µg adjuvanted dose group approximately two days after each vaccination. The subject was receiving chronic topical corticosteroid treatment for psoriasis and a tranquilizer for anxiety before and during the trial. These rash events were not examined or confirmed by the investigator, but were reported by the subject to be mild and resolve without any specific treatment within 48 hours. The subject was lost to follow-up. Overall, at all dose levels, the HAI-05 vaccine was generally well tolerated, with only mild local injection site reactions which did not appear to be dose related in the presence of Alhydrogel®, but were considerably lower without adjuvant even at the highest administered vaccine dose. 2.2. Safety and Reactogenicity b: Between Days 1 and 8. c: Between Days 22 and 29. Adjuvanted HAI-05 Unadjuvanted HAI-05 Placebo 15 µg N = 18 45 µg N = 20 90 µg N = 20 90 µg N = 20 N = 22 Vaccination n (%) a Dose 1 b Dose 2 c Dose 1 Dose 2 Dose 1 Dose 2 Dose 1 Dose 2 Dose 1 Dose 2 Pain at Injection Site 11 (61.1) 10 (55.6) 12 (60.0) 11 (55.0) 16 (80.0) 10 (50.0) 1 (5.0) 3 (15.0) 3 (13.6) 1 (4.5) Redness 0 0 0 0 0 0 0 0 0 0 Swelling 0 1 (5.6) 0 0 2 (10.0) 1 (5.0) 0 0 0 0 Table 4. Incidence of solicited local injection site reactions after the first and second vaccination. Table 5. Incidence of solicited systemic events after the first and second vaccination. a: Number (%) of subjects reporting symptom. Each subject was counted only once. b: Between Days 1 and 8. c Adjuvanted HAI-05 Unadjuvanted HAI-05 Placebo 15 µg N = 18 45 µg N = 20 90 µg N = 20 90 µg N = 20 N = 22 Vaccination n (%) a Dose 1 b Dose 2 b Dose 1 Dose 2 Dose 1 Dose 2 Dose 1 Dose 2 Dose 1 Dose 2 Fever 0 0 0 0 0 0 0 1 (5.0) 1 (4.5) 0 Tiredness 4 (22.2) 4 (22.2) 4 (20.0) 2 (10.0) 5 (25.0) 1 (5.0) 3 (15.0) 1 (5.0) 2 (9.1) 1 (4.5) Chills 2 (11.1) 1 (5.6) 1 (5.0) 0 1 (5.0) 0 1 (5.0) 0 2 (9.1) 1 (4.5) Malaise- Feeling Discomfort 1 (5.6) 2 (11.1) 0 0 3 (15.0) 4 (20.0) 1 (5.0) 0 3 (13.6) 1 (4.5) Joint Aches 2 (11.1) 2 (11.1) 1 (5.0) 1 (5.0) 1 (5.0) 1 (5.0) 2 (10.0) 0 1 (4.5) 1 (4.5) Muscle Aches 4 (22.2) 4 (22.2) 3 (15.0) 2 (10.0) 4 (20.0) 3 (15.0) 2 (10.0) 2 (10.0) 2 (9.1) 2 (9.1) Headache 4 (22.2) 4 (22.2) 4 (20.0) 2 (10.0) 7 (35.0) 6 (30.0) 4 (20.0) 4 (20.0) 4 (18.2) 0 Table 5. Incidence of solicited systemic events after the first and second vaccination. , y g b: GMT, geometric mean titer. c: CI, confidence interval. 2.3. Immunogenicity Furthermore, no seroconversion was observed in other placebo subjects after either the first or the second vaccination, and no subjects in the placebo group seroconverted based on HAI titers (the primary outcome measure). Taken together, these findings suggest that the single placebo subject who appeared to have seroconverted may be as a result of the MN assay variability. Table 6. HAI titers and seroconversion. Dose of HAI-05 A a N subjects per group Pre-vaccination (Day 1) Post-1st vaccination (Day 22) Post-2nd vaccination (Day 43) GMT b (95% CI c) GMT (95% CI) Seroconversion n (%) (95% CI) Titer ≥40 n (%) (95% CI) GMT (95% CI) Seroconversion n (%) (95% CI) Titer ≥40 n (%) (95% CI) Placebo – 22 5.0 (5.0, 5.0) 5.0 (5.0, 5.0) 0 0 5.0 (5.0, 5.0) 0 0 15 µg + 18 5.0 (5.0, 5.0) 5.0 (5.0, 5.0) 0 0 6.41 (4.16, 8.66) 0 0 45 µg + 20 5.0 (5.0, 5.0) 5.0 (5.0, 5.0) 0 0 6.16 (4.15, 8.18) 0 0 90 µg + 20 5.0 (5.0, 5.0) 5.0 (5.0, 5.0) 0 0 6.85 (3.83, 9.88) 1 (5.0) (0, 0.1) 1 (5.0) (0, 0.1) 90 µg – 20 6.75 (3.72, 9.78) 36.50 (0, 73.99) 2 (10.0) (0, 0.2) 2 (10.0) (0, 0.2) 43.23 (0, 89.35) 2 (10.0) (0, 0.2) 2 (10.0) (0, 0.2) a: A, Alhydrogel®. b GMT t i tit Viruses 2012, 4 3234 Table 7. MN titers and seroconversion. b e 7. N t te s a d se oco ve s o . , y g b: GMT, geometric mean titer. c: CI, confidence interval. 2.3. Immunogenicity Dose of HAI-05 A a N subjects per group Pre-vaccination (Day 1) Post-1st vaccination (Day 22) Post-2nd vaccination (Day 43) GMT b (95% CI c) GMT (95% CI) Seroconversion n (%) [95% CI] GMT (95% CI) Seroconversion n (%) [95% CI] Titer cut-off ≥20 Titer cut-off ≥10 Titer cut-off ≥20 Titer cut-off ≥10 Placebo – 22 5.78 (4.60, 6.96) 7.37 (4.62, 10.13) 1 (4.5) [0.0, 0.2] 1 (4.5) [0.0, 0.2] 5.53 (4.77, 6.30) 0 0 15 µg + 18 5.39 (4.88, 5.91) 5.21 (4.83, 5.59) 0 0 8.52 (5.04, 11.99) 1 (5.6) [0.0, 0.2] 3 (16.7) [0.0, 0.3] 45 µg + 20 5.00 (5.00, 5.00) 5.25 (4.82, 5.68) 0 1 (5.0) [0.0, 0.1] 11.26 (7.41, 15.11) 3 (15.0) [0.0, 0.3] 8 (40.0) [0.2, 0.6] 90 µg + 20 5.10 (4.92, 5.28) 5.21 (4.96, 5.45) 0 0 11.06 (4.54, 17.58) 2 (10.0) [0.0, 0.2] 4 (20.0) [0.0, 0.4] 90 µg – 20 8.33 (3.75, 12.91) 132.75 (0, 284.45) 2 (10.0) [0.0, 0.2] 3 (15.0) [0.0, 0.3] 133.77 (0.0, 285.35) 3 (15.0) [0.0, 0.3] 3 (15.0) [0.0, 0.3] a: A, Alhydrogel®. b Viruses 2012, 4 3235 Taken together, the results of the immunogenicity analyses demonstrate that the experimental HAI-05 vaccine generated low humoral immune responses, with no dose dependence and no enhancing effect of adjuvant. Taken together, the results of the immunogenicity analyses demonstrate that the experimental HAI-05 vaccine generated low humoral immune responses, with no dose dependence and no enhancing effect of adjuvant. Currently licensed influenza vaccines, manufactured by several companies globally, are made in embryonated eggs [13]. For the past four decades, these vaccines have been successfully used and were proven to be safe and effective [14‒16]. Recently, safety and efficacy of several avian H5N1 influenza A vaccines in healthy volunteers have been demonstrated [17‒23]. However, the H1N1 “swine influenza” pandemic of 2009 demonstrated that egg-based technologies do not have the capacity to satisfy the global need for an emerging pandemic influenza vaccine in a timely manner. Therefore, a number of countries, including the U.S., are making significant investments in developing alternative technologies that could satisfy this unmet need. In the last several decades, an alternative, recombinant protein-based approach has been introduced to vaccine development and manufacturing. In contrast to conventional vaccines, the recombinant, or subunit, vaccines represent individual immunogenic proteins derived from target pathogens. 2.3. Immunogenicity The subunit vaccine approach is, therefore, considered relatively safe compared to vaccination with whole organism and can be easily scaled up to meet healthcare needs. Some of these alternative manufacturing approaches are based on mammalian and insect cell expression systems for the production of recombinant proteins, including influenza vaccines [24‒31]. Over the past ten years, plants have emerged as a highly promising approach to economically manufacture recombinant proteins, including vaccine antigens [32‒35]. There have been numerous pre-clinical studies demonstrating the immunogenicity and protective efficacy of plant-produced vaccine candidates against a variety of pathogens [1,2,9‒11,36]. More recently, several groups have tested plant-produced vaccines and therapeutic products in humans and demonstrated safety and biological relevance of these products following parenteral administration in Phase 1‒3 clinical trials [37‒41]. We have engineered and produced HAI-05, a recombinant HA antigen from A/Indonesia/05/2005 (H5N1) strain of influenza A virus in a “launch vector”-based transient expression system. This was achieved in less than one month and was subsequently scaled up for cGMP manufacturing at FhCMB’s pilot plant facility [2]. Following successful pre-clinical evaluation, including a Good Laboratory Practice-compliant toxicology study in a rabbit model [2], the experimental vaccine entered into a Phase 1 human study. The vaccine was tested at 15 and 45 µg doses with Alhydrogel® adjuvant and at 90 µg dose with and without Alhydrogel® adjuvant and administered in a two-dose regimen three weeks apart. At these doses, the vaccine was generally shown to have a favorable safety profile in healthy volunteers, with no reported SAEs and no evidence of any dose-limiting or dose-related toxicity. As expected, the most frequent event was local injection site reaction after either dose, which was generally mild and self-limited. Despite the positive immunogenicity results in the pre-clinical studies [2], immune responses elicited by the HAI-05 vaccine in this study were low and somewhat variable with respect to both HAI and MN antibody titers, with the highest responses observed in the 90 µg unadjuvanted group, indicating no enhancement in the presence of Alhydrogel® adjuvant. By contrast, in pre-clinical studies, Alhydrogel® enhanced antibody responses elicited by HAI-05 and provided a dose-sparring Viruses 2012, 4 Viruses 2012, 4 3236 effect in animal models [2]. In addition, in a randomized, Phase 1 clinical trial, alum-adjuvanted whole-virus avian A/Hong Kong/1073/99 (H9N2) vaccine was shown to be more immunogenic than unadjuvanted vaccine [42]. 2.3. Immunogenicity The trial results reported here are consistent with findings from other randomized, controlled influenza vaccine clinical studies in different populations of human volunteers. Indeed, several groups evaluating split-virion 2009 pandemic influenza A H1N1 vaccines [43‒45] as well as split-virion or whole-virus influenza A H5N1 vaccines have reported that alum either failed to enhance or even decreased antibody production [18,20,46‒50]. The reason for Alhydrogel®’s inefficiency is not known, since potency of this adjuvant in stimulating both T helper 1 and 2 type responses and antibody production has been well documented [51]. As suggested in a previous study by Liang et al. [43], the absence of benefit may be due to delayed antigen release from alum-adjuvanted formulations. The low immunogenicity observed in this study is likely attributable to the use of a suboptimal dose of the H5 HA antigen in the HAI-05 vaccine. A post-hoc analysis of MN antibody responses using a cut-off titer of ≥10 revealed responses that were high among responders, which is suggestive of a threshold-like effect that may be further enhanced. Furthermore, the results of a completed Phase 1 trial of plant-produced HAC1 influenza vaccine (recombinant HA derived from A/California/04/2009 [H1N1] strain of influenza A virus) have demonstrated that HAC1 is immunogenic when compared with a licensed, egg-derived H1N1 vaccine [52]. Therefore, and as further supported by studies of conventional egg-derived H5N1 vaccines and recombinant H5N1 vaccines produced in other expression systems, the underwhelming results observed with the HAI-05 vaccine in this study appear to be related to the H5 antigen itself. 3.2. Vaccine 3.2. Vaccine The HAI-05 vaccine is a recombinant subunit HA antigen from the A/Indonesia/05/2005 (H5N1) strain of influenza A virus. The HA sequence encompassing amino acids 17–532 was optimized for expression in plants and synthesized by GENEART AG (Regensburg, Germany). To obtain the truncated HA molecule in the plant expression system, the transmembrane domain (a.a. 533–569) and signal peptide (a.a. 1–16) were removed from the entire HA sequence and the pathogenesis-related protein 1a (PR-1a) signal peptide was added to the N-terminus [1]. A poly-histidine (6 × His) affinity purification tag followed by the endoplasmic reticulum retention signal (KDEL) were added to the C-terminus [1]. The HA antigen has been cloned, expressed in N. benthamiana, and purified as described previously [2]. The purified HAI-05 protein has a monomeric solution state with a purity of >90% as determined by SDS-PAGE and reverse-phase chromatography [2]. Glycosylation analysis, as reported previously, indicated that the core glycan structure was followed by high mannose attachments as expected with use of the KDEL retention sequence. All six potential N-linked glycosylation sites (Asn10, Asn23, Asn154, Asn165, Asn286 and Asn484) showed the high mannose glycosylation [2]. The concentration of the HAI-05 formulated drug substance was 360 μg/mL in a formulation of 0.9% normal saline (United States Pharmacopeia, Hospira, Lake Forrest, IL) with trace amounts of phosphate buffered saline (PBS: 50 mM NaCl, 0.7 mM KCl, 3.6 mM Na2HPO4, and 0.7 mM KH2PO4) for intramuscular administration. Viruses 2012, 4 Viruses 2012, 4 3237 3.1. Study Design This study was a first-in-human, Phase 1 randomized, double-blind, placebo-controlled, dose-escalation clinical study conducted at two clinical centers in the U.S. The study was conducted in accordance with the Declaration of Helsinki and the Code of Federal Regulations of the United States Food and Drug Administration (FDA) and in compliance with the International Conference on Harmonization Guidelines for Good Clinical Practice. The study protocol, informed consent form and subject recruitment procedures were reviewed and approved by an Institutional Review Board and an Independent Ethics Committee. All participants provided written informed consent prior to screening and enrollment into the study. The primary objective of the study was to evaluate the safety, reactogenicity and tolerability of three escalating dose levels of the HAI-05 vaccine—15, 45 and 90 µg adjuvanted with Alhydrogel® and 90 µg without Alhydrogel®—and placebo (0.9% normal saline) delivered intramuscularly to healthy adults 18–49 years of age. The secondary objective was to evaluate and compare immunogenicity of these four HAI-05 vaccine formulations after two doses based on HAI and MN antibody titers. This study was registered under clinical trial reference identifier NCT01250795 [53]. 3.3. Study Population and Treatment Healthy male and non-pregnant female adults 18–49 years of age were excluded from enrollment if they received prior vaccination with any influenza vaccine containing H5 antigen; had any medical condition that may be associated with impaired immune responsiveness, including diabetes mellitus, cancer or treatment for cancer within the previous three years, excluding basal cell carcinoma or squamous cell carcinoma; had a history of anaphylactic type reaction to injected vaccines, positive serology for human immunodeficiency virus (HIV) types 1 and 2 (HIV-1 and HIV-2), hepatitis B surface antigen or hepatitis C virus antibodies or any acute or chronic pulmonary, cardiovascular, hepatic, neurologic or renal disease that might confound evaluation of the vaccine; or had recently taken or planned to take any other experimental vaccine within 30 days prior to vaccination. A total of 100 subjects were enrolled and randomized into 4 cohorts, each with 20 subjects to receive the HAI-05 vaccine formulation and 5 subjects to receive placebo (4:1 vaccine to placebo ratio) (Table 8). Subjects in the study vaccine groups received two doses of HAI-05 at 15, 45 or 90 μg administered with 0.3% aluminum hydroxide (Alhydrogel®) adjuvant (Brenntag Biosector, Denmark) or at 90 μg administered without adjuvant. Subjects in the placebo control group received two doses of saline (0.9% sodium chloride, United States Pharmacopeia). All doses were administered in a volume of 0.5 mL. Vaccine was administered in the deltoid muscle of the same non-dominant arm on Days 1 and 22. Progression to a next higher dose was determined by a Cohort Review Committee based on the safety of a lower dose and in accordance with specified halting rules. After the 15 and 45 μg dose levels were Viruses 2012, 4 Viruses 2012, 4 Viruses 2012, 4 3238 determined to be well tolerated, the two 90 μg dose levels (with and without Alhydrogel®) were administered simultaneously to subjects in the respective cohorts. Table 8. Study cohorts and treatments. Cohort HAI-05 treatment Placebo treatment Number of subjects Dose of HAI-05 (µg/0.5 mL) Dose of Alhydrogel® (mg aluminum/0.5 mL) Number of subjects Placebo 1 20 15 0.75 5 Saline 2 20 45 0.75 5 Saline 3 20 90 0.75 5 Saline 4 20 90 – 5 Saline 3.4. Safety Assessments Table 8. Study cohorts and treatments. Table 8. Study cohorts and treatments. 3.4. Safety Assessments 3.4. 3.3. Study Population and Treatment Safety Assessments Safety assessments (primary endpoints) consisted of reactogenicity events, AEs, clinical laboratory tests (serum chemistry, hematology and urinalysis), vital signs, and abnormal findings upon physical examination. At 30 minutes after each dosing (Days 1 and 22), any signs or symptoms of local and systemic reactions to the study medications (immediate complains) were recorded in an electronic Case Report Form. In addition, vaccinees were instructed to record any occurring reactogenic events for 7 days after each dosing (between Days 1 and 8 and Days 22 and 29, respectively) in a Subject Diary. The solicited immediate complaints and reactogenic events included: pain, redness or swelling at the injection site; fever; muscle or joint aches; headache; tiredness; chills; and malaise (feeling of discomfort or uneasiness). These symptoms were not recorded as AEs unless they were SAEs or persisted beyond Days 8 or 29. Any unsolicited symptoms (AEs) were recorded at 30 minutes after vaccination by the clinical site staff, and thereafter through Day 43 by subjects in a Supplemental Diary. A SAE was defined as any untoward medical occurrence that resulted in death, was life threatening, resulted in persistent or significant disability/incapacity, required hospitalization or prolongation of existing hospitalization, or was associated with a congenital abnormality/birth defect in the offspring of a study subject. The occurrence of any SAE was monitored throughout the course of the study. AEs were assessed for intensity using standardized criteria adapted from the September 2007 FDA Guidance entitled “Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials” [54]. The intensity was determined as mild, moderate or severe, based on the definitions provided in the Guidance. Vaccine-related AEs were those that the investigator judged as having a reasonable possibility that the vaccine contributed to the AE. AEs were coded to the Medical Dictionary for Regulatory Activities® (MedDRA) system organ class and preferred terms. 3.5. Immunogenicity Assessments Sera for immunogenicity assessments were collected on Days 1 (pre-vaccination; baseline), 22 (pre-vaccination; results for the first vaccination) and 43 (results for the second vaccination), Viruses 2012, 4 Viruses 2012, 4 3239 and analysed for HAI and MN antibody titers against A/Indonesia/05/2005 (H5N1) using validated assays at Southern Research Institute (Birmingham, AL). Immunogenicity assessments included determination of 1) GMTs of HAI and MN antibody titers; 2) the proportion of subjects in each group who seroconverted on Days 22 and 43 based on HAI and MN antibody titers; and 3) the proportion of subjects in each group who achieved HAI titers ≥40 on Days 22 and 43. For HAI titers, seroconversion was defined according to the FDA guidance as the percentage of subjects with either a pre-vaccination titer <10 and a post-vaccination titer ≥40 or a pre-vaccination titer ≥10 and a minimum 4-fold rise in a post-vaccination antibody titer. For MN titers, seroconversion was defined as the percentage of subjects with either a pre-vaccination titer at the limit of detection at the starting dilution of 1:20, designated to have a titer of 5, and a post-vaccination titer ≥20, or a pre-vaccination titer ≥20 and a minimum 4-fold rise in a post-vaccination antibody titer. In a post-hoc analysis of MN seroconversion, a post-vaccination MN titer of ≥10 was used as a cut-off value. 3.6. Statistical Analyses Statistical analyses were performed by PharmaNet Development Group, Inc. (Princeton, NJ). Safety analyses were based upon the intent-to-treat population defined as all volunteers who received at least one dose of vaccine or placebo and one post-vaccination visit. Immunogenicity analyses were based upon the per-protocol population defined as all subjects who received two doses of vaccine and who had provided serum specimens for antibody determinations. All statistical analyses were performed using SAS® Version 8.2 or higher. Categorical data were summarized using counts and percentages. Unless otherwise specified, descriptive statistics included: n, mean, standard deviation (SD), median, coefficient of variation (CV%), and minimum and maximum for continuous variables. In general, minimum and maximum were quoted to the number of decimal places as recorded in the electronic case report form; means, medians, SD, and CV% were quoted to one (1) further decimal place. Percentages were rounded to one decimal place. All statistical tests were two-sided and were performed using a 5% significance level, unless otherwise stated, leading to 95% (two-sided) confidence intervals (CIs). Demographic data of vaccinated subjects were summarized by treatment and analyzed by descriptive statistics. All AEs were summarized by numbers and percentages of subjects with corresponding AEs. Proportions of subjects in each treatment group with AEs within each body organ system were compared in the same manner. Immediate complaints and reactogenicity events were summarized by number of events and the percentage, and each dose group value was compared to the placebo group value using Fisher’s exact test. Clinical laboratory results were summarized using descriptive statistics. Proportions of subjects who developed a significant rise in antibody titers after the first dose (secondary endpoint) and second dose (primary endpoint) in each dose group were determined using standard statistical analyses. The HAI and MN GMTs and their 95% CIs were summarized for the two vaccination days (Days 1 and 22) and post-second dose (Day 43) with stratification by cohort. Point estimates and the two-sided 95% CIs for these evaluations were summarized for seroconversion Viruses 2012, 4 Viruses 2012, 4 3240 and subjects achieving titers ≥40 (for HAI) and for seroconversion (for MN) for the two vaccination days (Days 1 and 22) with stratification by cohort, along with the post-second dose (Day 43) results. 4. Conclusions The results of this first-in-human, Phase 1 randomized, double-blind, placebo-controlled, dose-escalation clinical study demonstrated that the experimental HAI-05 vaccine was generally safe at all dose levels. Mild local injection site reactions occurred across all dose levels, but at a considerably lower incidence rate in the absence of Alhydrogel® adjuvant. The vaccine generated low humoral immune responses in healthy adult subjects, with no dose dependence and no enhancing effect of adjuvant, suggesting the use of a suboptimal dose of the H5 HA antigen. Acknowledgments The authors would like to thank PharmaNet Development Group, Inc. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
https://openalex.org/W2312624875	https://zenodo.org/records/3462830/files/102913938%20Mutangi.pdf	English	null	Peri- Urban Livelihoods Strategies and Survival. River Sand Extraction in Mucheke River And Livelihoods Security in Masvingo Urban, Zimbabwe	Greener Journal of Social Sciences	2,013	cc-by	7,700	ABSTRACT The aim of the paper was to asses the contribution of river sand extraction to the livelihoods of the urban dwellers as well as the challenges which the extractors face in the activity. The paper was mainly grounded in qualitative methodology using such data gathering instruments as unstructured interviews, Focus Group Discussions and transect walks. The Sustainable Livelihoods Approach was used as a conceptual framework to understand the livelihoods of the urban poor. The paper reviewed that there are a lot of factors that have forced the urban poor to rely on river sand extraction as a livelihood strategy among these are low uptake of labor by the formal employment sector, dollarization of the economy and negative impacts of international policies. The extractors reviewed that they have resorted to the environment or natural resource use as the only form of the natural capital which they have and this has helped them to meet the day to day needs of urban life such as rent, electricity, water bills, payment of school fees and to buy food at household level. The paper reviewed that although the urban dwellers have used the activity to meet their basic needs, the business has its own challenges as those alluded to by the extractors. The issue of accessibility and gaining entry into the farm as well as constraints by the highway police were cited as the major constraints to the activity. However it was interesting to note that the extractors have not only sat down and relax but rather have devised different ways for them to meet their needs and among these strategies are the issue of social networks with the police as well as the school authority at Gokomere High School. Conclusions were drawn that the local authorities must find ways to integrate the activity into the mainstream development process because it is proving to be of paramount importance to the development process. Keywords: Livelihoods, informal economy, urbanization, dollarization, shocks, vulnerability, poverty, environment. Keywords: Livelihoods, informal economy, urbanization, dollarization, shocks, vulnerability ISSN: 2276-7800 Impact Factor 2012 (UJRI): 0.7863 ICV 2012: 5.99 Peri- Urban Livelihoods Strategies and Survival. River Sand Extraction in Mucheke River And Livelihoods Security in Masvingo Urban, Zimbabwe By Mutangi Gumisai Tinotenda Vol. 3 (10), pp. 496-503, December 2013. ISSN: 2276-7800 Greener Journal of Social Sciences INTRODUCTION The link between rural areas and urban areas in terms of livelihoods has been underestimated by many scholars on the belief that the two were not directly linked. There has been a tendency to link issue of livelihoods only to rural areas but as a result of the ever- persistent poverty in the Sub Saharan African urban areas the debate of livelihoods has become a cause of concern with scholars focusing on different strategies of livelihoods and their contribution both at individual and household level. The urban poor have been affected both by internal (politics, corruption and urban mistrust by politicians) and external factors (international policies, globalization) and these have pushed them to the fringe of the development process and some are left out as a result of the economic based life in urban areas. This however has led many urban dwellers to resort to different forms of livelihoods; some which are on farm and some non- farm activities. As a result of the ever- increasing poverty some have resorted to utilize peri urban resources to enhance their livelihoods and among these resources is river sand which is now on demand for purposes of construction in urban areas. This demand together with the persistent recurrent of poverty, loss of employment, changes in the economy (use of multicurrency) in Zimbabwe has forced some urban people to rely on resources which are close to rural areas for survival. Mutangi Gumisai Tinotenda Lecturer, Sociology Department, Great Zimbabwe University. Email: gtmutangi@gmail.com Email: gtmutangi@gmail.com Literature review Despite the uncertainty, large numbers of urban residents are clearly living in conditions of poverty and, in many countries (particularly sub- Saharan Africa) these numbers rose significantly during the economic restructuring of the 1980s (UNCHS, 1996). The livelihoods of the urban poor are defined in large part by the opportunities and constraints under which they are operating. p g Livelihoods may incorporate paid jobs but these are only one element, and not necessarily the most important, of the web of functional relationships which together comprise a living (UNDP, 1997; ISSD, 1999). Other elements include social networks and a variety of institutions which provide household support and access to resources (de Haan, 1997; Dersham and Gzirishvili, 1998; Douglass, 1998). A livelihood therefore draws on the wealth, knowledge, skills and adaptive strategies of the poor (UNDP, 1998). While sustainable livelihoods reflect the priorities of poor people it should be noted that there may be a difference between their short term, pragmatic survival-oriented priorities and longer term priorities which aim at the development of sustainable livelihoods. Further it recognises that the circumstances of the poor change constantly, and that they sustain themselves, despite precarious conditions, by employing a variety of assets (Sen, 1992; Rakodi, 1997; Beall and Kanji, 1999; Moser, 1996). It is therefore possible to improve their security, and thus contribute to the eradication of poverty, through a variety of wide ranging interventions which support their activities (Satterthwaite, 1997; Wratten, 1995) rather than merely attempting to provide paid employment (Korten, 1994). In most cases, cities are more culturally diverse, more socially fragmented, and less likely to be safe than rural areas. Furthermore, urban neighbourhoods contain a diversity of household types, which are often fluid in their structure, and income inequality is often at its most striking in cities. This social diversity is likely to create tensions and the need for different livelihood strategies from those practised in rural areas (Wratten, 1995; Rakodi, 1993; Moser, 1996). Furthermore urban economies are characterised by a greater degree of commercialisation, and most basic goods such as food and accommodation must be bought or rented through the market. Poor urban men and women therefore need higher cash incomes to survive, unlike their rural counterparts who may rely more heavily on subsistence agriculture or payment in kind and who are more likely to have access to free or common property resources (Wratten, 1995; Satterthwaite, 1997). Literature review According to Lanly (2002) most cities of the developing world are characterised by poor living standards, poor sanitation and service provision as well as congestion , overcrowding unemployment among numerous other 496 www.gjournals.org Greener Journal of Social Sciences Vol. 3 (10), pp. 496-503, December 2013. ISSN: 2276-7800 problems. Clearly, the livelihood opportunities offered by rural, peri-urban and urban areas are likely to be different from one another. While everywhere the livelihoods of the poor are diverse and complex, their content will vary according to the local opportunities available. Many authors suggest that livelihoods tend to be at their most complexes in urban areas, with households drawing on a wide variety of activities to capture income and other resources (Rakodi, 1999; Devas, 1999; Beall, 1997; Chambers, 1995). There is generally an explicit or implicit perception that livelihood strategies are household centred. (Moser, 1998; Beall and Kanji, 1999; Satterthwaite, 1997) and that each profile of social (household-individual) assets is distinctive (Friedman, 1996). problems. Clearly, the livelihood opportunities offered by rural, peri-urban and urban areas are likely to be different from one another. While everywhere the livelihoods of the poor are diverse and complex, their content will vary according to the local opportunities available. Many authors suggest that livelihoods tend to be at their most complexes in urban areas, with households drawing on a wide variety of activities to capture income and other resources (Rakodi, 1999; Devas, 1999; Beall, 1997; Chambers, 1995). There is generally an explicit or implicit perception that livelihood strategies are household centred. (Moser, 1998; Beall and Kanji, 1999; Satterthwaite, 1997) and that each profile of social (household-individual) assets is distinctive (Friedman, 1996). While it is clear that urban poverty is an extensive problem, differences in definitions and measures make the exact figures uncertain. World Bank estimates, for example, indicate that about a quarter of urban residents in developing countries are poor, while many national studies in Asia, Latin America and Africa indicate that more than half of their urban populations are living below poverty lines (UNCHS, 1996). Furthermore, factors such as poverty lines that are not weighted for higher living costs in urban areas and that fail to take account of non- income based aspects of poverty mean that poverty is frequently under-counted (Satterthwaite, 1999). Literature review Many urban households are facing a serious decline in their purchasing power. People have responded in various ways, most notably by diversifying their income sources. Poverty in urban areas is affected by a particular combination of factors which tend to produce a wide range of vulnerabilities. The most important vulnerability involves urban poor dwellers who are more immersed in the cash economy but earn incomes that are often erratic, unreliable and small (Smith, 1998). Most of the urban poor receive incomes that are too low to purchase what they need for long-term survival and they spend most of their household budgets on food (Mitlin, 1999). In 2008, the world food situation appeared to be in crisis, particularly in the developing world which is paralleled by high food prices and low food reserves. p y g p As a result of these challenges many in urban areas have resorted to certain income generating activities to meet their day- to- day needs. Some have resorted to urban agriculture (Kutiwa et al., 2010) as a panacea to the urban problems and some have resorted to cross border trade, street vending, some into firewood cutting and selling and some are venturing into small industries as a form of self empowerment. This implies that among the activities engaged in by the urban dwellers are both farm and non- farm activities. Chibisa and Sigauke (2008) state that, ‘post-2000 the informal sector had become the “lifeblood” of the nation.’ As a result, large sections of the population have been forced to engage in this sector because it has become a means of survival for many citizens, providing them with livelihoods. Cities in most countries are now expanding mainly as a result of natural growth rather than immigration. Nevertheless the economic opportunities they present means that they continue to attract migrants from rural areas or less developed towns in search of work and the chance to improve their lives. Such migrants are likely to be younger, more adventurous and entrepreneurial than those who remain in their home areas (Harris, 1992; Drakakis Smith, 1996). However, as well as representing opportunities, the city also represents costs - housing, basic infrastructure and other needs, such as food and clothing, are more expensive and less accessible in urban areas, and many migrants experience the burdens as well as the rewards of the city life. Literature review According to Clemens and Moss (2002) cited in Metcalf (2008) since 2000, Zimbabwe has been experiencing some amounts of shocks which has generated risks and shock such as drought, floods, economic and political crisis brought deep and sudden collapse in national input and shopping increased income poverty thus the livelihood situation in Zimbabwe has taken a turn for the worse. unemployment and increased inflation rate. According to Clemens and Moss (2002) cited in Metcalf (2008) since 2000, Zimbabwe has been experiencing some amounts of shocks which has generated risks and shock such as drought, floods, economic and political crisis brought deep and sudden collapse in national input and shopping increased income poverty thus the livelihood situation in Zimbabwe has taken a turn for the worse. p y In 2002, the Zimbabwean dollar was severely affected by inflation as result of severe droughts, which led to food shortages (Chakaodza, 2006). By this most companies both local and international companies closed down because it is said that the operation were not profitable as people have no money to purchase the goods and services provided by these companies, thus most people lost their jobs. By the mid 2005, the local currency had lost 99% of its value (Clemens and Moss 2005 in Metcalf, 2008), income in real terms had trembled by over 98 percent compare to the 1990 currency value. Unemployment, poverty and the cost of living had all skyrocketed to unprecedented levels (Gono, 2005). By the end of 2006, inflation was over1, 280 percent (Gono, 2005) and latest estimates put unemployment at 70 percent. As individuals, have agents according to Giddens in Ritzier (2008), the sought to come up with other ways to overcome such hardships. The victims of these stressful events devised coping strategies, in most cases are perceived as temporary reactions to transient setback. In the face of shock and stress, households forced to organize their labor and time to protect their consumption (de la Rocha and Grinspun, 2000 in Shoko and Veiga, 2004). g ) Africa’s urban areas have been hard hit by declining economies and the resulting structural adjustment policies, the cost of which have been disproportionately felt by the urban poor. Life in the urban areas has become more expensive while employment in the formal sector has de-creased and real wages have not kept pace with prices or have even declined in absolute terms. Literature review Another source of vulnerability for the urban poor relates to the linkages between specific cities and the global economy. Urban economies are part of a wider economic system and are affected by national and international macro policy (Douglass, 1998). Such policies and global forces frequently have mixed impacts on poor households and in particular on employment conditions (Elson, 1995 cited in Katepa-Kalala, 1997; Beall and Kanji, 1999; Moser, 1998). Previously, under policies of modernisation, formal employment was increased as a result of growth in the manufacturing industry in many countries. However, since the 1980s, policies of liberalisation and structural adjustment have affected employment in some areas. Losses of formal manufacturing jobs in some countries and sectors, as well as ‘down-sizing’ in the public sector have resulted in a large number of men and women looking for jobs in other areas such as part time services sector employment or the informal sector (Potter and Lloyd-Evans, 1998; UNCHS, 1996). This has increased the vulnerability of many poor men and women through loss of secure employment and its replacement with irregular or informal sources of income. p y p g Furthermore, the distinction between urban and rural is not only blurred by the ties between the two types of area – blurring between the two categories also stems from the fact that the spatial relationship between cities and rural areas is not a static one. Urbanisation as a process can have profound effects on people’s livelihoods, particularly, as noted above, in peri-urban areas, which tend to be most affected by urban growth. The effects of urbanisation on people’s livelihoods are often unequally felt, benefiting some and increasing the vulnerability of others. In Zimbabwe, the whole scenario of poverty and food insecurity was exacerbated by the collapse of larger scale commercial farming activities following the fast track reform programme of 2000 (Rukuni, 2002).Such governmental policy aiming for local indigenization , accelerated multiple problems including food insecurity , 497 497 www.gjournals.org Greener Journal of Social Sciences Vol. 3 (10), pp. 496-503, December 2013. ISSN: 2276-7800 unemployment and increased inflation rate. Sustainable Livelihoods Approach The term sustainable livelihoods relates to a wide set of issues which encompass much of the broader debate about the relationships between poverty and environment (Scoones, 2000). Chambers and Cornway (1980) state that livelihoods comprise of the capabilities, assets (both material and social resources) and activities required for a means of living. A livelihood is sustainable when it can cope with, and recover from, stresses and shocks and maintain or enhance its capabilities and assets both now and in the future. In an attempt to make a living, people use a variety of resources such as social networks, capital knowledge and markets to produce food and marketable commodities and to raise their incomes (Herbinck and Bourdillon, 2001). However when such resources are not available or when they are undermined people tend to go under stress and shock. This can be traced to Sen’s theory of entitlements, which postulates that the purpose of development is to improve human lives through expanding the range of things a person could do and be, for example, being healthy and well nourished, being knowledgeable and being able to participate in the life of the community. Development is thus about removing obstacles to what a person can do in life for example illiteracy, ill health, lack of access to income and employment opportunities, lack of civil and political freedoms (Zimbabwe Human Development Report, 2003). When linked to the issue of river sand extraction, it becomes imperative to understand the concept before one can delve into the lives of the urban people in detail. In this paper livelihoods will be conceptualized as the sum of ways in which households make ends meet from year to year, and how they survive (or fail to survive) through difficult times (UNDP, 2005). In the case of river sand extraction therefore, this framework will help to understand whether the activity is contributing to the betterment of the livelihoods of the urban dwellers in Masvingo or not and to evaluate the problems which these people are facing which can result in shock on the part of the people under study. Research objectives To examine the challenges that has forced the urban dwellers to engage in river sand extraction as a livelihood strategy To assess the contribution of proceeds from the activity to the livelihoods security of the extractors To analyze the challenges which they face in the extraction and transportation of the river s To analyze the challenges which they face in the extraction and transportation of the river sand to designated areas. Literature review The urban poor, whether or not they are migrants, survive through undertaking a variety of activities, which mainly take place in the informal sector. However, not all those working in the informal sector are poor, nor do all those working in the formal sector avoid poverty. Murwendo (2011) revealed that most urban dwellers obtain building materials from the peri-urban zones. The common building materials acquired from these zones are pit sand, river sand and stones which are used for construction of houses. In Masvingo City construction is a major activity. Unemployed youths engage in poaching pit sand from peri-urban zones for sale in the suburb. The Natural Resources Bulletin (2004), asserts that over the years materials like pit sand collected in and around some urban areas like Chitungwiza and Masvingo are a lucrative business. Unemployed youths who engage in pit sand vending indicated that they obtain the pit sand from the peri- urban forests near the City. They also indicated that they supply pit sand to developers. River sand extraction is carried out along the Mucheke River and the Shagashe River. River sand extraction from these rivers has an environmental benefit of reducing the siltation of the two rivers. Other types of building materials found on the peri- urban zones of Masvingo City include tree branches, poles, timber, bark and grass. These products are used within the City limits and outside the City boundary. However Murwendo (2011) looked at the environmental impacts of river sand extraction whilst this research is mainly focusing on economic , environmental and livelihoods security at 498 www.gjournals.org Greener Journal of Social Sciences Vol. 3 (10), pp. 496-503, December 2013. ISSN: 2276-7800 household level. A detailed analysis of the challenges faced by the urban poor, benefits driven from river sand extraction and the politics involved in extraction and transportation of river sand to designated areas were carried out. household level. A detailed analysis of the challenges faced by the urban poor, benefits driven from river sand extraction and the politics involved in extraction and transportation of river sand to designated areas were carried out. Research objectives Poverty, environment and livelihoods security The link between the environment and poverty has been a subject of debate for most scholars especially looking at how the environment or natural resources are linked to livelihoods enhancement. In this research it was established that the river sand extractors rely on river sand for their livelihoods. Most of the river sand extractors alluded to the fact that they cannot run away from the intricate relationship they have with their natural resources. Most of them alluded that poverty which has affected Zimbabwe since years back has forced them to extract river sand for them to meet their everyday needs such as food security at household level. In much of the developing world people are engaged in multiple activities rather than relying on a more limited range (e.g. one household member with full time paid employment) of livelihoods strategies to ensure their well being (UNDP, 1997). While such systems may incorporate paid employment this will not be the only, nor in many cases the most significant, strategy (Wratten, 1995; Chambers, 1995; Potter and Lloyd Evans, 1998; Korten, 1996). The more diverse and complex livelihood strategies become, the more they are likely to reduce vulnerability (Rakodi, 1997). They also said they extract river sand to sell to those who will be doing construction in both town and rural areas. It was interesting also to note that the extractors believed that since the environment was the only closer source of income generation for them they had nothing to look for except the environment. This also shows that development is closely linked to the environment in the sense that as a result of infrastructural development in Masvingo there is an increasing demand for construction resources which among them is river sand and pit sand. This shows that there is a close relationship between the environment and livelihood security. It is the environment on which the poor live and from which they draw their sustenance and it is important to see the links between environment, economy and society. The extractors were urban dwellers who also noted that the activity has helped them to cater for the day- to- day running of the house in terms of buying of basics and payment of rentals. Dollarization, vulnerability and shocks Dollarization, vulnerability and shocks Vulnerability is characterized as insecurity in the well-being of individuals, households, and communities in the face of changes in their external environment. People move in and out of poverty and the concept of vulnerability captures the processes of change better than poverty line measurements. Vulnerability has two facets: an external side of shocks, seasonalities, and critical trends; and an internal side of defenselessness caused by lack of ability and means to cope with these. The vulnerability context includes i)shocks, e.g., conflict, illnesses, floods, storms, droughts, pests, diseases, ii) seasonalities, e.g., prices, and iii) employment opportunities critical trends, e.g., demographic, environmental, economic, governance, and technological trends (Serrat 2008). g p , , , g , g ( ) The extractors noted that the use of multiple currencies in the Zimbabwean economy was a savior to some but a devil to others. They revealed that the dollarization of the economy led to some resorting to informal trade in order to earn out a living. The dollarization has witnessed a steady increase in the construction industry which relies on some materials from peri-urban areas. This has come as a savior to the extractors who said that they were lucky that although the dollarization of the economy affected some sectors, the construction sector has seen a sharp increase in the need for materials. It was also noted that the extractors engaged in the activity in order to recover from the shocks they experienced from the introduction of the multiple currency economy in 2009. The shock came in the sense that as a result of dollarization only the formally paid were now getting cash on a monthly basis whilst for the non formally employed the idea was to diversify livelihood strategies to meet the demands of the economy. It was noted that some of the extractors of river sand were formerly engaged in activities such as foreign currency exchange deals but as a result of the changes in the economy the activity was not at its peak since 2009. From this it can be deduced that people diversify livelihood strategies depending or suiting to the environment which is prevailing. After realizing the effects of dollarization the extractors decided to resort to river sand extraction to supply the booming construction sector. In a poor performing economy natural resource utilization becomes a key source of income generation for settlers and urban dwellers. METHODOLOGY Methodology is conceptualized by Begley (2008) as a body of methods used in a particular activity. It describes and analyses methods in a research. This research was grounded on qualitative methodology on the account of its efficacy in providing people’s own feelings from spoken words and observable phenomena on the implications faced by urban poor who engage in river sand extraction. Qualitative methodology, according to Palton (2002) is concerned with attempting to accurately describe, decode and interpret the occurring of phenomenon on their social contexts. Thus, in this case some contributions and constraints found in river sand extraction were clearly obtained through the involvement of the researcher at the places where the river sand was being extracted. Qualitative methodology describes problems from their contextual origin of those experiencing them. In this case, the people’s livelihoods were affected differently and with the use of qualitative methodology, the researcher was able to draw objective conclusions on whether river sand extraction as a livelihood strategy has positively or negatively affected urban people’s livelihoods. It is also flexible since it encompasses the use of unstructured response options as compared to quantitative methodology which are confined to documented literature. q gy In the study interviews, focus group discussions and transect walks were used to capture individual feelings on the kind of shocks which have affected them and how they have responded to such shocks as well as how they are using the proceeds from the activity. 499 www.gjournals.org Vol. 3 (10), pp. 496-503, December 2013. ISSN: 2276-7800 Greener Journal of Social Sciences Dollarization, vulnerability and shocks It is argued that in spite of its invisibility in the official statistics, most informal activities especially in Africa produce commodities that cannot be imported, substituted and are affordable to low income earners. Apart from yielding services to both high and low income earners, informal activities also provide useful business experience and improve the living standards for majority of the population in developing countries (Friedman, 1992). Lubell (1991) also notes the significance of the indigenous apprenticeship system in Africa which enhances human capital with minimal straining. www.gjournals.org Accessibility politics and challenges From the study it was noted that although the activity was working as a cushion for the informally employed, it was not without challenges. The challenges were coming from the issue of accessing the river sand because it is close to communal areas where the locals claim ownership as well as the school authorities at Gokomere High School because the river passes through the school farm. This brings forth the idea of conflicts found in resource ownership and utilization where everyone claims ownership. The locals are saying the river sand extractors are causing more harm than good and that they should not be allowed to utilize these resources because they belong to the locals. However in order to be safe in accessing the river sand the extractors get permission from the local school authorities and they are given an approval letter which they are supposed to use to gain access. It was revealed that if you do not have the letter of access from the school authorities it was difficult to extract the river sand hence issues of accessibility were marred by contradictions and conflicts. Chigumira (2010) also noted the fact that river sand extraction in Kadoma district was marred by conflicts in the sense that the people had to pay a certain amount of money to the headman for them to collect river sand to supply the booming construction industry in Kadoma. This however shows that there are always conflicts and contradictions in resource ownership and utilization especially peri-urban resources where there is no absolute owner of such resources. p The drivers also mentioned that sometimes they suffer constrains from highway patrol police who sometimes mount roadblocks between Gokomere and Masvingo town. However they revealed that since they have been long in the business they have established mutual relationships with the police officers and they escape without being fined. The paper noted that sometimes they use some form of bribes in order to escape the highway patrol police and this has helped them meet their daily needs for survival. This shows that human beings are calculative and reflexive, they think and device ways to circumvent constraints in order to meet their desired ends (livelihood security). All human beings are knowledgeable agents. That is to say, all social actors know a great deal about the conditions and consequences of what they do in their day-to-day lives. Poverty, environment and livelihoods security They alluded to the fact that they use the money to buy their basics as well as pay school fees for their children and relatives who some of them stay in town with them and some are in their rural homes. They cited the poor performing economy characterized by uncertainty of the GNU era where there was low uptake of labour in the labour market and low growth of industries and low investor confidence in the country’s economy. But however the extractors said they resorted to make use of natural resources found in peri-urban areas to help in the construction industry which they said is booming and the city is expanding. The Natural Resources Bulletin (2004) asserts that over the years materials like pit sand collected in and around some urban areas like Chitungwiza and Masvingo are a lucrative business. These natural resources have become their only source of income generation. Poverty, environment and livelihoods security Life in urban areas has more to do with money than social ties in rural areas hence they said the only option to survive was for them to engage in the business to make sure they meet the demands of urban context in a dollarized economy. Most of the drivers of the trucks and extractors said they get paid depending on the number of loads done per day. Cities in most countries are 500 Vol. 3 (10), pp. 496-503, December 2013. ISSN: 2276-7800 Greener Journal of Social Sciences now expanding mainly as a result of natural growth rather than immigration. Nevertheless the economic opportunities they present means that they continue to attract migrants from rural areas or less developed towns in search of work and the chance to improve their lives. Such migrants are likely to be younger, more adventurous and entrepreneurial than those who remain in their home areas (Harris, 1992; Drakakis Smith, 1995). They alluded to the fact that they use the money to buy their basics as well as pay school fees for their children and relatives who some of them stay in town with them and some are in their rural homes. They cited the poor performing economy characterized by uncertainty of the GNU era where there was low uptake of labour in the labour market and low growth of industries and low investor confidence in the country’s economy. But however the extractors said they resorted to make use of natural resources found in peri-urban areas to help in the construction industry which they said is booming and the city is expanding. The Natural Resources Bulletin (2004) asserts that over the years materials like pit sand collected in and around some urban areas like Chitungwiza and Masvingo are a lucrative business. These natural resources have become their only source of income generation. now expanding mainly as a result of natural growth rather than immigration. Nevertheless the economic opportunities they present means that they continue to attract migrants from rural areas or less developed towns in search of work and the chance to improve their lives. Such migrants are likely to be younger, more adventurous and entrepreneurial than those who remain in their home areas (Harris, 1992; Drakakis Smith, 1995). CONCLUSION The paper concluded that river sand extractors have resorted to the activity as a result of different shock and problems and these include the international policies, dollarization of the economy as well as a decline in labor uptake in the labor market. Conclusions were drawn however that although these factors were affecting the urban poor, they have resorted to river sand extraction as a livelihood strategy in order to improve their livelihoods. It was interesting to note that the extractors were using the money to meet the basic needs which are food, shelter, clothing as well as schooling for their children. Although the activity was seen as a panacea to the problems of the urban poor, it had challenges with regards to accessibility of the river sand as well as problems encountered in ferrying the river sand from Mucheke to urban areas. Though challenges were there the extractors said they had established social ties with the authorities and the highway patrol police. From the study it becomes clear that the activity has helped the urban poor to respond to their challenges thereby achieving livelihood security. Accessibility politics and challenges findings it can be seen that river sand as an informal activity has helped the urban poor to achieve livelihood security at household level. BIBLIOGRAPHY Beall, J. and Kanji, N. (1999) ‘Households, livelihoods and urban poverty.’ Conference paper on Urban Governance, Partnership and Poverty. Birmingham, UK: University of Birmingham. Beall, J. and Kanji, N. (1999) ‘Households, livelihoods and urban poverty.’ Conference paper on Urban Governance, Partnership and Poverty. Birmingham, UK: University of Birmingham. p y g , y g Beall J., 1997 “Assessing and Responding to Urban Poverty: Lessons from Pakistan” in IDS Bulletin Vol. 28 No. 2 p y g y g Beall J., 1997 “Assessing and Responding to Urban Poverty: Lessons from Pakistan” in p y g y g Beall J., 1997 “Assessing and Responding to Urban Poverty: Lessons from Pakistan” in IDS Bulletin Vol. 28 No. 2 Begley C.M. (2008); “Triangulation of communication skills and qualitative research instruments”; in journal of p y g y g Beall J., 1997 “Assessing and Responding to Urban Poverty: Lessons from Pakistan” in IDS Bulletin Vol. 28 No. 2 Begley C.M. (2008); “Triangulation of communication skills and qualitative research instruments”; in journal of advanced nursing 24:688-698 g p g y Begley C.M. (2008); “Triangulation of communication skills and qualitative research advanced nursing 24:688-698 Chakaodza G (2006) Structural Adjustment In Zambia and Zimbabwe; Constructive and Deconstructive, Harare; Sapes.Chambers R., 1995, “Poverty and Livelihoods; Whose Realities Count? “ in Urbanisation And The Environment Vol. 7, No.1 Chibisa, P and Sigauke, C. (2008) ‘Impact of Operation Murambatsvina (Restore Order) on Flea Markets in Mutare: Implications for Achieving MDG 1 and Sustainable Urban Livelihoods,’in Journal of Sustainable Development in Africa, Vol. 10, No. 1, 2008. Chigumira, E (2010) My Land, My Resource: Assessment of the Impact of the Fast Track Land Reform Programme on the Natural Environment, Kadoma District, Zimbabwe, Livelihoods after Land Reform in Zimbabwe Working Paper 14 p Devas, 1999, “Who Runs Cities? The Relationship between Urban Governance, Service Deliveryand Poverty” Conference paper on Urban Governance, Partnership and Poverty, University of Birmingham. p p , p y, y g de Haan, 1997, ‘Rural-urban migration and poverty: the case of India’, IDS Bulletin, Vol 28 No 2,Brighton: Institute of Development Studies p Dersham, L. and Gzirishvili, D. (1998) ‘Informal social support networks and household vulnerability: Empirical findings from Georgia.’ World Development 26 (10). g g p ( ) DFID (2001) ‘Meeting the challenge of poverty in urban areas.’ Strategy Paper. London: Department for International Development. Accessibility politics and challenges Such knowledge is not wholly propositional in character, nor is it incidental to their activities. Knowledgeability embedded in practical consciousness exhibits an extraordinary complexity. Actors are also ordinarily able discursively to describe what they do and their reasons for doing it. However, for the most part these faculties are geared to the flow of day-to-day conduct. The rationalization of conduct becomes the discursive offering of reasons only if individuals are asked by others why they acted as they did. Such questions are normally posed, of course, only if the activity concerned is in some way puzzling -- if it appears either to flout convention or to depart from the habitual modes of conduct of a particular person (Giddens, 1984). Thus although there are constraints in the business of river sand extraction and ferrying, these constraints are not only seen as external factors that have absolute control on individuals but rather these constraints are also acting as the informal sector is used to describe small scale, non-agricultural activities which provide a livelihood for people who make simple goods, such as chairs and handicrafts or who provide a range of basic services, like carrying water from a standpipe. Unlike those who work in factories, government offices or the larger commercial undertakings; informal activities are largely outside the countries’ legal and regulatory framework. Reasons to maintain informality are related to their marginal productivity, high initial capital investment and registration (that is often bureaucratic) required by the formal sector. At times, actors in the informal sector prefer to be clandestine so as to avoid taxes and government supervision (SLA) (Sida, 2004). The 2008 crisis cause a great havoc in the economy of Zimbabwe causing an increase in the informal sector industry. It is argued that in spite of its invisibility in the official statistics, most informal activities especially in Africa produce commodities that cannot be imported, substituted and are affordable to low income earners. Apart from yielding services to both high and low income earners, informal activities also provide useful business experience and improve the living standards for majority of the population in developing countries (Friedman, 1992). From the 501 www.gjournals.org Vol. 3 (10), pp. 496-503, December 2013. ISSN: 2276-7800 Greener Journal of Social Sciences findings it can be seen that river sand as an informal activity has helped the urban poor to achieve livelihood security at household level. BIBLIOGRAPHY Oxford University Pre , , q y y Serrat, O. (2008) The Sustainable Livelihood Approach. Philippines: Asian Development Bank. , , q y y Serrat, O. (2008) The Sustainable Livelihood Approach. Philippines: Asian Development q y y Serrat, O. (2008) The Sustainable Livelihood Approach. Philippines: Asian Development Bank. Serrat, O. (2008) The Sustainable Livelihood Approach. Philippine Shoko, D And Veiga, R (2004) Managing The Impact Of Gold Panning Activities Within The Context Of Intergrated Water Resources Management Planning In The Lower Manyame Sub-Catchment, Zambezi Basin, Zimbabwe. Harare; University Of Zimbabwe Shoko, D And Veiga, R (2004) Managing The Impact Of Gold Panning Activities Within The Context Of Intergrated Water Resources Management Planning In The Lower Manyame Sub-Catchment, Zambezi Basin, Zimbabwe. Harare; University Of Zimbabwe y Smith, Mitchell P. (1998) ‘Facing the market: Institutions, strategies and the fate of organised labour in Germany and Britain’, Politics and Society, 26, 1, 35-68. y Smith, Mitchell P. (1998) ‘Facing the market: Institutions, strategies and the fate of organised labour in Germany and Britain’, Politics and Society, 26, 1, 35-68. UNCHS, 1996, “An Urbanising World: Global Report on Human Settlements”. Oxford UniversityPress. UNDP (2005). 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Drakakis-Smith, 1995, Third World Cities: Sustainable Urban Development, In Urban Studies Vol.32, No. 4/5 Friedman J., 1996, “Rethinking Poverty: Empowerment and Citizens Right’s” in International Social Science Journal Vol. 148 No 2 Friedman J., 1996, “Rethinking Poverty: Empowerment and Citizens Right’s” in International Social Science Journal Vol. 148 No 2 Giddens, A. (1984) The Constitution of Society Oxford; Polity Press Gono, G(2005) Zimbabwe’s Casino Economy. Extraordinary Measures for Extraordinary Challenges, Harare; Zimbabwe Publishing House Giddens, A. (1984) The Constitution of Society Oxford; Polity Press Gono, G(2005) Zimbabwe’s Casino Economy. Extraordinary Measures for Extraordinary Challenges, Harare; Zimbabwe Publishing House g Harris. N (ed), 1992, “Cities in the 1990s: The Challenge for Developing Countries” UCL Press. Hebinck and Bourdollon (2001) Women men and work Rural Livelihoods in South Eastern Zimbabwe Gweru Harris. 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https://openalex.org/W2991304573	https://www.biodiversitylibrary.org/partpdf/202822	English	null	A LIST OF SPECIES OF MOLLUSCA FROM SOUTH AFRICA, FORMING AN APPENDIX TO G. B. SOWERBY'S “MARINE SHELLS OF SOUTH AFRICA.”	Journal of molluscan studies	1,903	public-domain	22,449	354 354 1 Jahrb. Deutsch. Malak. Gesell., 1874, pp. 119-146. A LIST OF SPECIES OF MOLLUSCA FROM SOUTH AFRICA, FORMING AN APPENDIX TO G. B. SOWERBY’S ‘MARINE SHELLS OF SOUTH AFRICA.” By Enear A. Suita, F.Z.S., ete. Read 12th June, 1903. Read 12th June, 1903. PLATE XY. Unrit the year 1848, when Dr. Ferdinand Krauss published his work on the Mollusca of South Africa, the fauna of that region had not received very special attention. In that work he quoted only about 365 marine forms, but subsequently, in a paper in Wiegmann’s Archiv fiir Naturgeschichte, 1852, he mentioned a few additional species. In 1874 Dr. E. von Martens’ gave a list of a collection made by Dr. G. Fritsch, in which he enumerated a considerable number of species which were either overlooked by Krauss or were described after the publication of that author’s work. In 1892 was published the ‘‘Marine Shells of South Africa,” by G. B. Sowerby, which was issued as ‘‘a catalogue of all the known species.” Unfortunately, the incompleteness of this list was very considerable, and therefore in 1897 he deemed it advisable to publish an Appendix, in which he gave not only descriptions of a considerable number of new species, but also references to many which had been omitted from his original list, and others which had been identified as coming from South Africa in the interval between the publication of the two parts of his book. Whilst admitting the usefulness of this work, it must be observed that its utility is in a considerable measure marred by its incom- pleteness. In the first place, it seems a pity that only the shell-bearing forms of Mollusca were treated of, so that anyone requiring information respecting the Cephalopoda, Nudibranchia, etc., has to seek it elsewhere. Then, again, there is a considerable number of species (about 80) which had been recorded from South Africa, that were entirely overlooked. To remedy this incompleteness, and as a further contribution to our knowledge of the fauna, the following list has been compiled. In it will be found over 300 species not in the ‘‘ Marine Shells of South Africa,” the majority of which are either new species or have been identified as South African since the publication of Mr. Sowerby’s work. The collection upon which this list has to a great extent been based has been presented to the British Museum by Mr J H Ponsonby To remedy this incompleteness, and as a further contribution to our knowledge of the fauna, the following list has been compiled. PLATE XY. In it will be found over 300 species not in the ‘‘ Marine Shells of South Africa,” the majority of which are either new species or have been identified as South African since the publication of Mr. Sowerby’s work. The collection upon which this list has to a great extent been based has been presented to the British Museum by Mr. J. H. Ponsonby, who has, for several years past, carried on an extensive correspondence with Mr. H. Burnup, Mr. F. Quekett, and various other collectors in The collection upon which this list has to a great extent been based has been presented to the British Museum by Mr. J. H. Ponsonby, who has, for several years past, carried on an extensive correspondence with Mr. H. Burnup, Mr. F. Quekett, and various other collectors in 300 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU South Africa who have sent specimens to him for identification. It is, therefore, entirely owing to his energy that the compilation of the present list has been possible. Besides the additional species which are catalogued, some criticisms are also offered respecting a few of the identifications i in Mr. Sowerby’s work, which may prove of some interest as clearing up points in nomenclature. Nine species are described as new. With regard to the range of the South African fauna, it should be stated that in the present list only those species are included which occur on the coasts of Cape Colony or Natal. The sequence of the genera, with the exception of Cavolinia, which is placed after Tornatina, is that adopted in Fischer’s ‘‘ Manuel de Conchyliologie.” Nine species are described as new. With regard to the range of the South African fauna, it should be stated that in the present list only those species are included which occur on the coasts of Cape Colony or Natal. The sequence of the genera, with the exception of Cavolinia, which is placed after Tornatina, is that adopted in Fischer’s ‘‘ Manuel de Conchyliologie.” DINGS OF THE MALACOLOGICAL SOCIETY. 9. Serra Jousseaumer, Rochebrune. 9. Serra Jousseaumer, Rochebrune. Sepia Jousseaum?, Rochebrune: Bull. Soc. Philom. Paris, sér. vit, vol, vili (1884), p. 117. Hab.—Cape of Good Hope (Rochebrune). 10. Serra Caprnsts, D’Orbigny. 10. Serra Caprnsts, D’Orbigny. Sepia Capensis, D’Orbigny: Céph. acét., p. 278, pl. vu, figs. 1-3 ; pl. xiijfies: 7—EL 5 pl xvit, figs. 18, 19: Hab.—Cape of Good Hope. 11. Serra vermrcunata, Quoy & Gaimard. Sepia vermiculata, Quoy & Gaimard: Voy. Astrolabe, Moll., vol. ii, p. 64, pl. i, figs. 1-5; D’Orbigny, Céph. acét., p. 279, pl. 11 des. Hab.—Cape of Good Hope (Q. & G.). 12. Sepia HIERREDDA, Rang. 12. Sepia HIERREDDA, Rang. Sepia hierredda, Rang: Mag. Zool., 1837, p. 75, pl. C; D’Orbigny, Jéph. acét., p. 268, pls. xiii, xviii. Sepia hierredda, Rang: Mag. Zool., 1837, p. 75, pl. C; D’Orbigny, Jéph. acét., p. 268, pls. xiii, xviii. Hab.—Cape of Good Hope (D’Orbigny). Hab.—Cape of Good Hope (D’Orbigny). 13. Sepra tusercutata, Lamarck. Sepia tuberculata, Lamarck: D’Orbigny, Céph. acét., p. 277, pls. iii, iv, v1, xvii, figs. 13-15. Hab.—Cape of Good Hope (D’Orbigny). 14. HemtsErius typicus, Steenstrup. 14. HemtsErius typicus, Steenstrup. Hemisepius typicus, Steenstrup: Dansk. Selsk. Skr. (5), vol. x (1875), p. 465, pl. i, figs. 1-10. Hab.—Table Bay, Cape of Good Hope (Steenstrup). 1. ARGONAUTA TUBERCULATA, Shaw. 1. ARGONAUTA TUBERCULATA, Shaw. Argonauta tuberculosa, Lamarck : Reeve, Conch. Icon., vol. xu, pl. 1. Hab.—Cape of Good Hope (Humphrey, Mus. Calonn., p. 6). 2. Potypus Carrnsis (Eydoux & Souleyet). Octopus Capensis, Kh. & 8.: Voy. Bonite, p. 11, pl. i, figs. 6, 7. Hab.—Cape of Good Hope (K. & 8.). 3. Potypus Horripus (D’Orbigny). Octopus horridus, D’Orbigny: Céph. acét., p. 51, Poulpes, pl. vu, fig. 3. Octopus argus, Krauss: Siidafrik. Moll., p. 132, pl. vi, fig. 26. Octopus horridus, D’Orbigny: Céph. acét., p. 51, Poulpes, pl. vu, fig. 3. Octopus horridus, D Orbigny: Céph. acét., p. 51, Poulpes, pl. vu, fig. 3. Octopus argus, Krauss: Siidafrik. Moll., p. 132, pl. vi, fig. 26. Hab.—Natal (Krauss). 4. Potypus rucosus (Bosc.). 4. Potypus rucosus (Bosc.). Octopus rugosus, Bose.: D’Orbigny, Céph. acét., p. 45, Poulpes, pls. vi, xxiii, fig. 2. Hab.—Simons Bay, Cape of Good Hope, 10-20 fathoms (Challenger Exped.). Octopus rugosus, Bose.: D’Orbigny, Céph. acét., p. 45, Poulpes, pls. vi, xxiii, fig. 2. b f d f h ( h ll pls. vi, xxiii, fig. 2. Hab.—Simons Bay, Cape of Good Hope, 10-20 fathoms (Challenger Exped.). 5. Lorigo Reynavupi, D’Orbigny. 5. Lorigo Reynavupi, D’Orbigny. Loligo Reynaudii, D’Orbigny :' Céph. acét., p. 315, pl. xxiv. Hab.—Cape of Good Hope (D’Orbigny). Loligo Reynaudii, D’Orbigny :' Céph. acét., p Hab.—Cape of Good Hope (D’Orbigny). 6. OmMAtostrePHES OUALANIENSIS (Lesson). Loligo Oualaniensis, Lesson: Voy. Coquille, Zool., p. 240, pl. 1, fioy 2: Hab.——Cape of Good Hope (Tryon); ? Cape of Good Hope (Gray). 7. SeprorruTHis masor, Gray. Sepioteuthis major, Gray: Spicil. Zool., p. 3, pl. iv, fig. 1. Hab.—Cape of Good Hope (Gray). Both Tryon and Hoyle have suggested that this species may be the same as Thysanoteuthis rhombus of Troschel. 8. Mucgatocrancura MAximA, Pfeffer. 8. Mucgatocrancura MAximA, Pfeffer. Megalocranchia maxima, Pfeffer: Abhandl. Ver. Hamburg, Bd. viii (1884), p. 24, fig. 32. Hab.—Cape of Good Hope (Pfeffer). QF 006 EDINGS OF THE MALACOLOGICAL SOCIETY. 15. SrPHONARIA NIGERRIMA, n.sp. Pl. XV, Figs. 4 and 5. Testa parva, nigra, irregulariter ovata, mediocriter conoidea, costis tenuibus circiter 44 subrugosis instructa; vertex centralis, pileiformis, postice versus; pagina interna omnino nigerrima, ad marginem sub- crenulata. Long. 10, diam. 8, alt. 4:5 mm. Hab.—Umbhlali, Natal (Burnup). R k bl f i l bl k Remarkable for its coal-black colour within and without. The cost are very small and somewhat rugose, being crossed by the lines of growth. Perhaps the young condition of S. carbo, Hanley, but the costz appear to be rather finer. GASTROPODA. 15. SrPHONARIA NIGERRIMA, n.sp. Pl. XV, Figs. 4 and 5. 16. SrpHONARIA TENUICOSTULATA, n.sp. Pl. XV, Figs. 14 and 15. Testa irregulariter ovata, depresse conoidea, cinerea, costis tenuibus inequalibus circiter 60 instructa, lineis incrementi subrugosa; vertex centralis, minutus, pileiformis, postice inclinatus, fuscus, levis ; pagina interna nigro-fusca, ad marginem crenulatum albo multiradiata, in profundo aut fusca aut albida. Long. 18, diam. 14, alt. 65 mm. Hab.—Umblali, Natal (Burnup). Hab.—Umblali, Natal (Burnup). , ( p) Characterized by the numerous and fine costelle. The respiratory groove is indicated within by a broadish pale ray; externally it is not very conspicuous, yet always traceable. Characterized by the numerous and fine costelle. The respiratory groove is indicated within by a broadish pale ray; externally it is not very conspicuous, yet always traceable. Characterized by the numerous and fine costelle. The respiratory groove is indicated within by a broadish pale ray; externally it is not very conspicuous, yet always traceable. 307 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU 17. Dorts Naratensts, Krauss. 17. Dorts Naratensts, Krauss. Doris Natalensis, Krauss: Siidafrik. Moll., p. 35. Hab.—Natal (Krauss). 18. Doris (Pratyports) cortacea (Abraham). Doris coriacea, Abraham: Proc. Zool. Soc., 1877, p. 247, pl. xxvii, figs. 1-4. Hab.—South Africa; Seychelles; Sir C. Hardy’s Islands (Abraham). 19. Patio(?) Capensts (Quoy & Gaimard). 19. Patio(?) Capensts (Quoy & Gaimard). Polycera Capensis, Quoy & Gaimard: Voy. Uranie & Physicienne, Zool., p. 417, pl. xvi, fig. 4. Hab.—Cape of Good Hope (Q. & G.). 20. Triopa Lucipa, Stimpson. 20. Triopa Lucipa, Stimpson. Triopa lucida, Stimpson: Proce. Acad. Nat. Sci. Philad., 1855, vol. vii, . 088, et ee Bay, Cape of Good Hope (Stimpson), et ee Bay, Cape of Good Hope (Stimpson), 21. Prevropuyriii1a Naratensis, Bergh. Pleurophyllidia Natalensis, Bergh: Naturhist. Tidssk., ser. m1, vol. iv (1866), p. 34, pl. vid. ( ) Diphythdea lineata, Otto: Krauss, Siidafrik. Moll., p. 35. Hab.—Natal (Krauss). 22. TriITonIA PALLIDA, Stimpson. 22. TriITonIA PALLIDA, Stimpson. Tritonia pallida, Stimpson: Proce. Acad, Nat. Sci. Philad., vol. vii (1855), p. 388. Hab.—¥alse Bay, Cape of Good Hope (Stimpson). 23. SorrpuLa TESSELLATA (Reeve). 23. SorrpuLa TESSELLATA (Reeve). Tornatella tessellata, Reeve: Conch. Icon., vol. xv, figs. 6a, 6. Hab.—Durban (Burnup); Red Sea (Reeve). 24. Hypatrna crecutata (Martyn). 24. Hypatrna crecutata (Martyn). 24. Hypatrna crecutata (Martyn). Bulla cireulata, Martyn: Univ. Conch., 1784, pl. 95. Bulla cireulata, Martyn: Univ. Conch., 1784, pl. 95. Hydatina velum, Gmelin (1789): Pilsbry, Man. Conch., vol. xv, p. 388, pl. xliv, figs. 7-10. Hab.—Oft Natal (Burnup). A single small specimen from this locality is rather less globular than usual, and the striping and transverse girdles are exceptionally dark-coloured. 25. Atys cytinprica (Helbling). Atys cylindrica, Helblng: Pilsbry, Man. Conch., vol. xv, p. 265, pl. xxxiul, figs. 60-64. Atys cylindrica, Helblng: Pilsbry, Man. Conch., vol. xv, p. 265, pl. xxxiul, figs. 60-64. Atys elongata, A. Adams : Sowerby, Marine Shells 8. Afr., Appendix, Pu Fae Dachan (Burnup). Atys elongata, A. Adams : Sowerby, Marine Shells 8. Afr., Appendix, Pu Fae Dachan (Burnup). Atys elongata, A. Adams : Sowerby, Marine Shells 8. Afr., Appendix, Pu Fae Dachan (Burnup). 26. Axera sotuta (Gmelin). Akera soluta (Gmelin): Pilsbry, Man. Conch., vol. xv, p. 378, pl. xlii, fig. 18. Hab.—Durban (Burnup). Widely distributed in the Indian Ocean and Australian waters. 358 EDINGS OF THE MALACOLOGICAL SOCIETY. EDINGS OF THE MALACOLOGICAL SOCIETY. 27. Cyzicuna nitENs, Smith. Cylichna nitens, Smith: Ann. & Mag. Nat. Hist., vol. ix (1872), p. 351. Hab.—Durban (Burnup) ; Fiji (Smith). Only young, thin specimens. 98. Cyricuna TuBULOSA, Gould. Cylichna tubulosa, Gould: Pilsbry, Man. Conch., vol. xv, p. 321. ( ) 98. Cyricuna TuBULOSA, Gould. 98. Cyricuna TuBULOSA, Gould. Cylichna tubulosa, Gould: Pilsbry, Man. Conch., vol. xv, p. 321. Hab.—Simons Bay, Cape of Good Hope (Gould). Cylichna tubulosa, Gould: Pilsbry, Man. Conch., vol. Hab.—Simons Bay, Cape of Good Hope (Gould). 29. ToRNATINA MERIDIONALIS, Smith. 29. ToRNATINA MERIDIONALIS, Smith. Tornatina meridionalis, Smith: Journ. Conch., vol. x (1902), p. 249, pl. iv, fig. 2. Hab.—Durban. 30. CavoLInia QuapRIDENTATA (Lesueur). The shell figured by Mr. Sowerby is not the Lobiger pellucidus of A. Adams, which, as I have already pointed out,’ cannot be separated from LZ. serradifalet (Calcara). It belongs, however, to the genus Oxynoe, and appears to be allied to O. Hargravesi of H. Adams, from the New Hebrides. It is ovate, very thin, pellucid, corneous, and marked with faint lines of growth. The aperture is somewhat pyriform and large. The columella is very slightly thickened, ascends with a spiral twist within, and is connected with the outer lip above by a very thin callosity, which spreads over the whorl. Length 9°5, diam. 6°75 mm. Hab.—Durban ; Port Elizabeth (Sowerby). The figure in Mr. Sowerby’s work is a very poor representation of the species. 0. olivacea, Rafinesque, is of a more globose form than the present species. Hab.—Durban ; Port Elizabeth (Sowerby). The figure in Mr. Sowerby’s work is a very poor representation of the species. 0. olivacea, Rafinesque, is of a more globose form than the present species. 39. TrREBRA MONILIS, Quoy & Gaimard (?). 39. TrREBRA MONILIS, Quoy & Gaimard (?). Terebra monilis, Quoy & Gaimard: Reeve, Conch. Icon., vol. xii, figs. 42a, b. Hab.—Durban (Burnup); New Ireland, Tahiti, ete. Terebra monilis, Quoy & Gaimard: Reeve, Conch. Icon., vol. xii, figs. 42a, b. Hab.—Durban (Burnup); New Ireland, Tahiti, ete. 40. TrREBRA ARCHIMEDES, Deshayes. Terebra archimedes, Deshayes: Proc. Zool. Soc., 1859, p. 314. Hab.—Durban (Burnup). 41. Trrepra RAPHANULA, Lamarck. 41. Trrepra RAPHANULA, Lamarck. Terebra raphanula, Lamarck: Reeve, Conch. Icon., vol. xii, fig. 77. Hab.—Durban (Burnup); Amboyna (Reeve). 42. Trrepra Evotuta, Deshayes. Terebra evoluta, Deshayes: Reeve, Conch. Icon., vol. xii, fig. 55. Hab.—Durban (Burnup) ; Japan (Deshayes). 42. Trrepra Evotuta, Deshayes. 43. TrresRA AFFINIS, Gray. 43. TrresRA AFFINIS, Gray. Terebra affinis, Gray : Reeve, Conch. Icon., vol. xii, fig. 39. Hab.—Durban (Burnup); also Philippines, Mauritius, Tahiti, , y , , , g Hab.—Durban (Burnup); also Philippines, Mauritius, Tahiti, ete. 44, Trresra TEXTILIS, Hinds, var. 44, Trresra TEXTILIS, Hinds, var. Terebra textilis, var.: Smith, Journ. Conch., vol. x (1901), p. 115. Hab.—Durban (Burnup). 45. Trrepra Livipa, Reeve. Terebra livida, Reeve: Conch. Icon., vol. xii, fig. 116. Hab.—Durban (Burnup); Philippines (Reeve). 45. Trrepra Livipa, Reeve. Terebra livida, Reeve: Conch. Icon., vol. xii, fig. Hab.—Durban (Burnup); Philippines (Reeve). 46. Trerepra Macanprewn, Smith. 46. Trerepra Macanprewn, Smith. Terebra Macandrewti, Smith: Ann. & Mag. Nat. Hist., 1877, vol. xix, p. 30. CavoLInia QuapRIDENTATA (Lesueur). 30. CavoLInia QuapRIDENTATA (Lesueur). 30. CavoLInia QuapRIDENTATA (Lesueur). Cavolinia quadridentata (Lesueur) : Pelseneer, Pteropoda Challenger Exped., p. 78. Cavolinia quadridentata (Lesueur) : Pelseneer, Pteropoda Challenger Exped., p. 78. p p THyalea quadridentata, Lesueur: Sowerby, Conch. Icon., vol. xx, figs. 14a, 6. Hab.—Umkomaas, Natal (Burnup). 31. Cavoninra Trisprinosa (Lesueur). Cavolinia trispinosa (Lesueur): Pelseneer, Pteropoda Challenger Exped., p. 76. Hab.—Durban (Burnup). 32. Cavortrnta uncrinaTa (Rang). 32. Cavortrnta uncrinaTa (Rang). Cavolinia uncinata (Rang): Pelseneer, Pteropoda Challenger Exped., p. 84. Hab.—Durban (Burnup). 33. CAavoLrNia LonerRostRis (Lesueur). Cawolinia longirostris (Lesueur): Pelseneer, Pteropoda Challenger Exped., p. 79. Hab.—Durban ; Umkomaas, Natal (Burnup). 34. APLYSIA DEPILANS, Linné. Aplysia depilans, Linn.: Pilsbry, Man. Conch., vol. xvi, p. 69 (as Tethys). Hab.—Simons Bay, Cape of Good Hope (Watson). This locality is doubted by Mr. Pilsbry (loc. cit.). 35. Paraptysta Lown, Gilchrist. 35. Paraptysta Lown, Gilchrist. Paraplysia Lowit, Gilchrist : Trans. 8. Afr. Soc., vol. xi (1900), p. 122, pl. xvi, figs. 1 and 2. Hab.—S. Africa. 36. DoLaBRIFERA TRIANGULARIS, Watson. Dolabrifera triangularis, Watson: Gasteropoda Challenger Exped., p. 673, pl. 1, fig. 7; Pilsbry, Man. Conch., vol. xvi, p. 119, pl. ixy, figs. 7, 8. Hab.—Simons Bay, Cape of Good Hope (Challenger Exped.). p y g .—Simons Bay, Cape of Good Hope (Challenger Exped.). 37. PrevroprancH#a Capensis, Vayssicre. Pleurobranchea Capensis, Vayssiére: Journ. de Conchyl., vol. xlviii (1900), p. 10. HTab.—Cape of Good Hope. 359 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU 38. Oxynor Nartatensis, n.sp. Pl. XV, Fig. 18. Oxynoe pellucidus, A. Adams”: Sowerby, Marine Shells 8S. Afr., p. 58, pl. v, fig. 99—bad ! Oxynoe pellucidus, A. Adams”: Sowerby, Marine Shells 8S. Afr., p. 58, pl. v, fig. 99—bad ! Oxynoe pellucidus, A. Adams”: Sowerby, Marine Shells 8S. Afr., p. 58, pl. v, fig. 99—bad ! The shell figured by Mr. Sowerby is not the Lobiger pellucidus of A. Adams, which, as I have already pointed out,’ cannot be separated from LZ. serradifalet (Calcara). It belongs, however, to the genus Oxynoe, and appears to be allied to O. Hargravesi of H. Adams, from the New Hebrides. It is ovate, very thin, pellucid, corneous, and marked with faint lines of growth. The aperture is somewhat pyriform and large. The columella is very slightly thickened, ascends with a spiral twist within, and is connected with the outer lip above by a very thin callosity, which spreads over the whorl. Length 9°5, diam. 6°75 mm. 1 Ann. & Mag. Nat. Hist., vol. iii (1889), pp. 308-310. VOL. V.—OCT VOL. V.—OCTOBER, 1903. 25 PROCEE ab.—Durba P i G 360 EDINGS OF THE MALACOLOGICAL SOCIETY. Hab.—Durban (Ponsonby Coll. in Brit. Mus.); also known from the Persian Gulf and the Mekran coast. Hab.—Durban (Ponsonby Coll. in Brit. Mus.); also known from the Persian Gulf and the Mekran coast. Hab.—Durban (Ponsonby Coll. in Brit. Mus.); also known from the Persian Gulf and the Mekran coast. 47. Trrepra Loism, n.sp. Pl. XV, Fig. 1. 47. Trrepra Loism, n.sp. Pl. XV, Fig. 1. Testa subulata, nitens, albo et pallide fusco strigata, infra suturam rufo-fusco punctata, et cirea peripheriam anfractus ultimi zona angusta pallida eincta; anfraectus circiter 16 plani, costis numerosis arcuatis instructi, supra sulco transverso impresso sculpti, et seriebus quatuor puncturarum inter costas ornati, ultimus ad medium rotundatus, cirea basim suleis 4—5 sculptus, ad caudam albus, fusco maculatus; columella rectiuscula, antice ad canalem obliqua. Long. 30, diam. 6 mm. Hab.—Umkomaas, Natal (Burnup). In colour very similar to 7. amewna, Deshayes, 7. Andamanica, Melvill and Sykes, and 7. Gotoensis, Smith, but differing from the first two in its flatter and shorter whorls and closer and more delicate riblets, and from the last by different spiral sculpture. Named after Miss Lois Trotter, of Durban. 47. Trrepra Loism, n.sp. Pl. XV, Fig. 1. Testa subulata, nitens, albo et pallide fusco strigata, infra suturam rufo-fusco punctata, et cirea peripheriam anfractus ultimi zona angusta pallida eincta; anfraectus circiter 16 plani, costis numerosis arcuatis instructi, supra sulco transverso impresso sculpti, et seriebus quatuor puncturarum inter costas ornati, ultimus ad medium rotundatus, cirea basim suleis 4—5 sculptus, ad caudam albus, fusco maculatus; columella rectiuscula, antice ad canalem obliqua. Long. 30, diam. 6 mm. H b U k N l (B ) Hab. Umkomaas, Natal (Burnup). In colour very similar to 7. amewna, Deshayes, 7. Andamanica, Melvill and Sykes, and 7. Gotoensis, Smith, but differing from the first two in its flatter and shorter whorls and closer and more delicate riblets, and from the last by different spiral sculpture. Named after Miss Lois Trotter, of Durban. 48. Trresra (ABRETIA) DiveRsA, Smith. Terebra (Abretia) diversa, Smith: Journ. Conch., vol. x (1901), p. 115, pl.a, fic. 6. Terebra rufopunctata, Sowerby: Marine Shells 8. Afr., Appendix, 1897, p. 12 (mec Smith). Hab.—Umazinto, Natal (Burnup). ( ) Terebra (Abretia) diversa, Smith: Journ. Conch., vol. x (1901), p. 115, pl.a, fic. 6. p Terebra rufopunctata, Sowerby: Marine Shells 8. Afr., Appendix, 1897, p. 12 (mec Smith). Hab.—Umazinto, Natal (Burnup). 49. Treresra (ABReTIA) Lieurroorr, Smith. 1 The river Kowie flows into the sea at Port Alfred, Cape Colony. 30. CavoLInia QuapRIDENTATA (Lesueur). 228; Melvill & Standen, Proc. Zool. Soc., vol. 11 (1901), p. 428, Dl. oext, fe. 6: 25 60 PROCEEDINGS OF THE PROCEE ab.—Durba P i G Terebra (Abretia) Lightfooti, Smith : Journ. Conch., vol. ix (1899), Dp. 2475 pl.v; fie. 1. Hab.—Table Bay, 10 fathoms (Lightfoot). 50. Trresra (Hasruza) aprcrrrncra, Sowerby. Terebra apicitincta, Sowerby : Proc. Malae. Soc., vol. iv, p. 5 (1900), pl. i, fig. 14. Hab.—The Kowie ' (Sowerby). 51. Terepra (Hastuna) casta, nov., var. NATALENSIS. Terebra casta, Hinds: Sowerby, Mar. Shells 8. Afr., Appendix, p. 13. Hab.—Umkomaas, Natal. The specimens from Natal differ from the typical form both in colour and sculpture. They are more copiously stained with reddish brown, and the riblets are more acute, not flattened. 7. bdbipartita, Deshayes, from the Sandwich Islands, which I regard as a variety of this species, has similar sharpish coste, but is somewhat differently coloured. 52, Trrepra (Impaces) cusprpata, Hinds. Terebra cuspidata, Hinds: Sowerby, Thes. Conch., vol. i, p. 181, pl. xlv, fig. 128; Reeve, Conch. Icon., vol. xii, pl. xxvi, fig. 141. Hab.—Cape Coast (Hinds). 52, Trrepra (Impaces) cusprpata, Hinds. 361 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLUS 53. Trrepra (Impaces) aprcina (?), Deshayes. Terebra apicina, Deshayes: Reeve, Conch. Icon., vol. xii, figs. 136a, d. Hab.—Umkomaas, Natal (Burnup). 54,. Terepra (Mazattanta) tTHyrzA, Melvill. 54,. Terepra (Mazattanta) tTHyrzA, Melvill. Terebra (Euryta) thyrea, Melvill: Mem. and Proc. Manchester Lit. and Phil. Soc., vol. xli, No. 7 (1897), p. 10, pl. vi, fig. 13. Hab.—Durban (Burnup); Mekran coast (Melvill). 55, Conus prreratos, Dillwyn. Conus piperatus, Dillwyn: Descriptive Cat., vol. i, p. 401. Conus punctatus, Hwass.: Reeve, Conch. Icon., vol. i, figs. 133¢-e. Hab.—Umkomaas (Burnup); Karachi, ete. 56. Conus Crytonicus, Chemnitz. Conus Ceylonicus, Chemnitz: Reeve, Conch. Icon., vol. i, Emendations, fig. 17 (as C. obesus). Hab.—Isipingo, Natal (Burnup). 57. Conus miILiaRis, Hwass. Conus miliaris, Hwass.: Reeve, Conch. Icon., vol. i, fig. 198. Hab. —Isipingo, Natal (Burnup); China, Annaa I., Lizard I., Tonga Is., Mozambique, ete. 58. Conus FLAvIpUs, Lamarck. 58. Conus FLAvIpUs, Lamarck. Conus flavidus, Lamarck: Reeve, Conch. Icon., vol. i, fig. 207 ; Kiener, Coq. Viv., pl. xxvi, fig. 4. Hab.—Pondoland, Cape Colony; ‘ off Durban.” With this species may be united C. Maltzanianus, Weinkauff, C. frigidus, Reeve, C. neglectus, Pease. The species is widely distributed in the Indian and Pacific Oceans. 59. Conus caprtanevs, Linn. (?). Conus capitaneus, Linn.: Reeve, Conch. Icon., vol. i, fig. 54. Hab.—Durban. ; 60. Conus vEexittuM, Gmelin. Conus vexillum, Martini: Reeve, Conch. Icon., vol. i, fig. Hab.—\sipingo (Burnup). co 61. Conus consors, Sowerby. Conus consors, Sowerby : Thes. Conch., vol. iii, fig. 492. var.= C. anceps, A. Adams: Sowerby, fig. 493. », =C. innexus, A. Adams: Proc. Zool. Soc., 1853, p. 118. 61. Conus consors, Sowerby. Conus consors, Sowerby : Thes. Conch., vol. iii, fig. 492. var.= C. anceps, A. Adams: Sowerby, fig. 493. », =C. innexus, A. Adams: Proc. Zool. Soc., 1853, p. 118. », =C. Daullet, Crosse: Rev. Mag. Zool., 1858, pp. 81, 119, pl. 1, figs. 2, 2a. Hab.— Natal (A. Adams for cnnexus). 62. Conus apLustRE, Reeve. Conus aplustre, Reeve: Conch. Icon., vol. i, fig. 170; Sowerby, Thes. Conch., vol. ii, fig. 448. Hab.—Cape of Good Hope (Brit. Mus.). 362 EDINGS OF THE MALACOLOGICAL SOCIETY. 63. Conus LinEatus, Chemnitz. Conus lineatus, Chemnitz: Reeve, Conch. Icon., vol. i, fig. 181. Hab.—Durban. 64. Conus pauprercuLus, Sowerby. 64. Conus pauprercuLus, Sowerby. Conus pauperculus, Sowerby: Thes. Conch., vol. iii, p. 30, pl. exevi, fir. 217, Hab.—Cape of Good Hope (Sowerby). 65. Conus stmetEex, Sowerby. 65. Conus stmetEex, Sowerby. Conus simplex, Sowerby: Thes. Conch., vol. ili, p. 31, pl. exev, fig. 199; Weinkauff, Conch. Cab., 2nd ed., p. 248, pl. xh, fig. 10. Hab.—S. Africa (Weinkauff). 66. Conus scrrutus, Reeve. 54,. Terepra (Mazattanta) tTHyrzA, Melvill. Conus scitulus, Reeve: Conch. Icon., vol. i, Conus, Suppl., pl. ix, fig. 283. HTab.—Cape Colony (Mus. Cuming). Synonyms of this species are C. jaspideus, Kiener nec Gmelin, C. pictus, Sowerby nee Reeve, and C. Daniel of Crosse. 67. Conus nruposus, Hwass. 67. Conus nruposus, Hwass. Conus nimbosus, Hwass.: Reeve, Conch. Icon., vol. i, pl. xiii, fig. 66. Hab.—Umkomaas, Natal (Burnup). Conus nimbosus, Hwass.: Reeve, Conch. Icon., vol. i, pl. xiii, fig. 66. Hab.—Umkomaas, Natal (Burnup). 68. Conus «tans, Bruguicre. 68. Conus «tans, Bruguicre. Conus glans, Bruguicre: Sowerby, Proc. Malac. Soe., vol. iv, p. 5 (1900). Hab.—Pondoland, Cape Colony (Sowerby). 69. CLAVATULA PARILIS, Smith. Clavatula parilis, Smith: Journ. Conch., vol. x (1901), p. 115, pl. i, fig. Ja Hab.—Ott Durban, 40 fathoms (Quekett). 70. PLevroromMA MARMORATA, Lamarck. 70. PLevroromMA MARMORATA, Lamarck. Pleurotoma marmorata, Lamarck: Reeve, Conch. Icon., vol. i, fig. 21a; Sowerby, Marine Invest. 8. Africa, 1892, p. 100, var. maculata. Hab.—Otf mouth of Tugela River, 55 fathoms (Sowerby). 71. Prevroroma Giicuristr, Sowerby. 71. Prevroroma Giicuristr, Sowerby. Pleurotoma Gilchristi, Sowerby: Marine Invest. 8. Africa, 1902, p. 99, pl. 1, fig. 9. HHab.—Off mouth of Tugela River, 55 fathoms (Sowerby). Very closely allied to P. monilifera of Pease from the Sandwich Islands, and probably recorded under that name in the Appendix to the ‘‘ Marine Shells of South Africa,” p. 2. 72. PxLevroroma BiyuBpaTa, Reeve. Pleurotoma bijubata, Reeve: Conch. Icon., vol. i, pl. x, fig. 87. ITab.—Durban. Pleurotoma bijubata, Reeve: Conch. Icon., vol. i, pl. x, fig. 87. ITab.—Durban. 363 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU 73. PLEUROTOMA VERTEBRATA, Smith. Pleurotoma vertebrata, Smith: Ann. & Mag. Nat. Hist., vol. xv (1875), p. 416. Hab.—Durban (Burnup); Japan (Smith); Persian Gulf (Melvill). Hab.—Durban (Burnup); Japan (Smith); Persian Gulf (Melvill). 74, Prevroroma (Surcura) anrerrpion, Watson. Pleurotoma (Surcula) anteridion, Watson: Gasteropoda Challenger Exped., p. 295, pl. xix, fig. 6. Hab.—Ott Cape of Good Hope, 150 fathoms. 75. Deira rvcrinata, Sowerby. 75. Deira rvcrinata, Sowerby. Pleurotoma inclinata, Sowerby: Proc. Zool. Soc., 1893, p. 488, pl. xxxviii, figs. 25, 26. Hab.—Isipingo, Natal (Burnup); Mauritius (Sowerby). 76. Dra pratystoma, Smith. Pleurotoma (Clionella) platystoma, Smith: Ann. & Mag. Nat. Hist., vol. xix (1877), p. 501. ( ) p Pleurotoma Wilkie, Sowerby : Journ. Conch., vol. vi (1889), p. 7, pled, figs 2. I can see no reasons for separating these two species. (See Sowerby, Marine Shells 8. Afr., pp. 4, 5.) 77. Dritt1a Rvueiscurpra, Sowerby. 77. Dritt1a Rvueiscurpra, Sowerby. Drillia rugisculpta, Sowerby : Proc. Malac. Soc., vol. iv, p. 214 (1901), pl. xa, fig: 20: Hab.—The Kowie. 78. Dera canruarts (Reeve). Pleurotoma cantharis, Reeve: Sowerby, Proc. Malac. Soe., vol. iv, p. 5 (1900). Hab.—Pondoland, Cape Colony (Sowerby). 79. Oxtva ELEGANS, Lamarck. Oliva elegans, Lamarck: Reeve, Conch. Icon., vol. vi, pl. xii, figs. 20a—c. Hab.—Durban. 80. Oxiva PAxILLUs, Reeve. Oliva paxillus, Reeve: Conch. Icon., vol. vi, pl. xxi, figs. 56a, 6. Hab.—Offt Durban. 81. Oxrva pricra, Reeve. 81. Oxrva pricra, Reeve. Oliva picta, Reeve: Conch. Icon., vol. vi, pl. xxvi, fig. 79. Hab.—Durban (Burnup); Philippines (Reeve). Oliva picta, Reeve: Conch. Icon., vol. vi, pl. xxvi, fig. 79. Hab.—Durban (Burnup); Philippines (Reeve). 82. Oxrva DacrytroLa, Duclos. 82. Oxrva DacrytroLa, Duclos. Oliva dactyliola, Duclos: Sowerby, Proc. Malac. Soc., vol. iv, p. 8 1900). Hob Pondoland, Cape Colony (Sowerby). Oliva dactyliola, Duclos: Sowerby, Proc. Malac. Soc., vol. iv, p. 8 1900). Hob Pondoland, Cape Colony (Sowerby). 83. Oxtva nana, Lamarck. Oliva nana, Lamarck: Tryon, Man. 71. Prevroroma Giicuristr, Sowerby. Conch., vol. v, p. 91, pl. xxxvi, figs. 96-100. Hab.—South Africa (Tryon). 364 EDINGS OF THE MALACOLOGICAL SOCIETY. EDINGS OF THE MALACOLOGICAL SOCIETY. 84. Ancitta Monrrovuziert, Souverbie. Ancillaria Montrouziert(, Reeve: Conch. Icon., vol. xv, fig. 9; Watson, Gasteropoda Challenger Exped., p. 229. Hab.—Twenty-five miles off Cape of Good Hope, 98 fathoms ( Watson). 85. Ancitta Hasta (Martens). Ancillaria hasta, Martens: Sitzungsb. naturf. Freunde Berlin, 1902, p- 241. Hab.—Agulhas Bank, 250 fathoms (Martens). 86. AncitLta oscuLata, Sowerby. 86. AncitLta oscuLata, Sowerby. Ancilla osculata, Sowerby: Proc. Malac. Soc., vol. iv, p. 3 (1900), pl. i, fig.:6: Hab.—Pondoland, Cape Colony. 87. Harpa conoratts, Lamarck. Harpa conoidalis, Lamarck: Reeve, Conch. Icon., vol. i, figs. 7a-e. Hab.—Durban (Burnup); Ceylon, Mauritius, Philippines, ete. 88. Marernerta Axncornsis, Smith. Marginella Algoensis, Smith: Journ. Conch., vol. x (1901), p. 114, pli a, fig. 4: Hub.—Algoa Bay, Cape Colony (Crawford). 89. MArGINELLA LINEATO-LABRUM, Gaskoin. 89. MArGINELLA LINEATO-LABRUM, Gaskoin. Marginella lineato-labrum, Gaskoin: Sowerby, Proc. Malac. Soe., vol. iv, p. 4 (1900). Hab.—Pondoland, Cape Colony (Sowerby). 90. MarGINELLA PUNCTILINEATA, Smith. 90. MarGINELLA PUNCTILINEATA, Smith. 90. MarGINELLA PUNCTILINEATA, Smith. Marginella punetilineata, Smith: Journ. Conch., vol. ix (1899), p- 249, pl. v, fig. 5. Hab.—Umkomaas, Natal (Burnup). 91. Marernetta prapocuus, Adams & Reeve. Marginella diadochus, Adams & Reeve: Reeve, Conch. Icon., vol. xv, pl. ix, figs. 35a, 6; Weinkauff, Conch. Cab., 2nd ed., p. 31, pl. v, figs. 2, 3, 5. Hab.—¥alse Bay (Lightfoot); Straits of Sunda (Ad. & Rve.); W. Africa (Marrat, Quart. Journ. Conch., vol. i, p 242); Cape of Good Hope, 150 fathoms ( Watson). I am inclined with Watson to believe that this form and JZ. musica, Hinds, are only variations of one and the same species. Both are quoted by Marrat as West African. 92. Marcrtvetta rustrormis, Hinds. Marginella fusiformis, Hinds: Reeve, Conch. Icon., vol. xv, fig. 79. Hab.—Port Elizabeth (Crawford) ; Straits of Malacca (Reeve). 93. Manreinetta Newcomst, Reeve. 93. Manreinetta Newcomst, Reeve. 93. Manreinetta Newcomst, Reeve. Marginella Newcombi, Reeve: Conch. Icon., vol. xv, pl. v, figs. 15a, b. Hab.— Lagulhas Bank, Cape of Good Hope” (Reeve). Marginella Newcombi, Reeve: Conch. Icon., vol. xv, pl. v, figs. 15a, b. Hab.— Lagulhas Bank, Cape of Good Hope” (Reeve). 365 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU 94. MarGInELLA PUELLA (Gould). Volvarina puella,.Gould: Proc. Boston Soc. Nat. Hist., 1860, vol. vii, p- 885; Otia, p. 139. Marginella puella, Gould: Sowerby, Marine Shells 8. Afr., Appendix, 9 This species is probably the same as IZ. Capensis, Dunker. This species is probably the same as IZ. Capensis, Dunker. 95. MarGINeLLA SEMINULA, Gould. 95. MarGINeLLA SEMINULA, Gould. Marginella seminula, Gould: Otia Conch., 1860, p. 139. Hab.—False Bay, Cape of Good Hope (Gould). Marginella seminula, Gould: Otia Conch., 1860, p. Hab.—False Bay, Cape of Good Hope (Gould). 96. Mareinetta cysriscus, Redfield. Marginella cystiscus Redfield: Tryon Man Conch vol v p 46 96. Mareinetta cysriscus, Redfield. Marginella cystiscus, Redfield: Tryon, Man. Conch., vol. v, p. 46. ( ) 96. Mareinetta cysriscus, Redfield. Marginella cystiscus, Redfield: Tryon, Man. Conch., vol. v, p. 46. Cystiscus Capensis, Stimpson: Amer. Journ. Conch., vol. i (1865), p. 55, pl. vii, fig. 2. Cystiscus Capensis, Stimpson: Amer. Journ. Conch., vol. i (1865), p. 55, pl. vii, fig. 2. Hab.—Cape of Good Hope (Redfield & Stimpson). Hab.—Cape of Good Hope (Redfield & Stimpson). 97. MarcGINnELLA FALLAX, n.sp. Pl. XV, Fig. 20. 97. MarcGINnELLA FALLAX, n.sp. Pl. XV, Fig. 20. 90. MarGINELLA PUNCTILINEATA, Smith. Testa alba, ovata, supra acuminata ; spira conica, ad apicem obtusa ; anfractus 4, primus conyexus, sequentes duo leviter concavi, infra suturam pellucido-marginati, ultimus convexiusculus, antice angus- tatus; labrum fere rectum, inflexum, intus tenuissime liratum, ad marginem acutum; columella quinque-plicata, plica suprema minima, vix conspicua. Long. 8 mm., diam. 4. 97. MarcGINnELLA FALLAX, n.sp. Pl. XV, Fig. 20. 97. MarcGINnELLA FALLAX, n.sp. Pl. XV, Fig. 20. Testa alba, ovata, supra acuminata ; spira conica, ad apicem obtusa ; anfractus 4, primus conyexus, sequentes duo leviter concavi, infra suturam pellucido-marginati, ultimus convexiusculus, antice angus- tatus; labrum fere rectum, inflexum, intus tenuissime liratum, ad marginem acutum; columella quinque-plicata, plica suprema minima, vix conspicua. Long. 8 mm., diam. 4. Hab.— Port Alfred, Cape Colony (W. H. Turton). p y ( ) This species has been confounded with MM. paxillus, Reeve, from which it differs in its smaller size, more conical spire, concave whorls, quite different columellar folds, and the lire within the outer lip, the labrum in paxillus being quite smooth within. 98. Votura (Fusrvotura) pyrraostoma (Watson). 98. Votura (Fusrvotura) pyrraostoma (Watson). Fusus (Sipho) pyrrkostoma, Watson: Gasteropoda Challenger Exped., p. 208, pl. xu, fig. 2. Voluta (Fusivoluta) pyrrhostoma, Watson: Martens, Sitzungsb. naturf. Freunde Berlin, 1902, p. 288. Hab.—Twenty miles off Cape of Good Hope, in 98 fathoms. The radula of this species is similar to that of the Volutide 98. Votura (Fusrvotura) pyrraostoma (Watson). Fusus (Sipho) pyrrkostoma, Watson: Gasteropoda Challenger Exped., p. 208, pl. xu, fig. 2. Voluta (Fusivoluta) pyrrhostoma, Watson: Martens, Sitzungsb. naturf. Freunde Berlin, 1902, p. 288. Hab.—Twenty miles off Cape of Good Hope, in 98 fathoms. The radula of this species is similar to that of the Volutide (Martens). 99. Votura (AtcirHoE) Ponsonsyr, Smith. Voluta (Alcithoe) Ponsonbyi, Smith: Proc. Malac. Soc., vol. iv, p. 231 (1901), fig. in text. The radula of this species is similar to that of the Volutide (Martens). 99. Votura (AtcirHoE) Ponsonsyr, Smith. Voluta (Alcithoe) Ponsonbyi, Smith: Proc. Malac. Soc., vol. iv, p. 231 (1901), fig. in text. Hab.—Off Durban, 40 fathoms (Quekett). 100. Votura (Lyrra) Quexerri, Smith. Voluta (Lyria) Queketti, Smith: Proc. Malac. Soc., vol. iv, p. 284 (1901), fig. in text. Hab.—Off Durban, 40 fathoms (Quekett). 101. Votura (Lyria) mirr#rormis, Lamarck. Lyria mitreformis, Lamarck: Reeve, Conch. Icon., vol. vi ( Voluta), pl. i, figs. 7a4,6; Tryon, Man. Conch., vol. iv, p. 103, pl. xxx1, fig. 143. Hab.—S. Africa (Tryon). EDINGS OF THE MALACOLOGICAL SOCIETY. 366 EDINGS OF THE MALACOLOGICAL SOCIETY. 102. Voturrirrmes ABysstcoLa, Adams & Reeve. Volutilithes abyssicola, Ad. & Rve.: M. F. Woodward, Proc. Malac. Soe., vol. iv, p. 121 (1900), pl. x, anatomy; Sowerby, Marine Invest. S. Africa, 1902, p. 97. Hab.—Lat. 34° 43’ 15” §., long. 18° 30’ E., in 125 fathoms (Sowerby). 103. Voturinirmes Griucreisti, Sowerby. 103. Voturinirmes Griucreisti, Sowerby. Volutilithes Gilchristit, Sowerby: Marine Invest. 8. Africa, 1902, p: 99, pl. un, fig. 5. Hab.—Cape Natal, 200 fathoms. 104. Neprunrorsis Gricnrist1, Sowerby. Neptuneopsis Gilchrist’, Sowerby: Marine Invest. S. Africa, vol. i (1898), p. 5, pl.; M. F. Woodward, Proc. Malac. Soc., vol. iv, p. 120 (1900). Hab.—Off Cape of Good Hope, 33 fathoms. 105. Mrrra episcopatis (Linn.). Mitra episcopalis, Linn. : Reeve, Conch. Icon., vol. ui, pl. i, fig. 5. Hab.—Durban (Burnup & Quekett); Ceylon, Philippines, Tahiti, ete. 106. Mirra FramMrieEraA, Reeve. 106. Mirra FramMrieEraA, Reeve. Mitra flammigera, Reeve: Conch. Icon., vol. i, figs. 1734, 6. Hab.—Durban; Kalk Bay (Burnup) ; ? (Reeve). 106. Mirra FramMrieEraA, Reeve. Mitra flammigera, Reeve: Conch. Icon., vol. i, figs. 1734, 6. Hab.—Durban; Kalk Bay (Burnup) ; ? (Reeve). 107. Mirra timptrera, Lamarck. Mitra limbifera, Lamarck: Sowerby, Journ. Conch., vol. vii (1894), p. 369. Hab.—Natal (Sowerby); Durban (Ponsonby Coll.). 108. Mirra puncrosrriata, A. Adams. Mitra punctostriata, A. Adams: Proe. Zool. Soe., 1854, p. 134. Hab.—Umkomaas, Natal (Burnup); Ceylon (Adams). 109. Mrrra pica, Reeve. Mitra pica, Reeve: Conch. Icon., vol. ii, pl. xxxi, fig. 247; Sowerby, Thes. Conch., vol. iv, p. 25, pl. 374, fig. 522. Hab.—Cape of Good Hope (Sowerby). 110. Mrrra patura, Reeve. 110. Mrrra patura, Reeve. Mitra patula, Reeve: Conch. Icon., vol. ii, pl. xxxix, fig. 333; Sowerby, Thes. Conch., vol. iv, p. 25, pl. 374, fig. 526; Tryon, Man. Conch., vol. iv, p. 125, pl xxxvu, fig. 91. Hab.—Cape of Good Hope (Sowerby & Tryon). 111. Mrrra (Cyztrypra) crenunata, Lamarck. 111. Mrrra (Cyztrypra) crenunata, Lamarck. Mitra erenulata, Lamarck: Reeve, Conch. Icon., vol. ii, figs. 190a, 0. HHab.—Umkomaas, Natal. 112. Mirra Kowteensis, Sowerby. Mitra Kowieensis, Sowerby: Proc. Malac. Soc., vol. iv, p. 213 (1901), Dl, ne Liz Tlab.—The Kowie. 367 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU 113. Mrrra rvzonata, Sowerby. Mitra euzonata, Sowerby: Proc. Malac. Soc., vol. iv, p. 4 (1900), ple i, fie. Li. Hab.—The Kowie. 114. Mirra canaricutata, Sowerby. 114. Mirra canaricutata, Sowerby. Mitra canaliculata, Sowerby: Proc. Malac. Soe., vol. iv, p. 4 (1900), pl. i, fig. 10. Hab.—The Kowie. Novit. Gane es xxxil, figs: 1, 2. Conch. Cab., Fasciolari ia, p. 143. 1 2 115. Mirra Batuyrapue, Sowerby. 115. Mirra Batuyrapue, Sowerby. Mitra bathyraphe, Sowerby: Proce. Malac. Soe., vol. iv, p. 4 (1900), plea, te; 9. Hab.—The Kowie. 116. Mirra opetiscus, Reeve. 116. Mirra opetiscus, Reeve. Mitra obeliscus, Reeve: Conch. Icon., vol. ii, pl. xv, fig. 107. Hab.—Durban (Burnup); Philippines (Reeve). Mitra obeliscus, Reeve: Conch. Icon., vol. ii, pl. xv, fig Hab.—Durban (Burnup); Philippines (Reeve). , , , p , Hab.—Durban (Burnup); Philippines (Reeve). 117. Mrrra ataupa, Sowerby. 117. Mrrra ataupa, Sowerby. Mitra alauda, Sowerby: Thes. Conch., vol. iv, pl. 361, figs. 184, 135 (non Quoy). FTab.—Durban; also Mauritius. 118. Fusus oceniirervs, Bory de St. Vincent. Fusus ocelliferus, Bory de St. V.: Reeve, Conch. Icon., vol. iv, fig. 3; Sowerby, Thes. Conch., vol. iv, fig. 64. var. = FP. robustior, Sowerby : op. cit., fig. 63. Hab —Cape of Good Hope (Sowerby Challenger var. = FP. robustior, Sowerby : op. cit., fig. 63. Hab.—Cape of Good Hope (Sowerby, Challenger Exped., Brit. Mus.); Port Elizabeth, for var. robustior (Sowerby). This species is very variable in form, some specimens being much more elongate than others, and much less angulated at the shoulder of the body-whorl. In the series of specimens examined all the connecting links are present. /. verruculatus, Lamarck, is the same as the typical form (Kiener, Coq. Viv., pl. xv, fig. 1). 119. Fusus rapratis, Watson. Fusus radialis, Watson: Gasteropoda Challenger Exped., p. 195, pl. xiv, fig. 2. Hab.—Off Cape of Good Hope, 150 fathoms. 120. Fosus toreuma (Martyn). Buccinum toreuma, Martyn: Uniy. Conch., pl. lvi. Lusus toreuma, Martyn: Reeve, Conch. Icon., vol. iv, fig. 27. Hab. — Pulo Condore (Martyn); Ceylon (Reeve); off Natal (Quekett). The single example from Natal is exceptionally slender, with the whorls less acutely angled at the middle. This results from two of the spiral ridges at this part being of equal size. The nodules upon the body-whorl are also produced below into more distinct costi than usual. 120. Fosus toreuma (Martyn). Buccinum toreuma, Martyn: Uniy. Conch., pl. lvi. Lusus toreuma, Martyn: Reeve, Conch. Icon., vol. iv, fig. 27. Buccinum toreuma, Martyn: Uniy. Conch., pl. lvi. Lusus toreuma, Martyn: Reeve, Conch. Icon., vol. iv, fig. 27. Hab. — Pulo Condore (Martyn); Ceylon (Reeve); off Natal (Quekett). DINGS OF THE MALACOLOGICAL SOCIETY. 368 121. Fusus suscontractus, Sowerby. 121. Fusus suscontractus, Sowerby. Fusus subcontractus, Sowerby : Marine Invest. 8. Africa, 1902, p. 97, pl. ui, fig. 2. Fusus subcontractus, Sowerby : Marine Invest. 8. Africa, 1902, p. 97, pl. ui, fig. 2. p g Hab.—Cape Natal, 200 fathoms (Sowerby). p g Hab.—Cape Natal, 200 fathoms (Sowerby). 122. Fusus Arricanus (Sowerby). Pl. XV, Fig. 19. Fulgur Africanus, Sowerby: Marine Shells 5. Afr., Appendix, p. 1, Dl. wi, ta: 589: Testa clavata, rimata, pallida, vel dilute fuscescens, periostraco fusco induta; inter tubercula nigro lineata; spira conica, eradata ; anfractus normales 5, supra declives, ~ leviter concavi, infra anculati, ad angulum tuberculati, superiores tres spiraliter fortiter striati, striis in ultimo fere ev anidis, ultimus cingulis latis transversis paulo elatis circiter 12 instructus, antice longe rostratus, rostro sinistrorsum verso, ad extremitatem subrecurvo ; apertura alba; labrum tenue, ad marginem denticulatum ; columella supra valde arcuata, callo albo infra partim libero, superne incrassato, induta. Long. 70, diam. 85 mm.; apertura sine canale 27 mm. longa, 14 lata. p g Hab.—Off Durban (Quekett) ; Port Elizabeth (Sowerby). Hab.—Off Durban (Quekett) ; Port Elizabeth (Sowerby). The type of this species which is in the Museum collection is a young half-grown shell in poor condition, and the figure of it conveys but a slight idea of the adult shell. A curious feature is the columellar callus. being detached from the whorl at the lower part, forming an umbilical rimation. A similar detaching of the callus also occurs in the following species, Fusus Adamsti of Kobelt. The position of the present species is uncertain, the animal being unknown, but the prolonged canal seems to recall the genus usus. Another specimen of F. Africanus received recently is 78 mm. in length, and has the protoconch preserved. This consists of two smooth globose whorls forming a papillose apex. (Q ) ; ( y) The type of this species which is in the Museum collection is a young half-grown shell in poor condition, and the figure of it conveys but a slight idea of the adult shell. A curious feature is the columellar callus. being detached from the whorl at the lower part, forming an umbilical rimation. A similar detaching of the callus also occurs in the following species, Fusus Adamsti of Kobelt. The position of the present species is uncertain, the animal being unknown, but the prolonged canal seems to recall the genus usus. Another specimen of F. 121. Fusus suscontractus, Sowerby. 7), the second row of nodules upon the body-whorl, mentioned by Mr. Sowerby in his description, is not indicated. Although not very conspicuous in some specimens they are generally more or less observable. The umbilicus is much more open in some examples than in others. The largest specimen examined, when perfect, probably measured as much as 75mm. in length. A comparison of a series of specimens in good condition with the type of L. imbricatus, now in the British Museum, shows that they all belong to one and the same species. Z. ambricatus was described from a somewhat immature specimen in which the labrum was undeveloped. In the figure of this species (Marine Shells 8. Afr., pl. vi, fig. 7), the second row of nodules upon the body-whorl, mentioned by Mr. Sowerby in his description, is not indicated. Although not very conspicuous in some specimens they are generally more or less observable. The umbilicus is much more open in some examples than in others. The largest specimen examined, when perfect, probably measured as much as 75mm. in length. A comparison of a series of specimens in good condition with the type of L. imbricatus, now in the British Museum, shows that they all belong to one and the same species. Z. ambricatus was described from a somewhat immature specimen in which the labrum was undeveloped. 126. Larirus PoLyGonus, var. Turbinella polygona (Gmelin): Reeve, Conch. Icon., vol. iv, pl. i, figs. la-c; Kobelt, Conch. Cab., pl. xvii, figs. 1-7; Kiener, Cog. Viy., pl. xiii, fig. 2. Hab.—Off Durban, in deep water (Quekett). The single example obtained differs from the typical form in having the body-whorl more prolonged anteriorly, and in the absence of the angulation at the middle of the body-whorl and in the less pronounced character of the longitudinal costation. The coloration is practically the same. 127. Latrrus aLsoapicatus, Smith. Latirus alboapicata, Smith: Journ. Conch., vol. x (1902), p. 250, pl. iv, fig. 5. Hab.— Durban. 128. PrristeRNIA LEUCOTHEA, Melvill. Peristernia leucuthea, Melvill: Mem. & Proc. Manchester Lit. & Phil. Soc., ser. Iv, vol. iv (1891), p. 399, pl. ui, fig. 15. Euthria eburnea, Sowerby: Proc. Malac. Soc., vol. iv, p. 1 (1900), plea, fig. 2. Hab.—Port Natal (Melvill); Isipingo and Umkomaas (Brit. Mus.) ; Pondoland (Sowerby). 121. Fusus suscontractus, Sowerby. Africanus received recently is 78 mm. in p g g Another specimen of F. Africanus received recently is 78 mm. in length, and has the protoconch preserved. This consists of two smooth globose whorls forming a papillose apex. 123. Fusus Apamsit, Kobelt. 123. Fusus Apamsit, Kobelt. 123. Fusus Apamsit, Kobelt. Fusus ventricosus, H. Adams: Proc. Zool. Soc., 1870, p. 110, woodcut. Fusus Adamsii, Kobelt: Conch. Cab., p. 152, pl. xlvii, fig. 3 Hab.—The Agulhas Bank, Cape of Good Hope (Adams). The name ventricosus, having already been employed in the genus, was changed by Kobelt to Adamsz. 124. Fascronarta Hrynemanni, Dunker. 124. Fascronarta Hrynemanni, Dunker. Fasciolaria Heynemanni, Dunker: Sowerby, Marine Shells 8. Afr., p. 17. Two very interesting specimens from deep water off Durban have 124. Fascronarta Hrynemanni, Dunker. Fasciolaria Heynemanni, Dunker: Sowerby, Marine Shells 8. Afr., p. 17. Two very interesting specimens from deep water off Durban have been sent by Mr. Quekett. They are much smaller and more slender than the type figured by Dunker,' and exhibit only the faintest traces of the transverse ridges upon the body-whorl. Consequently they have a much smoother general appearance. Their protoconch also is smaller. The Fuasciolaria purpurea of Jonas is not, as stated by Sowerby, the same as the present species. Its distinguishing features have alre ‘ady been Beanies out by uate 5 Fasciolaria Heynemanni, Dunker: Sowerby, Marine Shells 8. Afr., Fasciolaria Heynemanni, Dunker: Sowerby, Marine Shells 8. Afr., p. 17. p Two very interesting specimens from deep water off Durban have been sent by Mr. Quekett. They are much smaller and more slender than the type figured by Dunker,' and exhibit only the faintest traces of the transverse ridges upon the body-whorl. Consequently they have a much smoother general appearance. Their protoconch also is smaller. The Fuasciolaria purpurea of Jonas is not, as stated by Sowerby, the same as the present species. Its distinguishing features have alre ‘ady been Beanies out by uate 5 369 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU 125. Larirvus aBnormis, Sowerby. Latirus abnormis, Sowerby: Marine Shells 8. Afr., Appendix, pl. vi, ‘fig. 7. g Latirus imbricatus, Sowerby: Marine Invest. 8. Africa, 1902, p. 96, Dla tie. 1, H b N l Latirus imbricatus, Sowerby: Marine Invest. 8. Africa, 1902, p. 96, Dla tie. 1, Dla tie. 1, Hab.—Natal. Hab.—Natal. In the figure of this species (Marine Shells 8. Afr., pl. vi, fig. 121. Fusus suscontractus, Sowerby. Beneath the thick deciduous periostracum, which exhibits distinct lines of growth and has a silky appearance in certain lights, the shell is for the most part stained with brown, but where the surface is worn it is white. Hab.—Off Durban, in deep water (Quekett). The type of this species figured in the above work, although in fairly good condition, does not show all the characters of this interesting form. A specimen recently submitted to me from Port Shepstone (Burnup), and which Mr. Sowerby has identified as belonging to his species, may be thus described. It differs from the type in having ten or eleven nodosities at the angle of the body-whorl, and these protuberances upon the upper whorls produce a wavy sutural line. The body-whorl also exhibits four or five rather indistinct and somewhat nodose, transverse, raised, broad, rounded ridges. The aperture is white within, the outer lip being much thickened, slightly expanded anteriorly, blotched with brown within the margin, with a strong tubercular prominence within at the hinder end. The columella is covered with a thick brown-stained callus, which is broadly reflexed and appressed over the umbilical rimation, and thickened into a white prominence posteriorly a little distance above the plice. The upper two of these are about equal in size, the next one is rather smaller, and the foremost one still less conspicuous. Beneath the thick deciduous periostracum, which exhibits distinct lines of growth and has a silky appearance in certain lights, the shell is for the most part stained with brown, but where the surface is worn it is white. Since writing the above I have, through the kindness of Professor Poulton, had an opportunity of examining the type in the Oxford Museum. It is an immature shell, hence the undeveloped outer lip. Mr. Sowerby’s figure is far from accurate, the anterior part of the shell being represented much too broad, and the body-whorl above the columellar folds is too convex. The four or five rather indistinct and nodose transverse ridges on the body-whorl are just traceable in the type, and the suture of the spire is also wavy. The spire is a little flatter than in the present specimen, and the front fold on the columella is rather stronger than the second, thus showing that these are liable to variation. 121. Fusus suscontractus, Sowerby. ( y) The Euthria eburnea (the type of which is in the British Museum) was founded upon very much worn and bleached specimens of this species. So worn are the shells that the columellar folds are almost obliterated. Mr. Melvill’s figure does not well represent the species, which, moreover, is not always white, some specimens being stained with orange or rich brown below the suture and between the coste, with a colour-band round the middle of the body-whorl and another a little below it. The aperture and columella may be purplish or purplish brown, and the three columellar plice are distinct in some examples, whilst in others only one or two are present. EDINGS OF THE MALACOLOGICAL SOCIETY. 370 129. Vasum tRuNcatTUM (Sowerby). Pl. XV, Fig. 3. Turbinella truncata, Sowerby: Marine Shells 8. Afr., p. 17, pl. iv, fig. 85. Turbinella truncata, Sowerby: Marine Shells 8. Afr., p. 17, pl. iv, fig. 85. — jun. = Zurbinella triangularis, Smith: Journ. Conch., vol. x (1902), p. 249, pl. iv, fig. 6. g — jun. = Zurbinella triangularis, Smith: Journ. Conch., vol. x (1902), p. 249, pl. iv, fig. 6. Hab.—Off Durban, in deep water (Quekett). The type of this species figured in the above work, although in fairly good condition, does not show all the characters of this interesting form. A specimen recently submitted to me from Port Shepstone (Burnup), and which Mr. Sowerby has identified as belonging to his species, may be thus described. It differs from the type in having ten or eleven nodosities at the angle of the body-whorl, and these protuberances upon the upper whorls produce a wavy sutural line. The body-whorl also exhibits four or five rather indistinct and somewhat nodose, transverse, raised, broad, rounded ridges. The aperture is white within, the outer lip being much thickened, slightly expanded anteriorly, blotched with brown within the margin, with a strong tubercular prominence within at the hinder end. The columella is covered with a thick brown-stained callus, which is broadly reflexed and appressed over the umbilical rimation, and thickened into a white prominence posteriorly a little distance above the plice. The upper two of these are about equal in size, the next one is rather smaller, and the foremost one still less conspicuous. Melapium elatum, Schub. & Wag.: Smith, Journ. Conch., vol. x (1901), p. 110. Hab. —Ott Durban, 40 fathoms (Quekett). 130. VasumM TURBINELLUS (Linn.). Voluta turbinellus, Linn.: Hanley, Ipsa Linn. Conch., p. 233. Turbinella cornigera, Lamarck: Reeve, Conch. Icon., vol. iv, fig. 40. Hab.—Durban, Moluccas, Red Sea, Polynesia, ete. 131. Merarrum evatum (Schubert & Wagner). Melapium elatum, Schub. & Wag.: Smith, Journ. Conch., vol. x (1901), p. 110. Hab. —Ott Durban, 40 fathoms (Quekett). Melapium elatum, Schub. & Wag.: Smith, Journ. Conch., vol. x (1901), p. 110. f ( ) 371 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLUS 132. Trrronrpea inscuLpTa, Sowerby. Tritonidea insculpta, Sowerby: Proc. Malac. Soc., vol. iv, p. 2 (1900), pl. i, fig. 4. Hab.—The Kowie. 133. TrrronIDEA CARINIFERA (Kiister). 133. TrrronIDEA CARINIFERA (Kiister). EDINGS OF THE MALACOLOGICAL SOCIETY. 140. ComIneLLa BISERIALIS (Kiister). 140. ComIneLLa BISERIALIS (Kiister). 140. ComIneLLa BISERIALIS (Kiister). Buecinum biseriale, Kiister: Conch. Cab., p. 80, pl. xiv, fig. 12. Hab.—Cape Elim, 8. Africa (Kiister). 141. Enarna asrricta (Reeve). 141. Enarna asrricta (Reeve). Ricinula astricta, Reeve: Conch. Icon., vol. iii, pl. iv, fig. 30. Hab.—Umkomaas (Burnup). 142. Eneina pertata (Kiister). 142. Eneina pertata (Kiister). Buccinum perlatum, Kiister: Conch. Cab., 2nd ed., 1858, p. 61, pl. xii, figs. 5, 6 g Cantharus perlatus, Kiister: Tryon, Conch. Cab., vol. ii (1881), p. 158, pl. lxxiv, fig. 273. Engina Natalensis, Melvill: Proc. Malac. Soc., vol. i, p. 226 (1895), pl. xiv, fig. 12. Hab.—Natal (Kiister). 143. Prsanra cRENILABRUM, A. Adams. 133. TrrronIDEA CARINIFERA (Kiister). Buccinum cariniferum, Kiister: Conch. Cab., 1858, p. 63, pl. xu, figs. 9, 10. g Cantharus cariniferus, Kiister: Tryon, Man. Conch., vol. ii (1881), p- 155, pl. Ixxii, fig. 256. Tritonidea Natalensis, Smith: Journ. Conch., vol. x (1901), p. 111, ple i tie: 2:3. Hab.—Natal (Kiister, Burnup). 134, KEvrurra Carensis (Dunker). 134, KEvrurra Carensis (Dunker). Fusus Capensis, Dunker: in Philippi’s Abbild., vol. 1 (1844), p. 110, plig fey. p g y Cantharus Capensis, Dunker: Tryon, Man. Conch., vol. iti (1881), p. 156, pl. Ixxiu, fig. 260. Hab.—Cape of Good Hope (Dunker). E.. lacertina, Gould (1860), and #. Magellani, Vélain (1877), may prove synonymous with this species. p g y Cantharus Capensis, Dunker: Tryon, Man. Conch., vol. iti (1881), p. 156, pl. Ixxiu, fig. 260. Hab.—Cape of Good Hope (Dunker). E.. lacertina, Gould (1860), and #. Magellani, Vélain (1877), may prove synonymous with this species. 135. Eurarta Lacertina, Gould. 135. Eurarta Lacertina, Gould. Euthrya lacertina, Gould: Otia, p. 123; Martens, Jahrb. Deutsch. Malak. Gesell., vol. i (1874), p. 133, pl. vi, fig. 2. Hab.—Simons Bay, Cape of Good Hope (Gould), 136. Evruri Firmerx, Sowerby. 136. Evruri Firmerx, Sowerby. Euthria Filmere, Sowerby: Proc. Malac. Soe., vol. iv, p. 1 (1900), pli, fig.) 3. Hab.—Pondoland. 137. Evrae Qurxerti, Smith. 137. Evrae Qurxerti, Smith. Euthria Queketti, Smith: Journ. Conch., vol. x (1901), p. 110, pl. i, fee Hab.—Off Durban, 40 fathoms (Quekett). 138. Comrnetta Dunxert (Kiister). 138. Comrnetta Dunxert (Kiister). Cominella Dunkert, Kiister : Sowerby, Marine Shells 8. Afr., p. 10. Luthria lineolata, Dunker: Sowerby, l.c., p. 4. Mr. Sowerby appears to have overlooked the fact that only a single species was described under the above names, the latter merely having been altered to Dunkeri because it had already been employed. by Lamarck for a species of Yusus, in which genus Dunker also placed his species. It may be the young of C. papyracea, Brug. 139. ComINELLA (?) Protoneata, Smith. 139. ComINELLA (?) Protoneata, Smith. Cominella prolongata, Smith: Journ. Conch., yol. ix (1899), p. 248, 139. ComINELLA (?) Protoneata, Smith. Cominella prolongata, Smith: Journ. Conch., yol. ix (1899), p. 248, pl. v, fig. 3. Hab.—Cape Colony. Cominella prolongata, Smith: Journ. Conch., yol. ix (1899), p. 248, pl. v, fig. 3. Hab.—Cape Colony. 372 EDINGS OF THE MALACOLOGICAL SOCIETY. 143. Prsanra cRENILABRUM, A. Adams. Pisania crenilabrum, A. Adams: Proe. Zool. Soc., 1854, p. 188. Pisania Montrouziert, Crosse: Journ. de Conchyl., 1862, p. 251, pl. x, fig. 7; Sowerby, Proc. Malac. Soc., vol. iv, p. 2 (1900). Hab. — Pondoland coast (Sowerby); Umkomaas, Natal; New Caledonia (Crosse); Moreton Bay (Mus. Cuming). The locality (West Indies) given by Adams is probably a mistake. In adopting the name given to this species by Crosse, I presume Mr. Sowerby was misled by Tryon’s observation that it had priority (Man. Conch., vol. ii, p. 146). 144. Pos txvieatus, A. Adams. Phos levigatus, A. Adams: Proc. Zool. Soc., 1850, p. 155; Sowerby, Thes. Conch., vol. iii (1859), p. 90, pl. ccxxi, fig. 6; Tryon, Man. Conch., vol. ii (1881), p. 217, pl. 83, fig. 499. Hab.—Cape of Good Hope (Adams). 145. Nassarra @ractiis, Sowerby. 145. Nassarra @ractiis, Sowerby. Nassaria gracilis, Sowerby: Marine Invest. 8. Africa, 1902, p. 94, plat ne. 10; Hab.—Mouth of Tugela River, 40 fathoms (Sowerby). 146. Exsurna paprtiarts, Sowerby. p p , y Eburna papillaris, Sowerby: Reeve, Conch. Icon., vol. v, fig. 1; Sowerby, Marine Invest. 8. Africa, 1902, p. 93, pl. 11, fig. 3 Hab.—Oft Natal (Quekett) ; Algoa Bay, 24 fathoms (Sowerby). A single shell sent by Mr. Quekett differs from the type in having only a few scattered dots and some large oblique dark brown macu- lations below the suture. The umbilicus is open, the columellar callus being only partly developed, and the filled-up portion of the anterior canal is white, without the curved lines which are usually present. Mr. Sowerby gives some further remarks on the shell and perio- stracum, together with a figure of the animal. 373 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU 147. Nassa Evsutcata, Sowerby. 147. Nassa Evsutcata, Sowerby. Nassa eusuleata, Sowerby: Marine Invest. S. Africa, 1902, p. 94; pl. ii, fig. 8. Hab.—Mouth of the Tugela River, Natal, 46 fathoms (Sowerby). 148. Nassa Firmerx, Sowerby. Nassa Filmere, Sowerby : Proc. Malac. Soc., vol. iv, p. 2(1900), pl. i, fig. 5. Hab.—Pondoland. 149. Nassa cemmuLata, Lamarck. 149. Nassa cemmuLata, Lamarck. NVassa gemmulata, Lamarck: Reeve, Conch. Icon., vol. viu, fig. 29. Hab.—Durban ; Philippines (Reeve). 150. Nassa Frtosa, Gray. 150. Nassa Frtosa, Gray. Nassa filosa, Gray: Reeve, Conch. Icon., vol. viii, figs. 35a, b. Hab.—Durban (Burnup) ; Philippines (Reeve). 151. Nassa Natatensis, n.sp. Pl. XV, Fig. 6. Nassa Sturmii, Reeve (not of Philippi): Conch. Icon., vol. vin, figs. 148¢, b. The shell described and figured by Reeve as WV. Sturmii of Philippi is quite distinct from that well-known American species, which had previously been described by Say under the name of Nassa vibex. The present species is considerably larger, has a more turreted spire, is more coarsely nodose, differently coloured, has more lire within the labrum and a conspicuous eallosity above the posterior sinus. Reeve describes the columella as ‘but little callous.” This is true of the young specimen he had before him. 144. Pos txvieatus, A. Adams. Mature shells exhibit a well-developed reflexed yellowish callus, which may be sometimes smooth or more or less tubercular. A small transverse tubercle is always seen at the upper part. The outer lip is thickened with a strong varix, is acute at the edge, and is furnished within with about nine short lire. One of the two specimens from Natal presented to the Museum by Mr. J. H. Ponsonby is of a rich brown colour, with a white line above the middle of the body-whorl and passing up the spire above the suture. 152. Nassa cGauprosa, Hinds. Nassa gaudiosa, Hinds: Reeve, Conch. Icon., vol. viii, pl. viii, fig. 48. Hab.—Umkomaas, Natal (Burnup). 153. Nassa MARGARITIFERA (Dunker). 153. Nassa MARGARITIFERA (Dunker). Buccinum margaritiferum, Dunker: Philippi, Abbild., vol. ui, p. 47, pl. 1, fig. 12. p g Nassa costellifera, A. Adams: Reeve, Conch. Icon., vol. viii, figs. 58a, 5. Hab.— Durban. 154. Nassa Lentiernosa, A. Adams. , Nassa lentiginosa, A. Adams: Reeve, Conch. Icon., vol. viii, fig. 15. Hab.—Durban (Burnup); Philippines (Reeve). Nassa lentiginosa, A. Adams: Reeve, Conch. Icon., Hab.—Durban (Burnup); Philippines (Reeve). g , , , Hab.—Durban (Burnup); Philippines (Reeve). 374 DINGS OF THE MALACOLOGICAL SOCIETY. EDINGS OF THE MALACOLOGICAL SOCIETY. 155. Nassa mncrassata, Miller. Nassa incrassata, Miller: Sowerby, Proc. Malac. Soc., vol. iv, p. 3 1900). ay Beadotaal Cape Colony (Sowerby). 156. Nassa spurca, Gould, 156. Nassa spurca, Gould, Nassa spurca, Gould: Otia, p. 129. Hab.—Simons Bay (Gould). 157. Nassa quantoLa, Gould. 157. Nassa quantoLa, Gould. 157. Nassa quantoLa, Gould. 157. Nassa quantoLa, Gould. Nassa quantula, Gould: Otia, p. 128. Hab.—Simons Bay (Gould). Nassa quantula, Gould: Otia, p. 128. Hab.—Simons Bay (Gould). Nassa quantula, Gould: Otia, p. 128. Hab.—Simons Bay (Gould). 158. Burra (Buccrnanors) annutata, Lamarck. Bullia (Buceinanops) annulata, Lamarck: Sowerby, Marine Invest. S. Africa, 1902, p. 95, pl. 1, fig. 4. TTab.—Algoa Bay, 24 fathoms (Sowerby). A figure of the animal is given at the above reference. 159. Burra tevissmma (Gmelin). Bullia levigata, Martini: Sowerby, Marine Shells 8. Afr., p. 11. The name /@vissima should be substituted for that of levigata, since the latter was not given by a binomial writer. 159. Burra tevissmma (Gmelin). 159. Burra tevissmma (Gmelin). Bullia levigata, Martini: Sowerby, Marine Shells 8. Afr., p. 11. g y p The name /@vissima should be substituted for that of levigata, since the latter was not given by a binomial writer. 160. Burrta Mavritiana, Gray. Bullia Mauritiana, Gray : Reeve, Conch. Icon., vol. iii, pl. u, figs. 12a, 6; Martens, Jahrb. Deutsch. Malak. Gesell., 1874, p. lav, Hab.—Algoa Bay (Martens). 161. CoLumBELLA VARIANS, Sowerby. 161. CoLumBELLA VARIANS, Sowerby. Columbella varians, Sowerby : Thes. Conch., vol. i, p. 117, pl. xxxvu, figs. 47-50. Hab.—Durban (Burnup). 162. Cotumpetta Kravssri, Sowerby (1844). p , y ( ) Columbella cerealis, Menke (1848): Sowerby, Marine Shells 8S. Afr., pez. The name Araussii should be used for this species, having four years priority. 163. Cotumpetta BrcKxert, Sowerby. Columbella Beckeri, Sowerby: Proc. Malac. Soc., vol. iv, p. 3 (1900), ple. 7. Hab.—The Kowie (Sowerby). 164. Cotumpetta Firmer2, Sowerby. 164. Cotumpetta Firmer2, Sowerby. Columbella Filmere, Sowerby : Proc. Malac. Soc., vol. iv, p. 3 (1900), pl. 1, fig. 8. Hab.—Pondoland. 165. CotumBELLa arrata, Gould. ( ) ( ) 165. CotumBELLa arrata, Gould. Columbella (Anachis) atrata, Gould (1860): Tryon, Man. Conch., vol. v (1883), p. 169, pl. lvii, fig. 12. 375 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU Columbella pumila, Souverbie: Journ. de Conchyl., vol. xi (1863), p. 281, pl. xu, fig..4. p. 281, pl. xu, fig..4. Columbella regulus, Souverbie: op. cit., vol. xii (1864), p. 41. p p g Columbella regulus, Souverbie: op. cit., vol. xii (1864), p. 41. p p g Columbella regulus, Souverbie: op. cit., vol. xii (1864), p. 41. Hab.— Durban (Burnup); Hong Kong (Gould). Hab.— Durban (Burnup); Hong Kong (Gould). A widely distributed species occurring both in the Indian and Pacific Oceans. ( p) g g ( ) A widely distributed species occurring both in the Indian and Pacific Oceans. 166. CoLuMBELLA LEPTALEA, Smith. 166. CoLuMBELLA LEPTALEA, Smith. Columbella ( Anachis) leptalea, Smith: Journ. Conch., vol. x (1902), p. 250, pl. iv, fig. 4. Hab.—Umkomaas (Burnup). Columbella ( Anachis) leptalea, Smith: Journ. Conch., vol. x (1902), p. 250, pl. iv, fig. 4. 167. CoLtumpetta Burnurr, Smith. a Burnupt, Smith: Journ. Conch., vol. x (1901), NZ plea, fie. 2; ab. —Natal (Burnup). 168. CoLumMBELLA MiTRirorMIs (A. Adams). Zafra mitriformis, A, Adams: Ann. & Mag. Nat. Hist., 1860, vol. vi, 382, p- p Daphnella mitreformis, A. Ad.: Tryon, Man. Conch., vol. vi, p. 313. Hab.—Durban ; Japan (Adams). 169. CotumBetta Licutroorr, Smith. Columbella (Astyris) Lightfooti, Smith: Journ. Conch., vol. x (1901), pe Wp). a; fig. 3. Hab.—Kalk Bay, Cape Colony (Lightfoot). 170. Corumpetta apicata, Smith. 170. Corumpetta apicata, Smith. Columbella (Nitidella) ee Smith: Journ. Conch., vol. ix (1899), p. 247, pl. v, fig. 2 Hab.—Durban (Gap 171. Mera Dupontim, Kiener (emend.). Conus Dupontit, Kiener: Coq. Viv., p. 273, pl. xi, fig. 2 Conus Dupontit, Kiener: Coq. Viv., p. 273, pl. xi, fig. 2 Meta Dupontia, Kiener: Reeve, Conch. Icon., vol. xi, sp. 6, figs. 3¢, d. Hab.—Ichabo, South Africa (Reeve). Meta Dupontia, Kiener: Reeve, Conch. Icon., vol. xi, sp. 6, figs. 3¢, d. Hab.—Ichabo, South Africa (Reeve). 172. TropnHon Kowreensis, Sowerby. 172. TropnHon Kowreensis, Sowerby. Trophon Kowieensis, Sowerby: Proc. Malac. Soc., vol. iv, p. 218 (1901), pl. xxii, fig. 16. Hab.—The Kowie. 173. TropHon(?) instants, Sowerby. Trophon(?) insignis, Sowerby: Proc. Malac. Soc., vol. iv, p. 2 (1900), pl. tefien 1: Hab.—The Kowie. 174. Murex Fattax, Smith. 174. Murex Fattax, Smith. Murex fallax, Smith: Journ. Conch., vol. x (1901), p. 113, pl. i, fig. 9 Hab.—Off Durban, 40 fathoms (Quekett). VOL. V.—ocToBER, 1903. 26 26 376 EDINGS OF THE MALACOLOGICAL SOCIETY EDINGS OF THE MALACOLOGICAL SOCIETY 175. Murex (Cutcorrus) Banxstr, Sowerby. 175. Murex (Cutcorrus) Banxstr, Sowerby. Murex Banksti, Sowerby : Conch. Ilust., fig. 82; Reeve, Conch. Icon., vol. ii, pl. x, fig. 38; Kobelt, Conch. Cab., 2nd ed., p. 120, pl. xxxvi, fig. 6; Kiener, Cog. Viv., pl. xxi, fig. 1. ITab.—Durban (Burnup) ; Moluceas (Reeve, ete.) The specimen trom Durban is larger than any of the figured Murex Banksti, Sowerby : Conch. Ilust., fig. 82; Reeve, Conch. Icon., vol. ii, pl. x, fig. 38; Kobelt, Conch. Cab., 2nd ed., p. 120, pl. xxxvi, fig. 6; Kiener, Cog. Viv., pl. xxi, fig. 1. p , g ; , g , p , g ITab.—Durban (Burnup) ; Moluceas (Reeve, ete.) The specimen trom Durban is larger than any of the figured examples, having a length of 85 mm. Tab.—Durban (Burnup) ; Moluceas (Reeve, ete.) The specimen trom Durban is larger than any of the figured examples, having a length of 85 mm. The specimen trom Durban is larger than any of the figured examples, having a length of 85 mm. 176. Unrosatprnx (?) conrracta (Reeve). Buccinum contractum, Reeve: Conch. Icon., 176. Unrosatprnx (?) conrracta (Reeve). Buccinum contractum, Reeve: Conch. Icon., vol. ii, fig. 53 Hab.—Durban (Burnup) ; Philippines, ete. 176. Unrosatprnx (?) conrracta (Reeve). Buccinum contractum, Reeve: Conch. Icon., vol. ii, fig. 53 p ( ) ( ) Buccinum contractum, Reeve: Conch. Icon., vol. ii, fig. 53 Hab.—Durban (Burnup) ; Philippines, ete. 177. Latraxts Rosacevs, n.sp. Pl. XV, Fig. 16. Latiaxis nodosa, Sowerby: Marine Shells 8. Africa, p. 16 (nee A. Adams). Testa fusiformis, rimata, rosacea, spiraliter confertim squamose lirata et longitudinaliter plicata; anfractus 6-7, in medio angulati, supra declives, infra contracti, ultimus antice valde angustatus, in caudam brevem recurvam productus; apertura angulatim piriformis, cum canali longit. totius } adequans, intus rosacea; columella supra rectiuscula, antice obliqua, callo reflexo appresso roseo induta; labrum tenue, extus crematum. Long. 21, diam. 13 mm. Hab.—Port Elizabeth, Cape Colony. This species is uniformly rose coloured within and without, whereas ZL. nodosus, A. Adams, is dirty whitish. In the latter species the angulation of the whorls is rather more acute and rather lower down, or in other words less central. The body-whorl also is more suddenly contracted below the periphery than in the present species, and the umbilical fissure is rather broader. The longitudinal coste or plice are more conspicuous in the upper whorls than in the last. EDINGS OF THE MALACOLOGICAL SOCIETY The contrast of the colour of the aperture with the exterior is very striking. In addition to the punctate strie which cover the entire shell, the surface exhibits irregular and somewhat broken up, but distinct lines of growth. One specimen exhibits five interrupted brown lines, one below the suture and one upon each of the four spirals of the body-whorl. 181. Srstrum aspERuM (Lamarck). Ricinula asperum, Lamarck: Reeve, Conch. Icon., vol. iu, fig. 13. Hab.—Isipingo, Natal. 182. Srsrrum canceLLatum (Quoy & Gaimard). Ricinula cancellata, Quoy & Gaimard: Tryon, Man. Conch., vol. u, p. 188, pl lviii, tigs. 242, 250. Hab.—Umkomaas. Hab.—Umkomaas. 183. Sisrrum mMARGINATRUM (Blainyille). Sistrum marginatrum, Blainville: Tryon, Man. Conch., vol. 11, p. 186, pl. lvii, fig. 225. p g Sistrum affine, Pease: Sowerby, Marine Shells 8. Afr., Appendix, p. 6. Hab.—Durban (Burnup). 184. Srstrum morus (Lamarck). 184. Srstrum morus (Lamarck). Ricinula morus, Lamarck: Reeve, Conch. Icon., vol. 11. fig. 10. Hab.—Isipingo (Burnup). 185. Sistrum Ricinus (Linn.). 185. Sistrum Ricinus (Linn.). Ricinula arachnoides, Lamarck : Reeve, Conch. Icon., vol. iu, pl.i, fig. 5. Sistrum arachnoides Lamk : Sowerby Marine Shells 8 Afr p 15 Ricinula arachnoides, Lamarck : Reeve, Conch. Icon., vol. iu, pl.i, fig. 5. Sistrum arachnoides, Lamk.: Sowerby, Marine Shells 8. Afr., p. 15. Hab.—Natal. The older name ricinus should be used for this species. 186. Sistrum squamMosum, Pease, var. Sistrum squamosum, Pease, var.: Smith, Journ. Conch., vol. x (1901), 7 lls: ey rae and Durban (Burnup). 186. Sistrum squamMosum, Pease, var. Sistrum squamosum, Pease, var.: Smith, Journ. Conch., vol. x (1901), 7 lls: ey rae and Durban (Burnup). 187. Sisrrum squamitrRatuM, n.sp. Pl. XV, Fig. 17. Testa ovato-fusiformis, turrita, albida; anfractus 9, superiores tres leeves, convexi, ceteri in medio angulati, costis vel plicis circiter 8 ad angulum acute productis instructi, liris spiralibus squamatis 6-7 ornati, ultimus infra angulum liris circiter 14 (quarum 3—4 quam altis majores) instructus; apertura rosea, angusta, longit. totius } adequans; labrum intus denticulis 6-7 munitum; columella ecallo tenui roseo reflexo induta, prope medium subtuberculata. Long, 19, diam. 10 mm.; apertura cum canali 10 mm. longa, 3°5 lata. H b I i i (B ) Hab.—Isipingo (Burnup). 188. CorattiopHina RuBRococcINEA, Melvill & Standen. 88. Co att op a u ococc , e & Sta de . Coralliophila rubrococcinea, Melv. & Stand.: Proc. Zool. Soc , 1901, vol. um, p: 401, pl. xxi, fig. 2; Pseudomurex Meyendorfi, Sowerby : Marine Shells 8. Afr., p. EDINGS OF THE MALACOLOGICAL SOCIETY The species, judging from the shell alone, might with equal propriety be placed in the genus Coralliophila. Hab.—Port Elizabeth, Cape Colony. This species is uniformly rose coloured within and without, whereas ZL. nodosus, A. Adams, is dirty whitish. In the latter species the angulation of the whorls is rather more acute and rather lower down, or in other words less central. The body-whorl also is more suddenly contracted below the periphery than in the present species, and the umbilical fissure is rather broader. The longitudinal coste or plice are more conspicuous in the upper whorls than in the last. The species, judging from the shell alone, might with equal propriety be placed in the genus Coralliophila. 178. Ravana sButBosa (Solander). Pyrula bulbosa, Solander : Reeve, Conch. Icon., vol. iv, pl. iv, fig. 14. Hab.—Durban. 178. Ravana sButBosa (Solander). Pyrula bulbosa, Solander : Reeve, Conch. Icon., vol. iv, pl. iv, fig. 14. Hab.—Durban. 179. Purpura squamosa, Lamarck. The young of this species was described by Blainville (Nouv. Ann. Mus. Hist. Nat. Paris, vol. i, p. 251, pl. xii, fig. 6) under the name of Purpura elathrata. 180. Purpura pura, n.sp. Pl. XV, Fig. 21. Testa parva, alba, in apertura rufescens, fusiformi-ovata, spiraliter punctato-striata, et, costis spiralibus ad angulum anfractuum nodosis instructa ; anfractus circiter 6, supra concave declives, in medio nodose angulati, ultimus costis quatuor (quarum duo superiores nodosi sunt) cingulatus; apertura lris novem albis gracilibus, haud ad marginem labri productis, armata; labrum album, tenue; columella parum arcuata, callo albo levi antice ce induta, superne tuberculo parvo inconspicuo instructa. Long. 19, diam. 11 mm.; apertura 11mm. longa, 5:5 lata. old SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU Hab.—Umkomaas. Hab.—Umkomaas. The contrast of the colour of the aperture with the exterior is very striking. In addition to the punctate strie which cover the entire shell, the surface exhibits irregular and somewhat broken up, but distinct lines of growth. One specimen exhibits five interrupted brown lines, one below the suture and one upon each of the four spirals of the body-whorl. The contrast of the colour of the aperture with the exterior is very striking. In addition to the punctate strie which cover the entire shell, the surface exhibits irregular and somewhat broken up, but distinct lines of growth. One specimen exhibits five interrupted brown lines, one below the suture and one upon each of the four spirals of the body-whorl. EDINGS OF THE MALACOLOGICAL SOCIETY 16 (nec Caleara). Pseudomurex Meyendorfi, Sowerby : Marine Shells 8. Afr., p. 16 (nec Caleara). 78 PROCEEDINGS OF THE 378 PROC Hab.—Cap The shel 378 EDINGS OF THE MALACOLOGICAL SOCIETY. Hab.—Cape Colony: Port Elizabeth, East London, Pondoland. The shells which I associate with this species, with some hesitation, certainly do not belong to Pseudomurex Meyendorfi. The pinkish red colour alone is sufficient to distinguish them. Care, however, must be taken not to confound them with Zatiaxis rosaceus, which is somewhat similarly tinted but different in form. 189. Lororium Durpanense, Smith. Lotorium Durbanense, Smith: Journ. Conch., vol. ix (1899), p. 248, pl. v, fig. 4. Hab.—Durban (Burnup). 189. Lororium Durpanense, Smith. Lotorium Durbanense, Smith: Journ. Conch., vol. ix (1899), p. 248, pl. v, fig. 4. Hab.—Durban (Burnup). p Lotorium Durbanense, Smith: Journ. Conch., vol. ix (1899), p. 248, pl. v, fig. 4. ( ) p g Hab.—Durban (Burnup). 190. Lororrum ENcAusticum (Reeve). 190. Lororrum ENcAusticum (Reeve). Triton encausticus, Reeve: Conch. Icon., vol. i, fig. 43. Hab.—Durban ; Philippines (Reeve). Triton encausticus, Reeve: Conch. Icon., vol. i, fig. 43. Hab —Durban ; Philippines (Reeve) Hab.—Durban ; Philippines (Reeve). 191. Lorortum NAssARIFoRME, Sowerby. 191. Lorortum NAssARIFoRME, Sowerby. Lotorium nassariforme, Sowerby: Marine Invest. 8. Africa, 1902, p. Yo, plu, fig. 7 Hub.—Natal, 92 fathoms (Sowerby). 192. Lorortum RaNELLorpEs (Reeve). 192. Lorortum RaNELLorpEs (Reeve). Triton ranelloides, Reeve: Conch. Icon., vol. ii, figs. 10a, 6; Sowerby, Marine Invest. 8. Africa, 1902, p. 95, as Lotorivum. Hab.—Oft Natal, 92 fathoms (Sowerby). 193. Lorortom opscurum (A. Adams). Triton obscurus, A. Adams: Proc. Zool. Soc., 1854, p. 312. Hab.—Durban (Burnup); 8. Africa (A. Adams). 194. Lorortum rrironis (Linn.). Triton variegatus, Lamarck: Reeve, Conch. Icon., vol. ii, pls. 1, 1, figs. 3a, b. Hab.—Durban (Burnup). 195. Lorortom (Prrene) Mvurrayr (Smith). 195. Lorortom (Prrene) Mvurrayr (Smith). Lampusia (Priene) Murrayi, Smith: Proce. Zool. Soc., 1891, p. 486, pl. xxxiv, fig. 1. Hab.—Offt Cape of Good Hope, 150 fathoms (Challenger Expedition). 196. Lorortum (Disrorrrix) caNncetLtinum (Roissy), var. DECIPIENS, Reeve, Triton decipiens, Reeve: Conch. Icon., vol. ii, fig. 102. Hab.—Durban (Burnup) ; Philippines (Reeve). 197. Cotuprarta BractEaTa (Hinds). 197. Cotuprarta BractEaTa (Hinds). 197. Cotuprarta BractEaTa (Hinds). Epidromus bracteatus, Hinds: Sowerby, Proc. Malac. Soc., vol. iv, p. 3 (1900). Hab.—Pondoland (Sowerby). 198. Ranetta anceps, Lamarck. Ranella anceps, Lamarck: Reeve, Conch. Icon., vol. u, fig. 43 ; Kiener, Coq. Viv., pl. iv, fig. 2. Ranella lamellosa, Dunker: Sowerby, Marine Shells 8. Afr., p. 9. Hab.—Umkomaas; Port Elizabeth. 379 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU 199. Cassis nopuLosa (Gmelin), var. rorquata, Reeve. Cassis nodulosa (Gmelin) : Schroeter, Einleitung, vol.i, pl. 210. Srrompus LENTIGINosus, Linn. Strombus lentiginosus, Linn.: Reeve, Conch. Icon., vol. vi, fig. 31; Kiener, Coq. Viv., pl. xviii, fig. 1; Watson, Gasteropoda Challenger Exped., p. 415. Tub.—Simons Bay, Cape of Good Hope ( Watson). 378 PROC Hab.—Cap The shel ui, figs. 9a, b. Cassis torquata, Reeve: Conch. Icon., vol. v, figs. 1-Le. Hab.—Durban; New Holland (Reeve). Reeve’s figure le represents the typical form of C. nodulosa. 200. Dotrtum costarum, Menke. Dolium costatum, Menke: Synopsis, 2nd ed., 1830, p. 63. Dolium costatum, Menke: Deshayes, Anim. sans Vert., 2nd ed., vol. x (1844), p. 144; Reeve, Conch. Icon., vol. v, fig. 8. ( ) Dolium costatum, Menke: Deshayes, Anim. sans Vert., 2nd ed., vol. x (1844), p. 144; Reeve, Conch. Icon., vol. v, fig. 8. Hab.—Durban (Burnup); Mauritius, Maldives, Andamans, Philip- pines Hab.—Durban (Burnup); Mauritius, Maldives, Andamans, Philip- pines. 201. Dottum Donxert, Hanley. 201. Dottum Donxert, Hanley. Dolium Dunkeri, Hanley: Proc. Zool. Soc., 1859, p. 431. Hab.—Port Natal (Hanley). 202. Dotrum perpix (Linn.). 202. Dotrum perpix (Linn.). Dolium perdiz, Linn.: Sowerby, Proc. Malac. Soc., vol. iv, p. 5 (1900). Hab.—Pondoland (Sowerby). 203. Dotium vartEcAtoM, Lamarck. Dolium variegatum, Lamarck: Reeve, Conch. Icon., vol. v, pl. vy, figs. 7a, 6; Martens, Jahrb. Deutsch. Malak. Gesell., 1874, p. 134. Hab.—Algoa Bay (Martens). 204. AmpurpEeras Becxert, Sowerby. Amphiperas Beckeri, Sowerby: Proc. Malac. Soc., vol. iv, p. 5 (1900), plea fig. 13: Hab.—Pondoland (Sowerby). 205. Raprus eracitiimus, Smith. Radius gracillimus, Smith: Journ. Conch., vol. x (1901), p. 107, pl. i, figs. 20, 21. Hab.—Off Durban, 40 fathoms (Quekett). 206. CyprA MINORIDENS, Melvill. 206. CyprA MINORIDENS, Melvill. Cyprea minoridens, Melvill: Journ. Conch., vol. x (1901), p. 119. Cyprea fimbriata, partim, Sowerby: Thes. Conch., vol. iv, pl. 323, figs. 390, 391. Cyprea fimbriata, partim, Sowerby: Thes. Conch., vol. iv, pl. 323, figs. 390, 391. Hab. — Durban; Seychelles, Fiji Islands, and Sayage Island (Mus. Brit.). Less pyriform than fimbriata, and without the dark dots at the sides of the base. 207. Cyprma stormpa, Linn. 207. Cyprma stormpa, Linn. Cyprea stolida, Linn.: Reeve, Conch. Icon., vol. iii, figs. 67a, 6. Hab.—Durban (Burnup). 208. Erato eurrota, Sowerby. 208. Erato eurrota, Sowerby. Erato guttula, Sowerby : Conch. IIL. p. 18, fig. 50. Erato guttula, Sowerby : Conch. IIL. p. 18, fig. 50. Erato guttata, Sowerby: Thes. Conch., vol. ili, p. 82, pl. cexix, figs. 29, 30. Hab.—Durban (Burnup); Mauritius (Sowerby). 380 DINGS OF THE MALACOLOGICAL SOCIETY. 209. Srrompus Lamarcxu, Gray. Strombus Lamarckii, Gray : Sowerby, Thes. Conch., vol. i, p. 35, pl. ix, figs. 98, 99. Hab.—Durban (Burnup). 210. Srrompus LENTIGINosus, Linn. 211. Crertratum caHruLeumM, Sowerby. 211. Crertratum caHruLeumM, Sowerby. Cerithium ceruleum, Sowerby: Thes. Conch., vol. ii, p. 866, pl. clxxix, figs. 61, 62. Hab.—Durban (Burnup). 212. Crrrraium cireinum, Sowerby. 212. Crrrraium cireinum, Sowerby. Cerithium citrinum, Sowerby: Thes. Conch., vol. ii, p. 855, pl. clxxix, fig. 66; Conch. Icon., vol. xv, fig. 1. Hab.—Durban (Burnup) ; Philippines (Sowerby). 213. Cxrriraium piatrucum, Philippi. 213. Cxrriraium piatrucum, Philippi. Cerithium dialeuecun, Philippi: Sowerby, Conch. Icon., vol. xv, figs. 78a, b. Hab.—Durban (Burnup); Philippines (Reeve). 214. CrrirHium RuGosum, Wood. Cerithium rugosum, Wood: Sowerby, Conch. Icon., vol. xv, figs. 45a, b. Hab.—Durban (Burnup). 215. CeRITaiIuM ALBovaricosum, Smith. 215. CeRITaiIuM ALBovaricosum, Smith. Cerithium albovaricosum, Smith: Zool. Alert, p. 501, pl. xliv, fig. Q. Hab.—Durban (Burnup). 216. CrRIrHiuM rUFoNoDULosuM, Smith. 216. CrRIrHiuM rUFoNoDULosuM, Smith. Cerithium rufonodulosum, Smith: Journ. Conch., vol. x (1901), p. 108, pl. i, fig. 8: Hab.—Algoa Bay (Burnup). 217. CrrirHium zEBRUM, Kiener. Cerithium zebrum, Kiener: Kobelt, Conch. Cab., 2nd ed., p. 220, pl. xxxix, figs. 7-10. Cerithium zebrum, Kiener: Kobelt, Conch. Cab., 2nd ed., p. 220, pl. xxxix, figs. 7-10. Cerithium dvlectum, Sowerby : in Reeve’s Conch. Icon., vol. xv, pl. xvi, figs. 112a-c. p g Cerithium dvlectum, Sowerby : in Reeve’s Conch. Icon., vol. xv, pl. xvi, figs. 112a-c. Hab.—Mauritius (Kiener and Brit. Mus.); Gallapagos Is. (Sowerby); Samoa Is. (Tryon); Savage I., Oceania (Brit. Mus.) ; Durban (Burnup). This species is very variable in colour. Sometimes it is entirely white or purplish pink. The typical form is white with a dark brown band below the suture and a second band round the middle of the body-whorl. Other specimens are white with an interrupted brown line a little below the suture and a second on the lower part of the Hab.—Mauritius (Kiener and Brit. Mus.); Gallapagos Is. (Sowerby); Samoa Is. (Tryon); Savage I., Oceania (Brit. Mus.) ; Durban (Burnup). 381 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU body-whorl (Conch. Icon., fig. 1126). The latter is sometimes absent, as in one of the two Natal specimens now examined. The second example is totally white. 218, CrRrirHrum EGENUM, Gould. Cerithium egenum, Gould: Otia Conch., p. 62; Wilkes, U.S. Explor. Exped., p. 151, atlas, figs. 171-171); Tryon, Man. Conch., voltaix, ps lo7,, pl. xxy, fie: 70: Hab.—Durban (Burnup) ; Wilson’s I. (Gould); Solomon Is. (Smith). , p ,, p y, Hab.—Durban (Burnup) ; Wilson’s I. (Gould); Solomon Is. (Smith). 219. CrrirHivum LActeum, Kiener. Cerithium lacteum, Kiener: Sowerby in Reeve’s Conch. Icon., vol. EDINGS OF THE MALACOLOGICAL SOCIETY. 226. SprroeiypHus sprrorsis, Sowerby. 226. SprroeiypHus sprrorsis, Sowerby. Spiroglyphus spirorbis, Sowerby : Conch. Manual, 18389, p. 100, fig. 8; Tryon, Man. Conch., vol. viii (1886), p. 178, pl. li, fig. 58. Hab.—Cape of Good Hope? (Tryon). 227. Lrppisres cornu (Gmelin). Argonauta cornu, Gmelin: Syst. Nat., vol. vi, p. 8368. Cornu carinatum, Schumacher: Nouv. Syst., p. 255. Separatista Gray’, A. Adams: Proc. Zool. Soc., 1850, p.45; Sowerby, Marine Shells 8. Afr., p. 16. Hab —Cape of Good Hope (Gmelin and Adams) ; Port Elizabeth Argonauta cornu, Gmelin: Syst. Nat., vol. vi, p. 8368. Cornu carinatum, Schumacher: Nouv. Syst., p. 255. Separatista Gray’, A. Adams: Proc. Zool. Soc., 1850, p.45; Sowerby, Marine Shells 8. Afr., p. 16. Argonauta cornu, Gmelin: Syst. Nat., vol. vi, p. 8368. Cornu carinatum, Schumacher: Nouv. Syst., p. 255. Separatista Gray’, A. Adams: Proc. Zool. Soc., 1850, p.45; Sowerby, Marine Shells 8. Afr., p. 16. Hab.—Cape of Good Hope (Gmelin and Adams) ; Port Elizabeth (Sowerby). 228. Turrirerta Kowrensts, Sowerby. 228. Turrirerta Kowrensts, Sowerby. Turritella Kowiensis, Sowerby: Proc. Malac. Soc., vol. iv, p. 6 (1900), pl. i, fig. 12: Hab.—The Kowie (Sowerby). 211. Crertratum caHruLeumM, Sowerby. xv, pl. xii, figs. 85a, b. Hab.— Durban (Burnup) ; Philippines (Sowerby). 220. Brirrium quaDRIciNcruM, n.sp. Pl. XV, Fig. 12. Testa minima, elongata, pupoidea, ad apicem acuminata, fusca, tuberculis flavidis ornata; anfractus circiter 11, leviter convexiusculi, oblique plicati, et liris spiralibus (in anfractu penultimo 4, in ultimo 7-8) supra plicas granosis instructi, ultimus ad latus sinistrum varice pallido vix elato compositus ; apertura oblique ovalis, saturate fusca; columella arcuata, callo tenui fusco induta; canalis brevissimus, obliquus. Long. 5, diam. 1°5 mm.; apertura 1 mm. longa. Hab.—Durban (Burnup). The three or four lire around the lower half of the body-whorl are simple, and not granular like those above. Hab.—Durban (Burnup). The three or four lire around the lower half of the body-whorl are simple, and not granular like those above. 221, CrRITHIDEA INzxQuIscuLPTa (Kobelt). Cerithium ( Cerithidea) inequisculptum, Kobelt: Conch. Cab., 2nd ed., ps 158; pl. sox, fies. 10, 11. Hab.—Durban (Kobelt). 222. CertraipEA Naratensis (Kobelt). Cerithium ( Cerithidea) Natalense, Kobelt: Conch. Cab., 1893, p. 157, ply xxix, figs.8, 9. Hab.—Durban (Kobelt). 223. Crriruiopsis necLEcTA (C. B. Adams). Cerithium neglectum, C. B. Adams: Panama Shells, p. 154. Cerithiopsis neglecta (C. B. Adams): Smith, Proc. Zool. Soe., 1890, p. 293. Hab.—Durban (Burnup) ; St. Helena (Smith); Panama (Adams). 224. Puanaxis LinEatus (Da Costa). 224. Puanaxis LinEatus (Da Costa). Planaxis lineatus, Da Costa: Sowerby, Conch. Icon., vol. xx, pl. iii, fig. 20. Hab.—Durban (Burnup); West Indies. No notice should be taken of the grotesque lumping of species given in Tryon’s Man. Conch., vol. ix, p. 278. 225. Tuytacopes Nararensis, Morch. 225. Tuytacopes Nararensis, Morch. Thylacodes Natalensis, Morch: Proc. Zool. Soc., 1862, p. 70. Hab.—Natal (Morch). Thylacodes Natalensis, Morch: Proc. Zool. Soc., 1862, p. 70. Hab.—Natal (Morch). 382 EDINGS OF THE MALACOLOGICAL SOCIETY. 229. TurrItELLA PuNcTIcuLAtA, Sowerby. 229. TurrItELLA PuNcTIcuLAtA, Sowerby. Turritella puncticulata, Sowerby: Marine Invest. 8. Afr., 1902, p. 100. Lurritella punctulata, Sowerby: Marine Shells 8. Afr., p. 39, pl. v, fig. 102. Hab.—St. Francis Bay, 30-34 fathoms (Sowerby). 230. Lirrormna pintapo, Wood. 230. Lirrormna pintapo, Wood. Littorina pintado, Wood: Reeve, Conch. Icon., vol. x, figs. 54a, d. p g Littorina szigzac, Sowerby: Marine Shells 8. Afr., p. 386 (non Chemnitz). The shells quoted by Mr. Sowerby as Z. zigzae (a West Indian species) prove to belong to the present form. 231. Lrrrorrva Lavis, Philippi. 231. Lrrrorrva Lavis, Philippi. Littorina levis, Philippi: Abbild., vol. i, p. 10, pl. vi, fig. 6; Reeve, Conch. Icon., vol. x, fig. 95. Hab.—Umkomaas and Isipingo, Natal (Burnup). 232. Fossarus pusittus, Gould. Fossarus pusillus, Gould: Tryon, Man. Conch., vol. ix, p. 272, pl. ln, fig. 8. Hab —Umkomaas (Burnup); Liberia (Gould). Fossarus pusillus, Gould: Tryon, Man. Conch., vol. ix, p. 272, pl. ln, fig. 8. Hab —Umkomaas (Burnup); Liberia (Gould). 238. Fossarus Caprnsis, Pilsbry. 238. Fossarus Caprnsis, Pilsbry. Fossarus Capensis, Pilsbry: Proc. Acad. Nat. Sei. Philad., vol. liii (1901), p. 190. Hab —In ballast from South Africa, 234. Sorarium ca#i~atum, Hinds, 234. Sorarium ca#i~atum, Hinds, Solarium calatum, Hinds: Sowerby, Proc. Malac. Soc., vol. iv, p 5 (1900). JTab.—Pondoland (Sowerby). 383 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU 235. Sotarrum trocuorpEs, Deshayes. 235. Sotarrum trocuorpEs, Deshayes. Solarium trochoides, Deshayes: Hanley in Sowerby’s Thes. Conch., vol. ili, p. 243, pl. 254, figs. 89, 90. Hab.—Durban (Burnup) ; Philippine Is. (Hanley). 236. Rissora Crawrorpi, Smith. 236. Rissora Crawrorpi, Smith. Rissoia Orawfordi, Smith: Journ. Conch., vol. x (1901), p. 107, pl. 1, fig. 13. Hab.—Algoa Bay, 20 fathoms (Crawford). 237. Rrssomna (Irtvapra) annoutata (Dunker). 237. Rrssomna (Irtvapra) annoutata (Dunker). Rissoina annulata, Dunker: Moll. Japon., 1860, p. 12, pl. i, fig. 12. 237. Rrssomna (Irtvapra) annoutata (Dunker). Rissoina annulata, Dunker: Moll. Japon., 1860, p. 12, pl. i, fig. 12. Rissoina trochlearis, Gould: Otia, p. 144; Sowerby, Conch. Icon., vol. xx, pl xi, fig. 105. Hab.—Durban (Burnup); Japan (Dunker); China seas (Gouid) ; Bombay (Mus. Cuming); Hong Kong, Ceylon, Aden, Bombay, Andamans, Singapore, Arracan (Nevill Jk 238. Rissorna crassa, Angas. Rissoina crassa, Angas: Proc. Zool. Soc., 1871, p. 17, pl. i, fig. 16. Hab.—Durban (Burnup). Some of the South African specimens are more distinctly spirally striated than others and also than the types from Port Jackson. 229. TurrItELLA PuNcTIcuLAtA, Sowerby. The groove or excavation around the base of the body-whorl is also deeper and more evident than in the Australian shell. The size is rather variable, ranging from 8 to 5} mm. in length. 239. Rissorya ampreva (Gould), Pyramidella ambigua, Gould: Proc. Bost. Soc. Nat. Hist., 1849, vol. ii, p. 118; Otia, p. 60. p p Rissoa ambigua, Gould: U.S. Explor. Exped., p. 217, atlas, figs. 261-26le. Rissoina ambigua, Gould: Weinkauff, Conch. Cab., p. 25 (pl. ix, fig. 12). Rissoina ambigua, Gould: Weinkauff, Conch. Cab., p. 25 (pl. ix, fig. 12). Hab. — Durban (Burnup); Paumotu Is. (Gould); China seas (Mus. Cuming). I do not consider the shell figured by Schwartz von Mohrenstern (Rissoiden, pl. iui, fig. 27) under the name of Rissocna ambigua to belong to Gould’s species, as it differs both in form and sculpture. The spiral strize are most conspicuous around the base of the body- whorl, as shown in Gould’s figure. 2. fortis, C. B. Adams (Schwartz, l.c., fig. 25), seems to be the same as the present species. 240. AsstmrneEA Umtiaastana, Smith. Assiminea Umlaasiana, Smith: Journ. Conch., vol. x (1902), p. 248, pl. iv, fig. 3. Hab.—Mouth of Umlaas River, Natal (Burnup). 241. Feneiia cerrrurna (Philippi). Rissoa cerithina, Philippi: Zeitsch. Malak., 1849, p. 33; Nevill, Hand List Moll., ui, p. 114 (as Fenella). Hab.—Natal (N evil) ; Aden (Philippi). DINGS OF THE MALACOLOGICAL SOCIETY. EDINGS OF THE MALACOLOGICAL SOCIETY. 384 242. Frnetxa Futerpa, A. Adams (?). ? Fenella fulgida, A. Adams: MS. in Brit. Mus. ? Helix fulgida, J. Adams: Trans. Linn. Soc., 1800, p. 254. ? Rissoa fulgida, C. B. Adams: [ubi?]; Nevill, Hand List, i, p. 116. Hab.—Natal (Nevill). It is quite uncertain at present which author’s name is to be given to the name fulgida as representing the Natal shell. 248. Frenerta Natarensis, Smith. 248. Frenerta Natarensis, Smith. Fenella Natalensis, Smith: Journ. Conch., vol. ix (1899), p. 249, pl. v, fig. 6. Hab.—Cato’s Creek, near Durban (Burnup). Hab.—Cato’s Creek, near Durban (Burnup). 244. Creprputa Carensis, Quoy & Gaimard. Crepidula Capensis, Quoy & Gaimard: Voy. Astrolabe, vol. iii, p. 424, pl. Ixxu, figs. 13, 14; Tryon, Man. Conch., vol. vil, p. 125, pl. xxxvi, figs. 15, 16 (copy of Q. & G.). Hab.—Cape of Good Hope (Q. & G.). 245, CaLyprrma somipa (Reeve). 245, CaLyprrma somipa (Reeve). Trochita solida, Reeve: Conch. Icon., vol. xi (1859), pl. ii, figs. 10a, d. Hab.—False Bay (Martens, Jahrb. Deutsch. Malak. Gesell., vol. i, 1874, p. 127); Conchagua, Central America (Reeve). 246. Hrpponyx Barsatus, Sowerby. 246. Hrpponyx Barsatus, Sowerby. TTipponyx barbatus, Sowerby: Thes. Conch., vol. i, p. 369, pl. Lxxiii, figs. 26, 27; Tryon, Man. Conch., vol. viii, p. 135, pl. xl, fig. 2. Hab. — Durban and Umkomaas (Burnup); Cape of Good Hope (Tryon); Society Is. (Sowerby). 247. Vanikoro striata (D’Orbigny). 247. Vanikoro striata (D’Orbigny). Narica striata, D’Orbigny : Récluz, Mag. de Zool., 1845, p. 57, pl. exxxii, figs. 1, 2; Tryon, Man. Conch., vol. vii, p. lxix, fig. 80. Hab.—Cape Natal (Récluz); Cuba (D’Orb.). 248. Vanrkoro rieata (Récluz). . ( ). Vanikoro ligata, Récluz: Sowerby, Proc. Malac. Soe., vol. iv, p. 5 (1900). Hab.—Pondoland (Sowerby). 249. Vanikoro Guerrntana (Récluz). 249. Vanikoro Guerrntana (Récluz). Vanikoro Gueriniana, Récluz: Sowerby, Proc. Malac. Soc., vol. iv, p. 5 (1900). Hab.—Pondoland (Sowerby). 250. LametnartA Mavritrana, Bergh. 250. LametnartA Mavritrana, Bergh. Lamellaria Mauritiana, Bergh: Smith, Journ. Conch., vol. x (1901), 250. LametnartA Mavritrana, Bergh. Lamellaria Mauritiana, Bergh: Smith, Journ. Conch., vol. x (1901), p. 108. ITab.—Port Elizabeth (Crawford). 3885 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLUS 251. Natica Kraussi, Smith. 251. Natica Kraussi, Smith. Natica Kraussi, Smith: Journ. Conch., vol. x (1902), p. 248, pl. iv, fig. 1. Hab.—Durban (Burnup). 252. Natica psita, Watson. Natica psila, Watson: Gasteropoda Challenger Exped., p. 443, pl. xxviii, fig. 1. Hab.—Ott Cape of Good Hope, 150 fathoms. 253. EDINGS OF THE MALACOLOGICAL SOCIETY. Atlantica do not belong to that species. 263. Euroa Naratensis, Smith. 263. Euroa Naratensis, Smith. Eulima Natalensis, Smith: Journ. Conch., vol. ix (1899), p. 250, pl. v, fig. 8. Hab.—Durban. 264. Niso patreata, Sowerby. iso balteata, Sowerby: Proc. Malac. Soc., vol. iv, p. 6 (1900), pl. i, fig. 16. Hab.—The Kowie (Sowerby). 265. Opostomra cHrtonicota, Smith. EDINGS OF THE MALACOLOGICAL SOCIETY. Narica (Porrnices) Burnvri, n.sp. Pl. XV, Fig. 11. Testa parva, ovata, obtecte perforata, crassiuscula, fusco-cinerea vel rufescens; anfractus 5, convexi, superiores parvi, ultimus magnus, antice oblique descendens, oblique striatus; apertura parva, semi- circularis, intus fuscescens; columella obliqua, recta, callo crasso albo reflexo instructa. Long. 11, diam. 7 mm.; apertura 6 mm. longa, 3 lata. Hab.—Durban Bay (Burnup). Larger specimens are 17 mm. in length. 254. Tanruina nitens, Menke. Lanthina nitens, Menke: Reeve, Conch. Icon., vol. xi, figs. 16a, 6. Hab.—Kalk Bay ; Mediterranean, and Atlantic. Lanthina nitens, Menke: Reeve, Conch. Icon., vol. xi, figs. 16a, 6. Hab.—Kalk Bay ; Mediterranean, and Atlantic. 255. JANTHINA BALTEATA, Reeve. 255. JANTHINA BALTEATA, Reeve. Lanthina balteata, Reeve: Conch. Icon., vol. xi, pl. iii, figs. 11a, 8. Hab.—Durban ; Cape of Good Hope (Reeve). Lanthina balteata, Reeve: Conch. Icon., vol. xi, pl. iii, figs. 11a, 8. Hab.—Durban ; Cape of Good Hope (Reeve). 256. Scata Rosirtarpr (Sowerby). 256. Scata Rosirtarpr (Sowerby). Scalaria Robillardi, Sowerby: Proc. Malac. Soc., vol. i, p. 42 (1894), pl. iv, fig. 5. Hab.—Durban ; Mauritius (Sowerby). 257. Scata acuLEaTa (Sowerby). Scalaria aculeata, Sowerby: Thes. Conch., vol. i, p. 86, pl. xxxui, figs. 85-37. Hab.—Durban ; also Philippines, Hong Kong, Malacca, ete. 258. ScaLa MILLEcosTaTa (Pease). Scalaria millecostata, Pease: Smith, Journ. Conch., vol. x (1901), p. 109, pl. i, fig. 5. Hab.—Isipingo, Natal (Burnup); Sandwich Is. (Pease). 259. EvLIMA TRANSLUCIDA, Smith. 259. EvLIMA TRANSLUCIDA, Smith. Eulima translucida, Smith: Journ. Conch., vol. x (1901), p. 109, ple thie: 11. Hab.—Kast London, Cape Colony (Lavertine). 260. Eunima Atcornsis, Smith. 260. Eunima Atcornsis, Smith. Eulima Algoensis, Smith: Journ. Conch., vol. x (1901), p. 109, pl. i, fig. 10. Hab.—Algoa Bay, Cape Colony. 386 EDINGS OF THE MALACOLOGICAL SOCIETY. 261. Evrrma priecta, Smith. Eulima dilecta, Smith: Journ. Conch., vol. ix (1899), p. 250, pl. v, fig. 9. Hab.—Umkomaas, Natal. 262. Evrima munpa, Smith. 262. Evrima munpa, Smith. 262. Evrima munpa, Smith. Eulima munda Smith: Journ Conch vol ix (1899) p 249 pl v Eulima munda, Smith: Journ. Conch., vol. ix (1899), p. 249, pl. v, ite Ve ( ) Eulima Atlantica, Sowerby : Marine Shells 8. Afr., p. 26 (nec Smith). Hab.—Umkomaas (Burnup). Eulima Atlantica, Sowerby : Marine Shells 8. Afr., p. 26 (nec Smith). Hab.—Umkomaas (Burnup). The specimens referred by Mr. Sowerby to £. Atlantica do not belong to that species. The specimens referred by Mr. Sowerby to £. 265. Opostomra cHrtonicota, Smith. Odostomia chitonicola, Smith: Journ. Conch., vol. ix (1899), p. 250, ploy, tig. 10; Hab.—Umkomaas, on Chiton fossus (Burnup). 266. Oposrom1a Lavertinm, Smith. Odostomia Lavertine, Smith: Journ. Conch., vol. x (1901), p. 108, pl. i, fize 15. p Odostomia Angast, Sowerby: Marine Shells S. Afr., 1892, p. 26 (nec Tryon). Hab —East London Cape Colony (Lavertine) Hab.—East London, Cape Colony (Lavertine). 267. Opostom1a (Pyreurina) rosusta, Sowerby. Odostomia (Pyrgulina) robusta, Sowerby: Proc. Malac. Soc., vol. iv, p. 214 (1901), pl. xxii, fig. 19. Hab.—The Kowie. 268. Turponitta canpipa (A. Adams). Chemnitzia candida, A. Adams: Proc. Zool. Soc., 1853, p. 181. Chemnitzia lactea, Krauss: Siidafr. Moll., p. 88, pl. v, fig. 23. Turbonilla Krausst, Clessin: Conch. Cab., Pyramidellide, p. 1385, pl. xvi, fig. 6. Turbonilla candida, A. Adams: Sowerby, Marine Shells S. Afr., p. 26. Hab —Natal (Krauss); Port Elizabeth (Sowerby); Japan ? (Adams) Turbonilla candida, A. Adams: Sowerby, Marine Shells S. Afr., p. 26. Hab.—Natal (Krauss); Port Elizabeth (Sowerby); Japan ? (Adams). 269. TURBONILLA SIMILANS, n.sp. Pl. XV, Fig. 13. 269. TURBONILLA SIMILANS, n.sp. Pl. XV, Fig. 13. Testa Z. lactee@ similis, sed gracilior, minor, anfractibus magis rotundatis, costis magis flexuosis. Anfractus normales 9, perconyexi, costis gracilibus obliquis flexuosis (in anfractu penultimo circiter 18) , p , g Testa Z. lactee@ similis, sed gracilior, minor, anfractibus magis rotundatis, costis magis flexuosis. Anfractus normales 9, perconyexi, costis gracilibus obliquis flexuosis (in anfractu penultimo circiter 18) 387 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU instructi, sulcis intervenientibus infra haud ad suturam productis, ultimus infra medium levis; apertura parva, ovata: columella leviter arcuata, reflexa. Long. 5:5, diam. 1 mm. Hab.—S8. Africa. In 7. lactea, Linn., the costz and sulci reach from suture to suture, whereas in the present species they terminate inferiorly just above the suture. The shells here described are identified in ‘‘The Marine Shells of South Africa” as belonging to 7. lactea. 270. Tourponrtta tincta, Sowerby. Turbonilla tincta, Sowerby: Proc. Malac. Soc., vol. iv, p. 5 (1900), plea, tis, 15, Hab.—The Kowie (Sowerby). 271. TuRBoNILLA BATHYRAPHE, Sowerby. Turbonilla bathyraphe, Sowerby: Proc. Malac. Soc., vol. iv, p. 213 (i901); ply xx, fie, 18. Hab.—The Kowie (Sowerby). 272. TurBoniLLa pDiscutus, Vélain(?). Turbonilla disculus, Vélain: Tryon, Man. Conch., vol. vili, p. 336, pl ixxy, fig. 97. Hab.—Durban; St. Paul I., Indian Ocean (Vélain). 273. Mormuta Macanprem, A. Adams. Mormula Macandrea {sic}, A. Adams: Sowerby, Proc. Malac. Soc., vol. iv, p. 5 (1900). Hab.—Pondoland (Sowerby). 274. 286. Trocuus (InrunprsuLuM) TEXxTILIs, Reeve. 286. Trocuus (InrunprsuLuM) TEXxTILIs, Reeve. Trochus textilis, Reeve: Conch. Icon., vol. xiii (1861), pl. xiv, fig. 82 ; Pilsbry, Man. Conch., vol. xi, p. 38, pl. xli, fig. 7. ITab.—Cape of Good Hope (Reeve). 287. CrancuLys minraTus (Anton). 282. CycLosTREMA CINGULIFERA, A. Adams. 282. CycLosTREMA CINGULIFERA, A. Adams. Cyclostrema cingulifera, A. Adams: Sowerby’s Thes. Conch., vol. ii, p. 250, pl. 255, figs. 13, 14. Hab.—Durban (Burnup); Japan, Philippines (A. Ad.). 283. Trocuus (CarprNnaLiIA) vircatus, Gray. Trochus (Cardinalia) virgatus, Gray: Pilsbry, Man. Conch., vol. xi, p. 19, pl. v, figs. 48, 44. Hab.—Durban (Burnup). 284, Sonarrerta Becxert, Sowerby. 284, Sonarrerta Becxert, Sowerby. Solariella Beckert, Sowerby: Proc. Malac. Soc., vol. iv, p. 214 (1901), pl. xxu, fig. 21. Hab.—The Kowie (Sowerby). EDINGS OF THE MALACOLOGICAL SOCIETY. 279. Asrratium ANnprErsont, Smith. Astralium (Bolma) Andersoni, Smith: Journ. Conch., vol. x (1902), p. 248, pl. iv, fig. 7. Hab.—Off Durban (Quekett). This species attains fully three times the size of the shell figured. 280. Leprormyra BIcaARINATA (Martens). 280. Leprormyra BIcaARINATA (Martens). Collonia bicarinata, Martens: Sitzungsb. Gesell. naturf. Freunde Berlin, 1902, p. 241. HHab.—Agulhas Bank, 250 fathoms (Martens). p HHab.—Agulhas Bank, 250 fathoms (Martens). 281. Lrorra FuLGENs, Gould. 281. Lrorra FuLGENs, Gould. LInotia fulgens, Gould: Otia Conch., p. 114. Hab.—Simons Bay, Cape of Good Hope ( LInotia fulgens, Gould: Otia Conch., p. 114. LInotia fulgens, Gould: Otia Conch., p. 114. Hab.—Simons Bay, Cape of Good Hope (Gould). Hab.—Simons Bay, Cape of Good Hope (Gould). 285, Cynisca eranutosa (Dunker). 285, Cynisca eranutosa (Dunker). Delphinula granulosa (Dunker MS.), Krauss: Siidafr. Moll., p. 94, pl. v, fig. 28. Liotia granulosa, Dunker: Tryon, Man. Conch., vol. x, p. 109, pl. xxxvi, fig. 96. p g Cyclostrema granulata, A. Adams: Proc. Zool. Soc., 1858, p. 183. Cynisea granulata, H. & A, Adams: Genera, vol.i, p. 406; A. Adams, Ann. & Mag. Nat. Hist., 1861, vol. viii, p. 243 g p Gibbula granulosa, Dunker: Sowerby, Marine Shells S. Afr., p. 43. 265. Opostomra cHrtonicota, Smith. Neriva aterrma (Gmelin). 274. Neriva aterrma (Gmelin). Nerita nigerrima, Chemnitz: Reeve, Conch. Icon., vol. ix, figs. 11a, 6. Hab.—Umkomaas (Burnup). Nerita nigerrima, Chemnitz: Reeve, Conch. Icon., vol. ix, figs. 11a, 6. Hab.—Umkomaas (Burnup). 275. Neririva Natatensts, Reeve. Neritina Natalensis Reeve : Conch Icon vol ix pl xvi figs 75a 6 275. Neririva Natatensts, Reeve. 275. Neririva Natatensts, Reeve. Neritina Natalensis, Reeve : Conch. Icon., vol. ix, pl. xvi, figs. 75a, 6. Neritina ebra Kra ss: Siidafr Moll p 88 (nee Lamarck) 275. Neririva Natatensts, Reeve. Neritina Natalensis, Reeve : Conch. Icon., vol. ix, pl. xvi, figs. 75a, 6. Neritina Natalensis, Reeve : Conch. Icon., vol. ix, pl. xvi, figs. 75a, 6. Neritina zebra, Krauss: Siidafr. Moll., p. 88 (nee Lamarck). Hab.— Natal (Reeve); Durban (Burnup and others). 276. NERITINA CREPIDULARIA, Lamarck. 276. NERITINA CREPIDULARIA, Lamarck. Neritina erepidularia, Lamarck : Martens, Conch. Cab., 2nd ed., p. 37, pl. vii, figs. 1-14. Hab.—Natal (Krauss); Ashanti (Brit. Mus. ). For distribution and synonymy see Martens. 277. TursBo curysosromus, Linn. Turbo chrysostomus, Linn.: Pilsbry, Man. Conch., vol. x, p. 200, pl. x], fig. 19. Hab —Durban (Burnup). 278. Touro sPpLENDIDULUS, Sowerby. Turbo splendidulus, Sowerby: Thes. Conch., vol. vy, p. 229, pl. 506, figs. 180, 181. Hab.—Umzinto ; Durban ; ——? (Sowerby). 388 DINGS OF THE MALACOLOGICAL SOCIETY. 289. CxLancuLus mrxtus, n.sp. Pl. XV, Fig. 7. 289. CxLancuLus mrxtus, n.sp. Pl. XV, Fig. 7. Testa turbinata, profunde umbilicata, pallide fuscescens, granulis albidis et fuscis ornata infra suturam maculis saturate fuscis picta ; anfractus 5-6, ad suturam canaliculati, leviter convexi, seriebus granulorum (in anfractu penultimo plerumque sex, in antepenultimo tres) plus minus ineequalibus instructi, in interstitiis minute et oblique striati, ultimus ad peripheriam rotundatus, antice leviter descendens, seriebus granulorum 16-17 ornatus; umbilicus albus, levis, conicus, seepe intra marginem tuberculis paucis albis instructus; apertura obliqua, albida; labrum ad marginem acutum, intus incrassatum et lratum; columella obliqua, rectiuscula, leviter incrassata, supra et antice unidentata. Diam. maj. 12°56, min. 11 mm.; alt. 11 mm. Hab —Port Elizabeth Testa turbinata, profunde umbilicata, pallide fuscescens, granulis albidis et fuscis ornata infra suturam maculis saturate fuscis picta ; anfractus 5-6, ad suturam canaliculati, leviter convexi, seriebus granulorum (in anfractu penultimo plerumque sex, in antepenultimo tres) plus minus ineequalibus instructi, in interstitiis minute et oblique striati, ultimus ad peripheriam rotundatus, antice leviter descendens, seriebus granulorum 16-17 ornatus; umbilicus albus, levis, conicus, seepe intra marginem tuberculis paucis albis instructus; apertura obliqua, albida; labrum ad marginem acutum, intus incrassatum et lratum; columella obliqua, rectiuscula, leviter incrassata, supra et antice unidentata. Diam. maj. 12°56, min. 11 mm.; alt. 11 mm. Hab.—Port Elizabeth. This species has been confounded with C. Waltone of Sowerby. It differs in having finer and more closely set granules, in the last whorl descending in front, and in the thickened and lrate labrum. The latter in C. Waltone is scarcely thickened and smooth within. The apex of the spire in the present species is not so red as it is in Waltone. None of the specimens of the latter which have been examined have any tubercles within the edge of the umbilicus. This species has been confounded with C. Waltone of Sowerby. It differs in having finer and more closely set granules, in the last whorl descending in front, and in the thickened and lrate labrum. The latter in C. Waltone is scarcely thickened and smooth within. The apex of the spire in the present species is not so red as it is in Waltone. None of the specimens of the latter which have been examined have any tubercles within the edge of the umbilicus. 288. CLancuLus pEPREssUs (Gmelin). 288. CLancuLus pEPREssUs (Gmelin). Trochus depressus, Gmelin: Philippi, Conch. Cab., 2nd ed., p. 77, pl. xiv, fig. 6; Martens, Jahrb. Deutsch. Malak. Gesell., 1874, p. 129. Trochus depressus, Gmelin: Philippi, Conch. Cab., 2nd ed., p. 77, pl. xiv, fig. 6; Martens, Jahrb. Deutsch. Malak. Gesell., 1874, p. 129. Trochus depressus, Gmelin: Philippi, Conch. Cab., 2nd ed., p. 77, pl. xiv, fig. 6; Martens, Jahrb. Deutsch. Malak. Gesell., 1874, p. 129. p Hab.—False Bay (Martens). 287. CrancuLys minraTus (Anton). Trochus (Clanculus) miniatus, Anton: Pilsbry, Man. Conch., vol. xi, p. 58, pl. x, figs. 238, 24; pl. xiv, figs. 24, 25. This species appears in ‘“‘ The Marine Shells of South Africa,” p. 44, under the genus Gibbula. It is identical with Clanculus carinatus, A. Adams, quoted on the following page. 389 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU 288. CLancuLus pEPREssUs (Gmelin). DINGS OF THE MALACOLOGICAL SOCIEBTY. DINGS OF THE MALACOLOGICAL SOCIEBTY. Trochus meruloides, Krauss: Siidafr. Moll., p. 95; Martens, Jahrb. Deutsch. Malak. Gesell., 1874, p. 128. Hab.—Natal (Krauss); Algoa Bay (Martens). 295. OxysTELE zonaTa (Wood). Oxystele zonatus, Wood: Sowerby, Marine Shells S. Afr., p. 42. This species is placed in Gibbula by Pilsbry, Man. Conch., vol. xi, p. 258. Owing to its imperforate base Oxystele seems a better location for it. 295. OxysTELE zonaTa (Wood). Oxystele zonatus, Wood: Sowerby, Marine Shells S. Afr., p. 42. Oxystele zonatus, Wood: Sowerby, Marine Shells S. Afr., p. 42. This species is placed in Gibbula by Pilsbry, Man. Conch., vol. xi, p. 258. Owing to its imperforate base Oxystele seems a better location for it. 296. Ostrinus Tamsr (Dunker). Oxystele Tamsi, Dunker: Sowerby, Marine Shells S. Afr., p. 42. Hab.—Cape of Good Hope (from Krauss in Brit. Mus.). Th h t f th l ll l thi i i O li th Oxystele Tamsi, Dunker: Sowerby, Marine Shells S. Afr., p. 42. Oxystele Tamsi, Dunker: Sowerby, Marine Shells S. Afr., p. 42. Hab.—Cape of Good Hope (from Krauss in Brit. Mus.). The character of the columella places this species in Oszlinus rather than Oxystele. Oxystele Tamsi, Dunker: Sowerby, Marine Shells S. Afr., p. Hab.—Cape of Good Hope (from Krauss in Brit. Mus.). ( ) The character of the columella places this species in Oszlinus rather than Oxystele. 297. Gissputa Brcxert, Sowerby. Gibbula Beckeri, Sowerby: Proc. Malac. Soc., vol. iv, p. 214 (1901), pli xx, fir. 22. Hab.—The Kowie (Sowerby). 298. Grsputa (Enrpa) prerspectiva, Sowerby. Gibbula (Enida) perspectiva, Sowerby: Proc. Malac. Soc., vol. iv, p- 6 (1900), pl. i, fig. 17. Hab.—Pondoland (Sowerby); Port Shepstone (Burnup). A single young specimen from the latter locality may be different. It has a more prominent and wavy keel at the periphery, in which respect it resembles Hnida Townsendi, Sowerby. It is also dotted and spotted with pink and white, whereas the type, in the British Museum, is uniformly deep red, excepting the yellowish umbilicus and apex. 299. GissuLta (Enrpa) Townsenpi, Sowerby. Gibbula (Enida) Townsendi, Sowerby: Proc. Malac. Soc., vol. i, p. 279 (1895), pl. xviii, figs. 7-9. Gibbula (Enida) Townsendi, Sowerby: Proc. Malac. Soc., vol. i, p. 279 (1895), pl. xviii, figs. 7-9. Hab.—Durban (Burnup); Mekran coast (Sowerby); Thursday _ Island, Torres Straits (Brit. Mus.). ( ), p , g Hab.—Durban (Burnup); Mekran coast (Sowerby); Thursday _ Island, Torres Straits (Brit. Mus.). 300. 290. CALLIostoMA MULTILIRATUM (Sowerby). 290. CALLIostoMA MULTILIRATUM (Sowerby). Liziphinus multiliratus, Sowerby: Proc. Zool. Soc., 1875, p. 127, pl. xxiv, fig. 10; Marine Shells S. Afr., Appendix, p. 30; Pilsbry, Man. Conch., vol. xi, p. 342, pl. xv, figs. 45, 46. Calliostoma liratum, Sowerby : Proce. Malae. Soe., vol. iv, p. 6 (1900). Hab.—Cape of Good Hope and Pondoland (Sowerby). Liziphinus multiliratus, Sowerby: Proc. Zool. Soc., 1875, p. 127, pl. xxiv, fig. 10; Marine Shells S. Afr., Appendix, p. 30; Pilsbry, Man. Conch., vol. xi, p. 342, pl. xv, figs. 45, 46. y p p g Calliostoma liratum, Sowerby : Proce. Malae. Soe., vol. iv, p. 6 (1900). Hab.—Cape of Good Hope and Pondoland (Sowerby). 291. Cattiosroma Burnurr, Smith. Calliostoma Burnupi, Smith: Journ. Conch., vol. ix (1899), p. 250, pl. v, fig. 2. Hab.—Durban (Burnup). 292. Tuatorta Troscueti (Philippi). Trochus Troschelit, Philippi: Conch. Cab., 2nd ed., p. 131, pl. xxiii, fig. 2. Hab.—S. Atrica (Philippi). 293. OxysrELE tmpERvIA (Menke). Mr. Pilsbry (Man. Conch., vol. xi, p. 115) places this name as a synonym of sagittifera, Lamarck. If the figure of the latter given by Delessert (Recueil, pl. xxxvi, fig. 1) truly depicts Lamarck’s type, it is evident that this decision is incorrect, for the character of the columella shows that the species belongs to Oszlinus and not to Oxystele. 390 DINGS OF THE MALACOLOGICAL SOCIEBTY. Muiyotra tavissrma (Martens). Monilia (Minolia) levissima, Martens: Pilsbry, Man. Conch., vol. xi, p. 268. Hab.—South Africa, 8 fathoms (Martens). 301. Monoponra vermicuLata (Fischer). Trochus vermiculatus, Fischer: Coq. Viv., p. 228, pl. Ixxiv, fig. 3; Pilsbry, Man. Conch., vol. xi, p. 89, pl. xxi, figs. 25, 26; pl. Lxii, fig. 64. Hab.—Natal (Krauss). 302. Evcuxrtys (Hypocurtus) cancELLatus (Krauss). Stomatella cancellata, Krauss: Sowerby, Marine Shells 8. Afr., p. 47. Euchelus seems a better location for this species than Stomatella. SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU 391 303. Hatiorrs pertusa, Reeve. Haliotis pertusa, Reeve: Sowerby, Proc. Malac. Soc., vol. iv, p. 6 (1900). Hab.—The Kowie (Sowerby). 304. Mzcarapennus (AMBLYCHILEPAS) SCUTELLUM (Gmelin). Fissurellidea hiantula, Sowerby: Marine Shells 8. Afr., p. 48 (nec Lamarck), ) Fissurella scutella, Gray : Sowerby, l.c., p. 47. This well-known South African shell has be This well-known South African shell has been clearly shown by Pilsbry! not to be the F. hiantula of Lamarck. 305. Mercatrapennus (AMBLYCHILEPAS) SELLA (Sowerby). 305. Mercatrapennus (AMBLYCHILEPAS) SELLA (Sowerby). Fissurella sella, Sowerby: Thes. Conch., vol. iii, p. 203, pl. 243, fig. 305. Mercatrapennus (AMBLYCHILEPAS) SELLA (Sowerby). Fissurella sella, Sowerby: Thes. Conch., vol. iii, p. 203, pl. 243, fig. 197. Hab.—S. Africa (Sowerby). This will probably prove eventually to be a form of IL. scutellum. 806. Gtyruis spreta, Smith. 806. Gtyruis spreta, Smith. Glyphis spreta, Smith: Journ. Conch., vol. x (1901), p. 104, pl. i, fig. 18. Fissurella fimbriata, Sowerby: Marine Shells 8S. Afr., 1892, p. 48 (nec Reeve). Hab.—Mossel Bay, Cape Colony (Crawford). 307. GuypHis Exizasetua, Smith. Glyphis Elizabethe, Smith: Journ. Conch., vol. x (1901), p. 104, pl. i, fig. 12. Fissurella Sieboldii, Sowerby: Marine Shells 8. Afr., 1892, p. 47 (nec Reeve). Hab.—Port Elizabeth (Crawford). 308. GiypHis EveEvATA (Dunker). l h l k h h l ( ) 308. GiypHis EveEvATA (Dunker). 308. GiypHis EveEvATA (Dunker). Glyphis elevata, Dunker: Smith, Journ. Conch., vol. x (1901), p. 105. Hab —Cape of Good Hope (Dunker) Glyphis elevata, Dunker: Smith, Journ. Conch., vol. x (1901), p. 105. Hab.—Cape of Good Hope (Dunker). 309. GutypHis cruciFEeRA, Pilsbry. ’ ( ) 309. GutypHis cruciFEeRA, Pilsbry. Glyphis ecrucifera, Pilsbry:’ Man. Conch., vol. xii (1890), p. 225, pl. xxxii, figs. 27-31 [= Fissurella cruciata, Krauss, nee Gould }. Hab.—Natal. 310. Scurum unevis (Linn.). 310. Scurum unevis (Linn.). Scutus unguis (Linn.): Smith, Journ. Conch., vol. ii (1879), p. 261. Scutus unguis (Linn.): Smith, Journ. Conch., vol. ii (1879), p. 261. 1 Man. Conch., vol. xii, p. 184. VOL. V.—OCc 313. Paretta (?) pEcemMcostata, Smith. 313. Paretta (?) pEcemMcostata, Smith. Patella (?) decemcostata, Smith: Journ. Conch., vol. x (1901), p. 106, pl. i, fig. 22. Hab.—Algoa Bay. 314. PaTeLta sancuinans, Reeve. 314. PaTeLta sancuinans, Reeve. Patella sanguinans, Reeve: Conch. Icon., vol. vi, pl. vi, figs. 10a, 6; Pilsbry, Man. Conch., vol. xiii, p. 151, pl. xxx, figs. 53, 54. Hab.—Cape Natal (Reeve). DINGS OF THE MALACOLOGICAL SOCIEBTY. Scutum imbricatum, Quoy & Gaimard : Sowerby, Marine Shells 8S. Afr., p. 49. Hab.—Durban (Burnup). For distribution and synonymy see the first-named reference above. 311. Acmaa rosEorADIATA, Smith. Acmea roseoradiata, Smith: Journ. Conch., vol. x (1901), p. 106, pl. i, 311. Acmaa rosEorADIATA, Smith. ( ) 311. Acmaa rosEorADIATA, Smith. Acmea roseoradiata, Smith: Journ. Conch., vol. x (1901), p. 106, pl. i, fig. 19. H b C T (B i M ) Hab.—Cape Town (Brit. Mus.). 27 392 EDINGS OF THE MALACOLOGICAL SOCIETY. 312. Acma#a aLBonorata, Smith. Acmea albonotata, Smith: Journ. Conch., vol. x (1901), p. 107, pl. 1, figs. 14, 16. HTab.—Umkomaas, Natal (Burnup). 313. Paretta (?) pEcemMcostata, Smith. 315. Parerta parriarcua, Pilsbry. 315. Parerta parriarcua, Pilsbry. Patella patriarcha, Pilsbry: Man. Conch., vol. xiii (1891), p. 105, pl. lxiv, figs. 84, 85; pl. lxv, fig. 86. Hab.—Cape of Good Hope (Pilsbry). 316. PareLta TEstuprINARIA, Linn. Patella testudinaria, Linn.: Pilsbry, Man. Conch., vol. xin, p. 128, pl. xxv, figs. 16-19. Hab.—Cape of Good Hope (Pilsbry). 317. Parerta Naratensts, Krauss. This species is not referred to by Mr. Sowerby. It is regarded as a good species by Pilsbry (Man. Conch., vol. xiii, p. 103). Patella nigroalba, Blainville, Cape of Good Hope, is considered a doubtful species by Professor Pilsbry. 818. ParTetta MorBrpa, Reeve. Patella morbida, Reeve: Conch. Icon., vol. viii, pl. xxv, figs. 64a, 6; Pilsbry, Man. Conch., vol. xiii, p. 100, pl. xv, figs. 3, 4. Hab.—Cape of Good Hope (Reeve). 319. Curron Crawrorpt, Sykes. 319. Curron Crawrorpt, Sykes. 319. Curron Crawrorpt, Sykes. Chiton Crawfordi, Sykes: Proc. Malac. Soc., vol. ii, p. 279 (1899), fig. ii. Hab.—Algoa Bay (Crawford), 320. IscHnocurron ontscus (Krauss). Ischnochiton oniseus, Krauss: Sykes, Nautilus, vol. xii, p. 41. It is distinct from L. Elizabethensis of Pilsbry. Hab.—Table Bay, Simons Bay, Port Elizabeth, ete. ; also Mauritius (Tryon). 321. Iscunocuiton Exizaseruensis, Pilsbry. Ischnochiton Elizabethensis, Pilsbry: Nautilus, vol. viii (1894), p. 9 Sykes, Nautilus, vol. xu, p. 41. Hab.—Port Elizabeth (Pilsbry) ; Natal (Brit. Mus.). 393 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU 322, Cumropieura peEstiruta (?), Sykes. 322, Cumropieura peEstiruta (?), Sykes. 322, Cumropieura peEstiruta (?), Sykes. Chetopleura destituta, Sykes: Proc. Malac. Soc., vol. v, p. 195 (1902), fig. Hab.— Durban (Burnup). 323. Onrraocuiton (?) Isrprncornsts, Sykes. 323. Onrraocuiton (?) Isrprncornsts, Sykes. Onithochiton (?) Isipingoensis, Sykes: Proc. Malac. Soc., vol. iv, p. 259 (1901), fig. Hab.—Isipingo, 8. Africa (Burnup). 324. Dinoprax Fossus, Sykes. Dinoplax fossus, Sykes: Proc. Malac. Soc., vol. iii, p. 277 (1899), fig. i. Hab.—Umkomaas (Burnup). 325. Acanrnocauires Carpentert, Pilsbry. Acanthochites Carpentert, Pilsbry: Man. Conch., vol. xv (1893), p. 35, pl. i, figs. 14-22. Hab.—Port Elizabeth (Pilsbry). 326. CrypropLax striatus (Lamarck) (?). 1 Marine Shells of South Atrica,’’ p. 65. 326. CrypropLax striatus (Lamarck) (?). Cryptoplax striatus, Lamk.(?): Sykes, Journ. Malac., vol. vii (1900), p. 164, figs. 2-5. Hab.—Umkomaas. 3827. Dentatium stricaAtum, Gould. Dentalium strigatum, Gould: Otia Conch., p. 119; Pilsbry, Man. Conch., vol. xvii, p. 13, pl. v, figs. 69, 70. Hab.—F alse Bay, Cape of Good Hope (Gould). 328. DernrALIuM LoneiTRorsuM, Reeve. 328. DernrALIuM LoneiTRorsuM, Reeve. Dentalium longitrorsum, Reeve: Sowerby, Conch. Icon., vol. xviii, pl. ii, figs. 9a, b. Dentalium longitrorsum, Reeve: Sowerby, Conch. Icon., vol. xviii, pl. ii, figs. 9a, b. Hab.—Durban (Burnup) ; Philippines (Sowerby). Hab.—Durban (Burnup) ; Philippines (Sowerby). 329. DentaLium DENTALIS, Linné. Dentalium dentalis, Pilsbry: Man. Conch., vol. xvii, p. 53. Hab.—Simons Bay, Cape of Good Hope ( Watson). 330. DENTALIUM REGULARE, n.sp. Pl. XV, Fig. 2. Testa brevis, leviter arcuata, rubescens, antice pallida, longitudinaliter striata, striis postice profundis, interstitus costuliformibus. Long. circiter 38, diam. 4mm. 330. DENTALIUM REGULARE, n.sp. Pl. XV, Fig. 2. Testa brevis, leviter arcuata, rubescens, antice pallida, longitudinaliter striata, striis postice profundis, interstitus costuliformibus. Long. circiter 38, diam. 4mm. Hab.—Port Shepstone (Burnup). The strie are about twenty-six in number, and extend the whole length of the shell, but are quite superficial towards the anterior end and gradually become deeper posteriorly, so that the intervening spaces are rib-like, some being stouter than others. The posterior end exhibits a narrow oblong orifice, set m a pale pink circle which is margined with a darker red ring. Outside this, the shell is pale pink or whitish. The lines of growth are not strongly marked. EDINGS OF THE MALACOLOGICAL SOCIETY. 394 PELECYPODA. 331. OsTrEaA LENTIGINOSA, Sowerby. 331. OsTrEaA LENTIGINOSA, Sowerby. Ostrea lentiginosa, Sowerby : Conch, Icon., vol. xviii, figs. 58a, 6. Hab.—Durban (Burnup) ; —— ? (Sowerby). 332, Awnomma, sp. (?), unnamed in B.M. 332, Awnomma, sp. (?), unnamed in B.M. Hab.—S. Africa. 333. Pricatruta squamosissima, Smith. 333. Pricatruta squamosissima, Smith. Plhicatula squamosissima, Smith: Journ. Conch., vol. ix (1899), p. 241, pl. v, ‘figs. 15, 16. Hab.—Umkomaas, Natal (Burnup). 334, Marearitirera Natarensis (Jameson). Pteria (Margaritifera) Natalensis, Jameson: Proc. Zool. Soe., 1901, vol. i, p. 383, fig. Hab.—Umkomaas, near Durban (Burnup). 335, MARGARITIFERA VULGARIS, var. (?). Margaritifera vulgaris (Schumacher): Jameson, Proce. Zool. Soc., 1901, vol. i, p. 384. Avicula (Meleagrina) flabellum, Sowerby: Marine Shells 8. Afr., Appendix, p. 27 (nec Reeve). Hab.—Durban (Burnup). The specimens named Avicula flabellum by Mr. Sowerby are considered by Dr. Jameson to belong rather to the common Ceylon species. A. flabellum appears to be synonymous with 4. radiata, Leach, a West Indian form. 336, Pinna Kravssi, Hanley. Pinna Kraussvi, Hanley: Proc. Zool. Soc., 1858, p. 226. Hab.—Natal (Hanley). 337. Pinna squamirera, Sowerby. Pinna squamifera, Sby.: Reeve, Conch. Icon., vol. xi, fig. 12; Clessin, Conch. Cab., 2nd ed., pl. xxxv, fig. 2. Hab.—Knysna Harbour, south coast of Cape Colony (J. F. Davis) ; Conch. Cab., 2nd ed., pl. xxxv, fig. 2. Hab.—Knysna Harbour, south coast of Cape Colony (J. F. Davis) ; Port Elizabeth (Sowerby). Hab.—Knysna Harbour, south coast of Cape Colony (J. F. Davis) ; Port Elizabeth (Sowerby). Hab.—Knysna Harbour, south coast of Cape Colony (J. F. Davis) ; Port Elizabeth (Sowerby). This species is quite distinct from P. pernula (Chemnitz ?), with which Mr. Sowerby has united it.'. It differs in form, colour, and sculpture. The P. pernula (Reeve, Conch. Icon., fig. 22) is a well- known Mediterranean shell, and occurs also at Madeira, the Canary Islands, and on the West African coast. It is of a reddish colour with paler scales, and appears to be devoid of the numerous squamulee on the ventral side of the valves which are present in P. squamulifera. Mr. Davis found his specimens, a fine series of which he has kindly sent to the Museum, within the harbour, which is landlocked, the entrance from the sea being narrow and between high cliffs. The largest specimen is nine inches in length, 399 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLUS 338. Lirmopomus mucRONATA (Philippi). Modiola mucronata, Philippi: Sowerby, Marine Shells 8. Afr., p. 346. Nucutana Betcnerr (Hinds). 346. Nucutana Betcnerr (Hinds). Leda Belcheri, Hinds: Hanley in Sowerby’s Thes. Conch., vol. iu, p. 116, pl. cexxvil, fig. 53. Hab.—Agulhas Bank, off Cape of Good Hope (Hinds). Quoted in the ‘‘ Marine Shells of South Africa,” p. 6, as a Nucula. 347. Las#a RuBRA (Montagu). PELECYPODA. 64. Hab.—Natal (Krauss) ; Java (Philippi). This species belongs to Lzthodomus. 339. LirHopomus cyLinpRIca (Krauss). 339. LirHopomus cyLinpRIca (Krauss). Modiola cylindrica, Krauss: Sowerby, Marine Shells S. Afr., p. 64. Thi i b l t th Lith d d t t M di l Modiola cylindrica, Krauss: Sowerby, Marine Shells S. Afr., p. 64. This species belongs to the genus Lithodomus, and not to Modiola. This species belongs to the genus Lithodomus, and not to Modiola. 840. Mopronarra Cuminerana (Dunker). Modiola Cumingiana, Dunker: Reeve, Conch. Icon., vol. x, pl. ix, figs. 63a, 6, sp. 58. Hab.—Umkomaas, Natal (Burnup); Red Sea and Australia. 341. Seprrrrr Kxavssr (Kiister). Tichogonia Kraussi, Kiister: Kobelt, Conch. Cab., Mytilide, p. 14, pl. vi, figs. 1-6. Hab.—Natal (Kiister). Perhaps the same as S. delocularis. 342. Arca NAvicuLaRIs, Bruguiére. 342. Arca NAvicuLaRIs, Bruguiére. Area navicularis, Bruguicre: Reeve, Conch. Icon., vol. ii, pl. xi, fig. 70. Hab.—Durban (Burnup); China (Reeve); E. and N. Australia; Amboina. 343, Arca (ScapHaRca) INZQUIVALVIs, Bruguicre. Areca inequivalvis, Bruguiére: Reeve, Conch. Icon., vol. ui, fig. 54. Hab.—Durban. 344, Arca (ScapHarca) Eryrarensis, Jonas (emend.). Arca Erythreensis, Jonas: Dunker, Novit. Conch., Suppl., ii, p. 123, pl. xl, figs. 6-8. Hab.—S. Africa; Red Sea (Jonas), 345, Arca (DapHnopErMA) Domineensts, Lamarck. Arca Domingensis, Lamarck: Lischke, Japan. Meeres-Conch., ii, p. 143, for references and synonymy. Arca squamosa, Lamarck: Sowerby, Marine Shells 8. Afr., p. 65. Hab.—Natal (Krauss), Cosmopolitan ! EDINGS OF THE MALACOLOGICAL SOCIETY. 348. Trextimya pauta (A. Adams), var. pecuLiaris, A. Ad. Pythina paula, A, Adams: Proce. Zool. Soc., 1856, p. 47. Montacuta paula, A. Ad, : Smith, Challenger Lamellibranchiata, p. 203, pl. xii, figs. 1-16. Pythina peculiaris, A. Adams: 1.e. Pythina peculiaris, A. Adams: 1.e. Tellimya paula and T. peculiaris, A. Ad.: Smith, Ann. & Mag. Nat. Hist., vol. xii (1891), p. 235. H b D b Tellimya paula and T. peculiaris, A. Ad.: Smith, Ann. & Mag. Nat. Hist., vol. xii (1891), p. 235. Hab.—Durban. 349. Monracura Naratensts, Smith. 349. Monracura Naratensts, Smith. Montacuta Natalensis, Smith: Journ. Conch., vol. ix (1899), p. 251, pl. v, fig. 14. Montacuta Natalensis, Smith: Journ. Conch., vol. ix (1899), p. 251, pl. v, fig. 14. Hab.—Umkomaas (Burnup). 350. Kerra Naratensts, n.sp. Pl. XV, Fig. 8. 350. Kerra Naratensts, n.sp. Pl. XV, Fig. 8. Testa oblonga, valde convexa, mediocriter tenuis, inequilateralis, alba, versus marginem virescens, nitida, pellucida, lineis incrementi tenuibus striata; margo dorsi anticus brevis, obliquus, curvatus, posticus longior, leviter arcuatus, parum declivis; margo ventris vix curvatus; umbones acuti, longe antemediani; dens cardinis anticus in utraque valva validus, infra umbonem situs, posticus elongatus, prominens, subyalidus ; cicatrices magne. Long. 7, alt. 5, diam. 4 mm. Hab.—Durban (Burnup). Like X. solida, Angas, from Port Jackson, but thinner, not quite the same shape, being more obtuse at the ends, and the posterior dorsal margin less oblique. Testa oblonga, valde convexa, mediocriter tenuis, inequilateralis, alba, versus marginem virescens, nitida, pellucida, lineis incrementi tenuibus striata; margo dorsi anticus brevis, obliquus, curvatus, posticus longior, leviter arcuatus, parum declivis; margo ventris vix curvatus; umbones acuti, longe antemediani; dens cardinis anticus in utraque valva validus, infra umbonem situs, posticus elongatus, prominens, subyalidus ; cicatrices magne. Long. 7, alt. 5, diam. 4 mm. ( ) Hab.—Durban (Burnup). Hab.—Durban (Burnup). Like X. solida, Angas, from Port Jackson, but thinner, not quite the same shape, being more obtuse at the ends, and the posterior dorsal margin less oblique. 3851. Scrntmta turerpa, Deshayes (?). 3851. Scrntmta turerpa, Deshayes (?). Scintilla turgida, Deshayes: Sowerby, Thes. Conch., vol. iu, p. 178, pl. 285, figs. 51, 52. Hab.—Natal (Burnup); Samar, Philippines (Deshayes). Scintilla turgida, Deshayes: Sowerby, Thes. Conch., vol. iu, p. 178, pl. 285, figs. 51, 52. Hab.—Natal (Burnup); Samar, Philippines (Deshayes). 3852. Lrsrrmva ancutata (Lamarck). Cypricardia angulata, Lamarck: Reeve, Conch. Icon., vol. i, fig. 2; Sowerby, Marine Shells 8. Afr., Appendix, p. 26. Hab.—Durban (Burnup). 353. Dosryta consoprina, Deshayes. 353. Dosryta consoprina, Deshayes. Dosinia consobrina, Deshayes: Cat. Veneridee Brit Mus., p. 10. Hab.—Cape of Good Hope. 354, Dosryia pubescens (Philippi). Dosinia pubescens, Philippi: Romer, Novit. Conch., Suppl., i, p. 79, ple cys ie a Hab.—Durban. 3855. Mererrix zonarta (Lamarck). 347. Las#a RuBRA (Montagu). Lasea rubra, Montagu: Jeffreys, Brit. Conch., vol. ii, p. 219, vol. v, pl. xxxii, fig. 1. Kellia (Poronia) rubra, Montagu: Forbes & Hanley, Brit. Moll., vol. ii, p. 94, pl. O, fig. 3; pl. xxxvi, figs. 5-7. ( ) Kellia (Poronia) rubra, Montagu: Forbes & Hanley, Brit. Moll., vol. ii, p. 94, pl. O, fig. 3; pl. xxxvi, figs. 5-7. Hab.—Umkomaas (Burnup). 396 EDINGS OF THE MALACOLOGICAL SOCIETY. 3855. Mererrix zonarta (Lamarck). Cytherea (Meretriz) zonaria, Lamarck: Sowerby, Journ. Conch., vol. vii (1894), p. 377. LHab.—Natal (Sowerby). SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU 397 356, Mererrix (CaLzisra) FLORIDELLA (Gray). Venus (Callista) floridella, Gray: Romer, Novit. Conch., Suppl., i, p- 55, pl. xvi, fig. 3. Tapes (Hemitapes) Africana, Mihlfeldt: Sowerby, Marine Shells S. Afr., p. 60. p The Chione floridella of Gray has about four years priority over the Venus Africana (Mihlfeldt) of Philippi. 357. Trveta Natatensts, Dunker. Tivela (Trigona) Natalensis, Dunker: Malak. Blatt., vol. xi (1864), p. 101; Romer, Novit. Conch., Suppl., iti, p. 10, pl. iv, fig. 3. Cytherea ( Tivela) alucinans, Sowerby : Marine Shells 8. Afr., Appendix, 1897, p. 24, pl. vii, figs. 5, 6. H b —P t N t l (D k ) Cytherea ( Tivela) alucinans, Sowerby : Marine Shells 8. Afr., Appendix, 1897, p. 24, pl. vii, figs. 5, 6. Hab.—Port Natal (Dunker). In some specimens the posterior dorsal outline is rather curved, whilst in others it is decidedly straighter. Hab.—Port Natal (Dunker). In some specimens the posterior dorsal outline is rather curved, whilst in others it is decidedly straighter. 308, VENUS DECLIvIs, Sowerby. Venus declivis, Sowerby: Smith, Journ. Conch., vol. x (1901), p. 116. Hab.—Durban Bay (Quekett). 399. Venus (Trvoctea) AraKanensis, Nevill. Venus (Timoclea) Arakanensis, Nevill: Journ. Asiat. Soc. Bengal, voOkexir( 1871), p. 10; pl. 1, fig: 16. ( ) Venus (Anomalocardia) Malonei, Vanatta: Proc. Acad. Nat. Sci. Philad., vol. liu (1901), p. 185, pl. v, figs. 4, 5. Venus Arakana, Nevill: Sowerby, Marine Shells 8. Afr., Appendix, p- 33. Hab.—Port Elizabeth and Durban; also Arakan and Ceylon (Brit. Mus.). Venus Arakana, Nevill: Sowerby, Marine Shells 8. Afr., Appendix, p- 33. Hab.—Port Elizabeth and Durban; also Arakan and Ceylon p Hab.—Port Elizabeth and Durban; also Arakan and Ceylon (Brit. Mus.). 360. CuronE ampicva, Deshayes. Chione ambigua, Deshayes: Cat. Veneride Brit. Mus., p. 153. Venus paupercula, var., Krauss: Siidafr. Moll., p. 11; Philippi, Abbild., vol. 1, p. 638, pl. iv, fig. 5. Hab.—Bay of Natal (Krauss). 361. Tapes (Hemrrares) Kravsst (Deshayes). Chione Kraussi, Deshayes: Cat. Conchif. Brit. Mus., p. 154. Venus paupercula, var. abbreviata, Krauss: Siidafr. Moll., p. 11, pl. i, fig. 10. Hab.—Natal (Krauss). This species seems to be different from the Tapes paupercula, with which it has been united by Romer and Pfeiffer. It certainly is quite distinct from the Zapes Araussi as figured by Reeve (Conch. Icon., vol. xiv, fig. 1 “¢ Marine Shells of South Africa,’’ p. 54. 366, Psammosia corrueata, Deshayes. 366, Psammosia corrueata, Deshayes. Psammobia corrugata, Deshayes: Reeve, Conch. Icon., vol. x, fig. 9 var. = P. rubicunda, Deshayes: Reeve, fig. 34. var. = P. ornata, Sowerby, nee Deshayes: Sowerby, Marine Shells S. Afr., Appendix, p. 22. Hab. — Durban ; Philippine Islands (Reeve) ; Muscat, Arabia (Dr. Jayakar in Brit. Mus.). var. = P. ornata, Sowerby, nee Deshayes: Sowerby, Marine Shells S. Afr., Appendix, p. 22. var. = P. ornata, Sowerby, nee Deshayes: Sowerby, Marine Shells S. Afr., Appendix, p. 22. Hab. — Durban ; Philippine Islands (Reeve) ; Muscat, Arabia (Dr. Jayakar in Brit. Mus.). Variable in colour, but always characterized by the wrinkles on the posterior area. Hab. — Durban ; Philippine Islands (Reeve) ; Muscat, Arabia (Dr. Jayakar in Brit. Mus.). Variable in colour, but always characterized by the wrinkles on the posterior area. 3855. Mererrix zonarta (Lamarck). 41), which probably is not a South African shell at all. Venus paupercula, var. abbreviata, Krauss: Siidafr. Moll., p. 11, pl. i, fig. 10. 362. Tapers pisrupra, Romer. Tapes disrupta, Romer: Novit. Conch., Suppl., ui, p. 70, pl. xxiv, fig. 4. Hab.—Cape of Good Hope (Romer). 398 EDINGS OF THE MALACOLOGICAL SOCIETY. 363. Drproponra rotunpaTa (Montagu) ? 363. Drproponra rotunpaTa (Montagu) ? 363. Drproponra rotunpaTa (Montagu) ? Diplodonta rotundata, Montagu: Forbes & Hanley, Brit. Moll., vol. 1i, p- 66, pl. xxxv, fig. 6; pl. M, fig. 7. Hab.—Durban. 364. Donax Bertini, Pilsbry. 364. Donax Bertini, Pilsbry. ies Bertini, Pilsbry: Proc. Acad. Nat. Sci. Philad., vol. liii (1901), 189. Hab. —In ballast from 8. Africa. 365. Psammopra PALLIDA, Deshayes. Psammobia pallida, Deshayes: Proc. Zool. Soc., 1854, p. 323; Smith, Challenger Lamellibranchiata, p. 93. Psammobia pallida, Deshayes: Proc. Zool. Soc., 1854, p. 323; Smith, Challenger Lamellibranchiata, p. 93. Hab.—Durban. Other localities are Red Sea, Aden, Suakim, off Delta of Ganges (Brit. Mus.). Only a single right valve was sent from Durban, but it is remarkable as being the largest specimen yet examined, having a length of 50mm. It is brownish white, with a few remote bluish concentric zones and numerous radiating arcuate brownish rays. Smith, Challenger Lamellibranchiata, p. 93. Hab.—Durban. Other localities are Red Sea, Aden, Suakim, off Delta of Ganges (Brit. Mus.). Only a single right valve was sent from Durban, but it is remarkable as being the largest specimen yet examined having a length of Hab.—Durban. Other localities are Red Sea, Aden, Suakim, off Delta of Ganges (Brit. Mus.). 366, Psammosia corrueata, Deshayes. 367. Psammorma ponacrorpeEs (Reeve). 367. Psammorma ponacrorpeEs (Reeve). Soletellina donacioides, Reeve: Conch. Icon., vol. x, pl. iii, fig. 11. Hab.—Durban (Burnup) ; 8. Australia (Reeve). Hab.—Durban (Burnup) ; 8. Australia (Reeve). 868. Psammorma LunuLATA (Deshayes). 868. Psammorma LunuLATA (Deshayes). 868. Psammorma LunuLATA (Deshayes). Capsella lunulata, Deshayes: Reeve, Conch. Icon., vol. x, fig. 12. Capsella lunulata, Deshayes: Reeve, Conch. Icon., vol. x, fig. 12. fab.— Durban (Burnup); Philippines (Reeve). Capsella lunulata, Deshayes: Reeve, Conch. Icon., vol. x, fab.— Durban (Burnup); Philippines (Reeve). 869. Soren Capensis, Fischer. 869. Soren Capensis, Fischer. Solen Capensis, Fischer: Man. Conch., p. 145. Solen marginatus, Koch: Philippi, Abbild., vol. i, p. 37, pl. i, fig. 6 (nec Pulteney) ; Krauss, Siidafr. Moll., p. 1. Hab.—Mouth of Kromme River, south of Cape Colony (Krauss) ; Port Elizabeth (Sowerby). Port Elizabeth (Sowerby). Mr. Sowerby! has confounded this. species with the British Solen marginatus of Pulteney. A reference to Philippi’s description will at once clear up this mistake. Krauss was right in quoting it as the S. marginatus, Koch. 399 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU 370. Srireva porira (Wood). Solen politus, Wood: Index Test., Suppl., p. 3, pl. 1, fig. 6 Siliqua Japonica, Dunker: Sowerby, Marine Shells 8. Afr., p. 54. Siliqua polita Wood: Sowerby Marine Invest 8S Africa 1902 p 100 Siliqua polita, Wood: Sowerby, Marine Invest. 8S. Africa, 1902, p. 100. Hab.—Off mouth of Amatikulu River, 24 fathoms (Sowerby). 371. Papurta eLaBrata (Lamarck). Mesodesma glabrata, Lamarck: Reeve, Conch. Icon., vol. viii, fig. 20. Hab.—S. Africa; Australia (Reeve); also Mauritius and other parts of the Indian Ocean. 372. Ervitia siscutpra, Gould. 372. Ervitia siscutpra, Gould. Ervilia biseulpta, Gould: Otia Conch., p. 166. Ervilia biseulpta, Gould: Otia Conch., p. 166. Hab.—Durban (Burnup); Japan (Gould). 373. CoRBULA RUGIFERA, n.sp. Pl. XV, Figs. 9 and 10. 373. CoRBULA RUGIFERA, n.sp. Pl. XV, Figs. 9 and 10. Corbula rugifera, A. Adams MS. in Coll. Cuming. 373. CoRBULA RUGIFERA, n.sp. Pl. XV, Figs. 9 and 10. Corbula rugifera, A. Adams MS. in Coll. Cuming. Testa oblonga, antice rotundata, postice acuminata, ineequilateralis, inequivalvis, alba, versus umbones rosea, radiis paucis pellucidis seepe picta; valvee postice oblique carinate, ante carinam concentrice irregulariter costulate, pone carinam corrugate ; margo dorsi postica leviter obliqua, recta, antica magis declivis, brevior, ante umbones leviter concava; umbones ante medium siti, incurvati, haud contigui; pagina interna albida, flavescens vel fusco tincta, postice rosacea. Long. 18, alt. 8, diam. 5°5 mm. ( ) Testa oblonga, antice rotundata, postice acuminata, ineequilateralis, inequivalvis, alba, versus umbones rosea, radiis paucis pellucidis seepe picta; valvee postice oblique carinate, ante carinam concentrice irregulariter costulate, pone carinam corrugate ; margo dorsi postica leviter obliqua, recta, antica magis declivis, brevior, ante umbones leviter concava; umbones ante medium siti, incurvati, haud contigui; pagina interna albida, flavescens vel fusco tincta, postice rosacea. Long. 18, alt. 8, diam. 5°5 mm. ( ) ( p) This is the only species apparently that has the posterior area behind the umbonal angle peculiarly obliquely wrinkled. This feature alone will separate it from allied forms. The few pellucid rays upon the central portion of the valves are much more distinct in some specimens than in others. This is the only species apparently that has the posterior area behind the umbonal angle peculiarly obliquely wrinkled. This feature alone will separate it from allied forms. The few pellucid rays upon the central portion of the valves are much more distinct in some specimens than in others. 374. SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU Panopea Natatensts, Woodward. Panopea Natalensis, Woodward: Proc. Zool. 374. Panopea Natatensts, Woodward. Panopea Natalensis, Woodward: Proc. Zool. Soc., 1855, p. 220. p Panopea Natalensis, Woodward: Proc. Zool. Soc., 1855, p. 220. Panopea australis, Valenciennes: in Chenu’s Ill. Conch., pl. viii, figs. 1-9 (nec Sowerby). ( ) Glycimeris: es, Bosc.: H. & A. Adams, Gen. Moll., vol. iu, pl. xciv, fig. 2. ( ) p g Hab.—Port Natal (Valenciennes). p g Hab.—Port Natal (Valenciennes). 875. Lucrna (Drvaricerta) Datiiana, Vanatta. Lucina (Divaricella) Dalliana, Vanatta: Proc. Acad. Nat. Sci. Philad., vol. liii (1901), p. 184, pl. v, figs. 10, 11. ( ) p p g Lucina (Divaricella) quadrisuleata, Sowerby : Marine Shells 8, Afr., Appendix, p. 26 (nee D’Orbigny). Hab.—Natal. This species differs from Z. guadrisulcata not only in the characters pointed out by Mr. Vanatta, but also in being flatter and in the kind of crenulation of the margin of the valves. Lucina (Divaricella) quadrisuleata, Sowerby : Marine Shells 8, Afr., Appendix, p. 26 (nee D’Orbigny). Hab.—Natal. This species differs from Z. guadrisulcata not only in the characters pointed out by Mr. Vanatta, but also in being flatter and in the kind of crenulation of the margin of the valves. 376. Lorrpgs rosacea, Smith. 376. Lorrpgs rosacea, Smith. ( ) Loripes rosacea, Smith: Journ. Conch., vol. ix (1899), p. 251, pl. v, figs. 12, 13. Hab.—Durban (Burnup),. 400 EDINGS OF THE MALACOLOGICAL SOCIETY. EDINGS OF THE MALACOLOGICAL SOCIETY. 877. Trettina nosrtis, Hanley. 877. Trettina nosrtis, Hanley. Tellina nobilis, Hanley : Thes. Conch., vol. i, p. 300, pl. lx, fig. 148 ; Sowerby, Conch. Icon., vol. xvu, pl. xv, fig. 74. Hab.—Cape Colony (Martens, Jahrb. Deutsch. Malak. Gesell., vol. 1, 1874, p. 122). Tellina nobilis, Hanley : Thes. Conch., vol. i, p. 300, pl. lx, fig. 148 ; Sowerby, Conch. Icon., vol. xvu, pl. xv, fig. 74. Sowerby, Conch. Icon., vol. xvu, pl. xv, fig. 74. Hab.—Cape Colony (Martens, Jahrb. Deutsch. Malak. Gesell., vol. 1, 1874, p. 122). 378. Tettina (AncuLUs?) RHoMBorDES, Quoy & Gaimard. Tellina rhomboides, Quoy & Gaimard : Sowerby, Conch. Icon., vol. xvii, figs. 1l4a, 6; Smith, Challenger Lamellibranchiata, p. 103. Hab.—Durban. 379. TrLtina seMEN, Hanley. Tellina semen, Hanley: in Sowerby’s Thes. Conch., vol. 1, p. 249, pl. lvi, fig. 8. g Hab.—Durban (Burnup); N. Australia and Fiji (Challenger Coll.). 380. TeLiina semitzvis, Martens. 380. TeLiina semitzvis, Martens. 380. TeLiina semitzvis, Martens. Tellina semilevis, Martens: Romer, Conch. Cab., 2nd ed., p. 63, pl. xviii, figs. 1-4. Tellina semilevis, Martens: Romer, Conch. Cab., 2nd ed., p. 63, pl. xviii, figs. 1-4. g Tellina tenwilirata, Sowerby : Conch. Icon., vol. xvi, fig. 253. Hab.—Durban (Burnup) ; Red Sea and Querimba Island (Romer). 381. TxLtina venusta, Deshayes. 381. TxLtina venusta, Deshayes. Tellina venusta, Deshayes: Proc. Zool. Soc., 1854, p. 368. Hab.—Durban (Burnup) ; Sandwich Islands (Deshayes). 382. Tetra crucieera, Lamarck, var. Excutra, Gould. Tellina exculta, Gould: U.S. Explor. Exped., vol. xii, p. 407, atlas, figs. 517a, 6; Otia Conch., p. 80; Sowerby, Conch. Icon., vol. xvii, figs. 178a, 6. Hab.—Durban (Burnup); Fiji (Gould) ; Torres Straits (Sowerby). 383. Macoma reTrorsa (Sowerby). 383. Macoma reTrorsa (Sowerby). Tellina (Macoma) candidata, Sowerby : Journ. Conch., vol. vii (1894), p- 375: Marine Shells S. Afr., Append., 1897, p. 23, pl. vi, fig. 25. Macoma retrorsa, Sowerby : Smith, Journ. Conch., vol. x (1901), p. 116. Hab.—Durban (Burnup). The following species appear to have been erroneously or doubtfully considered South African. The following species appear to have been erroneously or doubtfully considered South African. The following species appear to have been erroneously or doubtfully considered South African. 384. MaroInecna quaprrrascrata, Marrat. 390. PrraLoconcuus ocrosectus, Carpenter. 390. PrraLoconcuus ocrosectus, Carpenter. Petaloconchus octosectus, Carpenter: Proc. Zool. Soc., 1856, p. 317. Hab.—? 8. Africa (Carpenter). P.S.—Since the preceding paper was written a report by Mr. G. B. Sowerby upon a collection of Mollusca dredged off the shores of South Africa has been published in the ‘ Marine 386. CERITHIDEA RissorpEA (Sowerby). 386. CERITHIDEA RissorpEA (Sowerby). 386. CERITHIDEA RissorpEA (Sowerby). Cerithium rissoideum, Sowerby: Thes. Conch., vol. ii, p. 889, pl. elxxxvi, fig. 289; in Reeve’s Conch. Icon., vol. xv ( Cerithidea), pl. ii, fig. 11. Cerithium rissoideum, Sowerby: Thes. Conch., vol. ii, p. 889, pl. elxxxvi, fig. 289; in Reeve’s Conch. Icon., vol. xv ( Cerithidea), pl. ii, fig. 11. Hab.—Port Natal (Sowerby). This species, which is the same as Cerithidea crassilabrum of A. Adams, was originally described by Sowerby without any locality. Subsequently, in the Conch. Iconica, he placed in its synonymy the Cerithium erassilabrum of Krauss, and from this source was evidently derived the locality ‘‘ Port Natal.”” When he quoted erassilabrum he apparently had in his mind the Cerithidea described by Adams under that name, which is a West Indian form. 387. Cattiostroma EUeLYPtum (A. Adams). Calliostoma euglyptus \| sic], A. Adams: Sowerby, Marine Shells S. Afr., p. 42. This species occurs on the east coast of North America. The specimens so named by Mr. Sowerby belong to C. ornatum, Lamarck. 388. Curron LABECULATUS, Reeve. 384. MaroInecna quaprrrascrata, Marrat. 384. MaroInecna quaprrrascrata, Marrat. Marginella quadrifasciata, Marrat: Sowerby, Marine Shells 8. Afr., Appendix, p. 9. Hab.—S. Africa (Sowerby). This species was described originally as from Kabenda, 30 miles north of the Congo, and I am inclined to believe that it is not South African. 385. MARGINELLA CINEREA, Jousseaume. 385. MARGINELLA CINEREA, Jousseaume. Marginella cinerea, Jousseaume : Sowerby, Marine Shells S. Afr., p. 9. The specimens so named by Mr. Sowerby are distinct from this species, and are in my opinion small examples of his JL Burnupi. UU. cinerea therefore cannot yet be regarded as a South African form. 385. MARGINELLA CINEREA, Jousseaume. Marginella cinerea, Jousseaume : Sowerby, Marine Shells S. Afr., p. 9. The specimens so named by Mr. Sowerby are distinct from this species, and are in my opinion small examples of his JL Burnupi. UU. cinerea therefore cannot yet be regarded as a South African form. Marginella cinerea, Jousseaume : Sowerby, Marine Shells S. Afr., p. 9. The specimens so named by Mr. Sowerby are distinct from this species, and are in my opinion small examples of his JL Burnupi. UU. cinerea therefore cannot yet be regarded as a South African form. 401 SMITH: LIST OF SOUTH AFRICAN MARINE MOLLU 388. Curron LABECULATUS, Reeve. Chiton labeculatus, Reeve: Martens, Jahrb. Deutsch. Malak. Gesell., vol. 1 (1874), p. 125. Hab.—Algoa Bay (Martens) ; ——? (Reeve). This species, according to P. P. Carpenter’s note with the type- specimen in the British Museum, is identical with Say’s C. apiculata, a well-known North American form. Martens is therefore evidently mistaken in his identification of a South African shell with this species. (See also Pilsbry, Man. Conch., vol. xiv, p. 36.) 3889. ApLysIA MARMORATA, Blainville. — Aplysia marmorata, Blainville: Sowerby, Conch. Icon., vol. xvii, tigs. 2la, b. Hub.—Cape of Good Hope (Sowerby) ; coast of France (Blainville). Mr. Sowerby appears to have only known this species from Blainville’s figures (which he has copied) and description. It is therefore all the more remarkable that he has given a South African locality. Oncipium Peroni (Savigny ?). Cancellaria imbricata, Watson. Astralium Andersoni, Smith. Minolia levissima, Martens. Ancilla contusa, Reeve. Neptuneopsis pyrrhostoma, Watson. ( ) Ancilla contusa, Reeve. Tritonidea Natalensis, Smith. Pleurotoma (Clavatula) gravis, Hinds. Cyprea similis, Gray. Turritella declivis, Adams & Reeve. Cancellaria imbricata, Watson. Astralium Andersoni, Smith. Minolia levissima, Martens. Neptuneopsis pyrrhostoma, Watson. Voluta (Lyria) Queketti, Smith. Marginella diadochus, Adams & Reev Murex fallax, Smith. Fasciolaria rutila, Watson. Latirus abnormis, Sowerby. Fusus rubrolineatus, Sowerby. Nassa trifasciata, A. Adams, Pseudoliva aneilla, Hanley. p p py Voluta (Lyria) Queketti, Smith. ( y ) Marginella diadochus, Adams & Reeve. Pleurotoma (Clavatula) gravis, Hinds. Murex fallax, Smith. Cyprea similis, Gray. Fasciolaria rutila, Watson. y Fusus rubrolineatus, Sowerby. Nassa trifasciata, A. Adams, Minolia levissima, Martens. Minolia levissima, Martens. Pseudoliva aneilla, Hanley. Oncipium Peroni (Savigny ?). Fischer in his Manual quotes an Oneidiwm Peroni without any author’s name as from South Africa. Two species have been described under this name, one by Savigny and one by Cuvier. The former is probably the same as Semper’s O. Savignyt (Reisen im Archipel der Philippinen, Theil ii, vol. 11, p. 260). Fischer in his Manual quotes an Oneidiwm Peroni without any author’s name as from South Africa. Two species have been described under this name, one by Savigny and one by Cuvier. The former is probably the same as Semper’s O. Savignyt (Reisen im Archipel der Philippinen, Theil ii, vol. 11, p. 260). P.S.—Since the preceding paper was written a report by Mr. G. B. Sowerby upon a collection of Mollusca dredged off the shores of South Africa has been published in the ‘ Marine 402 PROCEEDINGS OF THE MALACOLOGICAL SOCIETY. Investigations in South Africa,” vol. ii, pp. 213-232, pls. lii-y. It contains the following new species :— Investigations in South Africa,” vol. ii, pp. 213-232, pls. lii-y. It contains the following new species :— Pleurotoma (Surcula) lobata. eo (Drillia) fossata. ~) scitecostata. ’% (Clavatula) turriplana. ae (Clavus) lignaria. ae (Genotia) Beleformis. Mangilia (Eucythara) Africana. Conus eucoronatus. », Gilchristi. Conus patens. Cyprea Fultoni. Nassa Desmoulioides. 9, analogica. Cancellaria producta. Epidromus erebriliratus. Scala tenebrosa. Dentalium exasperatum. Chiton (Hanleya) Sykesi. Conus patens. Cyprea Fultoni. Nassa Desmoulioides. 9, analogica. Cancellaria producta. Epidromus erebriliratus. Scala tenebrosa. Dentalium exasperatum. Chiton (Hanleya) Sykesi. ( ) Mangilia (Eucythara) Africana. Conus eucoronatus. », Gilchristi. In addition to the new species, thirty-six forms, already described, are enumerated, of which the following are new to the South African fauna :— Vanikoro cancellata, Lamarck. Cyprea Barclayi, Reeve. Pedicularia sicula, Swainson, Puncturella Noachina, Linn. Dentalium politum, Linn. ms Belchert, Sowerby. 5 novemcostatum, Lamarck. 5 plurifissuratum, Sowerby. Scaphander puncto-striatus, Mighels. Marginella fusiformis, Hinds. Mitra cylindracea, Reeve. 5, aedala, Reeve. Murex axicornis, Lamarck, var. ? Trophon carduus, Broderip. Latiaxis tortilis, A. Adams. Ancilla bulloides, Reeve. », angustata, Sowerby. Oniscia Mac Andrewi, Sowerby. Natica Sagraiana, D’ Orbigny, var. Marginella fusiformis, Hinds. Mitra cylindracea, Reeve. Trophon carduus, Broderip. Latiaxis tortilis, A. Adams. », angustata, Sowerby. g y Oniscia Mac Andrewi, Sowerby. y Natica Sagraiana, D’ Orbigny, var. Other species referred to by Mr. South African, are :— Sowerby, but already known as Neptuneopsis pyrrhostoma, Watson. ( ) Ancilla contusa, Reeve. Tritonidea Natalensis, Smith. Pleurotoma (Clavatula) gravis, Hinds. Cyprea similis, Gray. Turritella declivis, Adams & Reeve. Smith, E. A. 1903. "A LIST OF SPECIES OF MOLLUSCA FROM SOUTH AFRICA, FORMING AN APPENDIX TO G. B. SOWERBY'S "MARINE SHELLS OF SOUTH AFRICA."." Proceedings of the Malacological Society of London 5, 354–402. View This Item Online: https://www.biodiversitylibrary.org/item/52297 Permalink: https://www.biodiversitylibrary.org/partpdf/202822 This file was generated 29 March 2024 at 06:45 UTC EXPLANATION OF PLATE XV. Fic. 1. TZerebra Loise, n.sp. Fig. 12. Bittiwm quadricinctum, n.sp. 9, 18. TZurbonilla similans, n.sp. », 2. Dentalium regulare, n.sp. b ll b 5, 14, 15. Siphonaria tenuicostulata, n.sp. », 38 Turbinella truncata, Sowb. Latiaxis rosaceus, 0.sp. Sistrum squamiliratum, D.sp. 5, 6. Nassa Natalensis, n.sp. 18. Oxynoe Natalensis, n.sp. f b »» 7 Clanculus mixtus, n.sp. p 8. Kellia Natalensis, n.sp. b l f y p 19. Fusus Africanus, Sowb. i ll f ll p is, n.sp. 19. Fusus Africanus, Sowb. », 8. Kellia Natalensis, n.sp. », 8. Kellia Natalensis, n.sp. », 9, 10. Corbula rugifera, n.sp. ( ) », 9, 10. Corbula rugifera, n.sp. ll i ( li i ) i Marginella fallax, n.sp. », ll. WNatica(Polinices) Burnupi,n.sp. Purpura pura, D.sp. Copyright & Reuse Copyright Status: Public domain. The BHL considers that this work is no longer under copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's largest open access digital library for biodiversity literature and archives. Visit BHL at https://www.biodiversitylibrary.org. This file was generated 29 March 2024 at 06:45 UTC
https://openalex.org/W2138259354	https://europepmc.org/articles/pmc3923329?pdf=render	English	null	A Case of Tracheal Adenoid Cystic Carcinoma in a Worker Exposed to Rubber Fumes	Annals of occupational and environmental medicine	2,013	cc-by	4,048	* Correspondence: toxneuro@kosha.net Occupational Safety and Health Research Institute, KOSHA, Incheon, Korea © 2013 Kim 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. Kim et al. Annals of Occupational and Environmental Medicine 2013, 25:22 http://www.aoemj.com/content/25/1/22 Kim et al. Annals of Occupational and Environmental Medicine 2013, 25:22 http://www.aoemj.com/content/25/1/22 Kim et al. Annals of Occupational and Environmental Medicine 2013, 25:22 http://www.aoemj.com/content/25/1/22 Open Access Abstract Background: Primary tracheal tumors occur infrequently, accounting for less than 0.1% of all tumors. Adenoid cystic carcinoma (ACC) is the second most common type of malignancy of the trachea after squamous cell carcinoma (SCC). Little has been reported on the risk factors for tracheal ACC. The purpose of this study is to describe a case of tracheal ACC in a patient who had been exposed to rubber fumes, and to review the relationship between tracheal ACC and rubber fumes. Case report: A 48-year-old man who had been experiencing aggravation of dyspnea for several months was diagnosed as having ACC of the trachea on the basis of a pathologic examination of a biopsy specimen obtained via laser microscopy-guided resection. The patient had been exposed to rubber fumes for 10 years at a tire manufacturing factory where he worked until ACC was diagnosed. His job involved preheating and changing rubber molds during the curing process. Conclusion: ACC of both the trachea and the salivary glands show very similar patterns with regard to histopathology and epidemiology and are therefore assumed to have a common etiology. Rubber manufacturing is an occupational risk factor for the development of salivary gland tumors. Further, rubber fumes have been reported to be mutagenic. The exposure level to rubber fumes during the curing process at the patient’s workplace was estimated to be close to or higher than British Occupational Exposure Limits. Therefore, tracheal ACC in this case might have been influenced by occupational exposure to rubber fumes. Keywords: Adenoid cystic carcinoma, Occupational exposure, Rubber fumes, Curing A Case of Tracheal Adenoid Cystic Carcinoma in a Worker Exposed to Rubber Fumes Dohyung Kim, Yang-In Hwang, Sungyeul Choi, Chulyong Park, Naroo Lee and Eun-A Kim* Background Although the specific carcinogen(s) have not yet been identified, several reports have described the mutagenic potential of rubber fumes [12-14]. The purpose of this study is to describe a case of tracheal ACC in a patient who had been exposed to rubber fumes while performing rubber curing in a tire manufacturing factory and to review the relationship between ACC development and exposure to rubber fumes. Although the specific carcinogen(s) have not yet been identified, several reports have described the mutagenic potential of rubber fumes [12-14]. The purpose of this study is to describe a case of tracheal ACC in a patient who had been exposed to rubber fumes while performing rubber curing in a tire manufacturing factory and to review the relationship between ACC development and exposure to rubber fumes. The incidence of primary tracheal carcinoma is 0.10 to 0.26 per 100,000 persons [1-5], accounting for 0.1–0.4% of total malignancy [6]. Adenoid cystic carcinoma (ACC) is the second most common type of primary tracheal tu- mors after squamous cell carcinoma (SCC) [7]. Due to the low incidence rate, little has been reported on the risk factors for tracheal ACC [8,9]. The only risk factors that have been investigated with regard to tracheal ACC are age, sex, and smoking status. Tracheal ACC has been found to develop more frequently in the fourth and fifth decades of life with no gender preference. Further, smoking status was not found to be associated with the development of tracheal ACC [6,10,11]. Case presentation Clinical course A 48-year-old man first visited an otolaryngology clinic in May 2005 because of exertional dyspnea. His symp- toms persisted and deteriorated over several months, and he was found to have a tracheal tumor. At this point, he was transferred to a university hospital. The patient did not have hypertension, diabetes, tuberculosis, viral hepatitis, or HIV infection. Several years earlier, he had undergone an operation for appendicitis. He did not Rubber fumes consist of respirable dust generated during the compounding or curing processes of tire manufacturing. Page 2 of 5 Kim et al. Annals of Occupational and Environmental Medicine 2013, 25:22 http://www.aoemj.com/content/25/1/22 have any known drug allergies. His family did not have a history of respiratory cancer. The patient reported that he had smoked approximately 4 cigarettes a day since he was 23 years old and had drunk 50 mL of Korean dis- tilled spirits per day since he was 20 years old. He had no dyspepsia or resting dyspnea. Visual inspection of the larynx did not reveal any remarkable findings. The Epstein- Barr virus (EBV) antigen/antibody status was not available from the medical records. Figure 1 A worker is transferring and placing a mold by using a forklift. Computed tomography (CT) of the patient’s neck, conducted on May, 2005, revealed a 3.5-cm polypoid soft-tissue tumor at the inner side of the right posterior wall of the subglottic region, extending to the outer side. The tumor blocked the inner cavity of the trachea. Laser microsurgery was conducted for partial excision of the tumor and frozen section biopsy. The final diagnosis on the basis of the frozen biopsy was tracheal ACC, with no extra-tracheal metastasis. Adjuvant radiation therapy was administered twice during next year. A follow-up medical examination was performed 5 years later, during which, chest radiography revealed multiple metastatic nodules in both lung fields. The patient subsequently received palliative chemotherapy. Figure 1 A worker is transferring and placing a mold by using a forklift. close to 95% of the British OEL (0.60 mg/m3) [16]. Other potential respiratory carcinogens that the patient might have been exposed to included N-nitrosamines and poly- cyclic aromatic hydrocarbons (PAHs) [17]. N-nitrosamines had not been measured until 2000 and had always been less than the detection limit. PAHs were measured to range from 1.4 to 22.6 μg/m3, which was less than 10% of the Korean OEL (200 μg/m3) [15]. Case presentation Clinical course Temperatures from 1998 to 2001 exceeded the limits for medium-grade labor intensity (75% of working, 25% of rest, 28°C) [15]. Occupational history h d Annals of Occupational and Environmental Medicine 2013, 25:22 http://www.aoemj.com/content/25/1/22 Page 3 of 5 Table 1 Levels of chemicals in the work environment during the rubber curing process OEL (TWA) 1997* 1998 2000 2001 2002 2003 2004 2005 Total dust 10 mg/m3 NA 0.38 –0.64 0.09–0.91 0.03–0.31 NA NA NA NA Rubber fumes† None‡ NA NA NA NA 0.12–0.31 0.27–0.57 0.05–0.08 0.03–0.30 PAHs 200 μg/m3 NA ND 3.8–11.6 1.4–22.6 3.2–4.4 NA NA NA SO2 2 ppm ND NA NA NA NA 0.04–0.10 0.04–0.09 0.01–0.08 N-Nitrosamine None NA NA NA NA NA ND ND ND 1,3-Butadiene 10 ppm NA NA NA NA ND ND ND ND Heat 28°C NA 32 29.3 30.2 NA 23.0 27.1 26.9 NA: Not available. ND: Non-detectable. OEL: Occupational exposure limit. TWA: Time weighted average. PAHs: Polycyclic aromatic hydrocarbons. Data not available for 1995, 1996, and 1999. †Measurement unit is mg/m3. ‡The Korean OEL is not defined, but the British OEL is 0.60 mg/m3 [16]. Table 1 Levels of chemicals in the work environment during the rubber curing process Table 1 Levels of chemicals in the work environment during the rubber curing process OEL (TWA) 1997 1998 2000 2001 2002 2003 2004 2005 Total dust 10 mg/m3 NA 0.38 –0.64 0.09–0.91 0.03–0.31 NA NA NA NA Rubber fumes† None‡ NA NA NA NA 0.12–0.31 0.27–0.57 0.05–0.08 0.03–0.30 PAHs 200 μg/m3 NA ND 3.8–11.6 1.4–22.6 3.2–4.4 NA NA NA SO2 2 ppm ND NA NA NA NA 0.04–0.10 0.04–0.09 0.01–0.08 N-Nitrosamine None NA NA NA NA NA ND ND ND 1,3-Butadiene 10 ppm NA NA NA NA ND ND ND ND Heat 28°C NA 32 29.3 30.2 NA 23.0 27.1 26.9 NA: Not available g he Korean OEL is not defined, but the British OEL is 0.60 mg/m3 [16 25619-56-1, 7%); however, when these chemicals were ex- posed to temperatures similar to those used during the mold preheating process (180‒195°C ), PAHs were not detected, as assayed by the gas chromatography–mass spectrometry (GC-MSD) analytical method. explanation for higher incidence of salivary gland cancer in rubber industrial workers. Vermeulen et al. [14] used Salmonella typhimurium strains, which overproduce specific enzymes such as nitroreductase and acetyltrans- ferase in the presence of mutagenic material, and pro- posed that some components of rubber fumes may have mutagenic potential, and these may include aromatic amines. Kim B et al. Conclusion Tracheal ACC is known to originate from the salivary glands of the trachea [6]. Therefore, it seems natural that tracheal ACC resembles salivary gland ACC of the head and neck (HNACC) in many ways. The histological simi- larity of tracheal ACC and HNACC has been thoroughly described [8]. Both of them consist of epithelial cells in cribriform, tubular, and solid growth patterns with hya- line or mucoid material-filled extracellular matrix, and show differentiation characteristics such as ductal-lining or myoepitheilal cells [9,18]. These two different cancers are thought to have very similar epidemiological charac- teristics with regard to age, sex, and smoking status, as mentioned earlier in the introduction [18]. The present case was potentially exposed to carci- nogens including PAHs and N-nitrosamines. PAHs are generated by incomplete combustion of organic material and are known to increase the risks of lung cancer and skin cancer [23]. N-nitrosamines are formed in the curing process when chemicals such as tetramethylthiuram disul- fide, zinc diethyldithiocarbamate, or morpholino merca- ptobenzothiazole are used [17]. N-nitrosamines are con- sidered to be carcinogenic to humans on the basis of many experimental animal studies [24]. Among these carcinogens, it seems that the patient had been signifi- cantly exposed only to rubber fumes. Through the working period, the concentrations of other carcinogens such as PAHs and N-nitrosamines were non-detectable or less than 10% of the Korean OEL or even not avail- able (Table 1). However, the unavailable rubber fume concentrations during the first 6 years (1996–2001) could be estimated by using a regression equation pro- posed by Lee et al [13]. Indeed, the estimated maximal value for the concentration of rubber fumes in this period was close to or higher than the British OEL (0.60 mg/m3) [16] (Table 2). These similarities indicate that tracheal ACC and HNACC may have common risk factors. Because HNACC is one of many subtypes of salivary gland tumors of the head and neck, risk factors for such a specific subtype are not well- known [18]. Therefore, it seems reasonable to assume the general risk factors of salivary gland tumors to be the same as those of HNACC. The general risk factors of salivary gland tumors are human immunodeficiency virus infection, Epstein-Barr virus infection, and ionizing ra- diation. It has been shown that workers from a variety of industries have an increased incidence of salivary gland tumors. Occupational history h d [22] reported that once the nano- particles in rubber fumes are deposited in the human body, they can easily translocate to other tissues and have a high toxicological effect due to their small size. Occupational history h d The patient was discharged from the army after 3 years of service (1977–1980) and worked in rice farming (1981–1994). In 1995, he entered tire manufacturing, and was assigned to the curing process, where he worked for 10 years, until 2005. The curing process in- volved the use of 72 vulcanizers (installed with 144 molds). His main duty was preheating and changing molds and bladders. Groups of 3 workers were required to perform the mold-changing job. The workers moved the molds and bladders by using a forklift after the molds had been used several times or if the size of the tire being made needed to be changed (Figures 1 and 2). Molds were changed at a frequency of 3 to 5 times a day, and each change required approximately 1 to 2 hours. Preheating the mold required a high temperature of 180°C to 190°C for 4 to 6 hours. The patient was oc- casionally asked to clean the molds using an airbrush and apply anti-rust agents afterwards. There was a possibility of exposure to PAHs vaporized from anti-rust agents applied to molds due to the high temperature. In addition, cleaning the molds with an air- brush might have generated dust from pipe insulation materials. We analyzed a sample of those materials for the presence of asbestos and PAHs. The insulation mater- ial was found to contain glass fiber. Major components of the anti-rust agents were found to be mineral oil (CAS NO. 64742-55-8, 90%) and barium sulfate (CAS No. Figure 2 A worker is assembling a mold without any respiratory protective equipment. Available environmental measurement data provided by the company included information from 1997 to 2005, but information for 1999 was missing. The risk factor mea- sured by the company was total dust until 2002. The com- pany then changed to measuring rubber fumes (Table 1). During this period, the total dust and rubber fumes were measured to range from 0.03 to 0.91 mg/m3 and 0.03 to 0.57 mg/m3, respectively. The exposure level to total dust was less than 10% of the Occupational Exposure Limits (OEL) determined by the Korean Ministry of Employment and Labor [15]. The exposure level to rubber fumes was Figure 2 A worker is assembling a mold without any respiratory protective equipment. Kim et al. Annals of Occupational and Environmental Medicine 2013, 25:22 http://www.aoemj.com/content/25/1/22 Kim et al. References 7. Gaissert HA: Primary tracheal tumors. Chest Surg Clin N Am 2003, 13(2):247–256. 7. Gaissert HA: Primary tracheal tumors. Chest Surg Clin N Am 2003, 13(2):247–256. 8. Gaissert HA, Mark EJ: Tracheobronchial gland tumors. Cancer Control J Moffitt Cancer Cent 2006, 13(4):286–294. 8. Gaissert HA, Mark EJ: Tracheobronchial gland tumors. Cancer Control J Moffitt Cancer Cent 2006, 13(4):286–294. Despite the above limitations, this study is the first case report that suggests a relationship between expos- ure to rubber fumes and the development of tracheal ACC. However, in order to definitively prove the rela- tionship between rubber fumes and occupational cancer, further epidemiological and experimental studies are necessary. 9. Travis WD, Brambilla E, Muller-Hermelink HK, Harris CC: World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Lung, Pleura, Thymus and Heart. 3rd edition. Lyon: IARC Press; 2004:65. 9. Travis WD, Brambilla E, Muller-Hermelink HK, Harris CC: World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Lung, Pleura, Thymus and Heart. 3rd edition. Lyon: IARC Press; 2004:65. 10. Bhattacharyya N: Contemporary staging and prognosis for primary tracheal malignancies: a population-based analysis. Otolaryngol Head Neck Surg 2004, 131(5):639–642. 10. Bhattacharyya N: Contemporary staging and prognosis for primary tracheal malignancies: a population-based analysis. Otolaryngol Head Neck Surg 2004, 131(5):639–642. 11. Eduardo M, Diaz J, Sturgis EM, Laramore GE, Sabichi AL, Lippman SM, Clayman G: Neoplasms of the Head and Neck [Internet]. 2003 [cited 2013 May 15]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK12895/. In conclusion, tracheal ACC and salivary gland tumor of head and neck have very similar patterns with regard to their histopathology and epidemiology, and may therefore share a common etiology. Rubber manufacturing is con- sidered to be a risk factor of salivary gland tumors, and rubber fumes have been reported to have mutagenic and toxicological properties. Exposure levels of rubber fumes for this case are estimated to have been close to or higher than the British OEL, but those of other potential carcino- gens such as PAHs and N-nitrosamines are not. Although the patient was an ex-smoker, tracheal ACC is not known to be associated with a smoking habit. Besides, this patient was not concluded to have been exposed to other risk factors of salivary gland tumors such as HIV infection or ionizing radiation. Therefore, it is suggested that the development of tracheal ACC in this case may have been influenced by occupational exposure to rubber fumes. Competing interests Competing interests Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. References 12. Kim E-A, Park J, Kim K-H, Lee N, Kim D-S, Kang S-K: Outbreak of sudden cardiac deaths in a tire manufacturing facility: can it be caused by nanoparticles? Saf. Heal. Work 2012, 3(1):58–66. 13. Lee N, Lee B-K, Jeong S, Yi GY, Shin J: Work environments and exposure to hazardous substances in Korean tire manufacturing. Saf Heal Work 2012, 3(2):130–139. 14. Vermeulen R, Bos RP, de Hartog J, van Drooge H, Kromhout H: Mutagenic profile of rubber dust and fume exposure in two rubber tire companies. Mutat Res 2000, 468(2):165–171. 15. Notification of Ministry of Employment and Labor, 2011–13 [Internet]; 2011. Available from: http://www.kosha.or.kr/www/. 15. Notification of Ministry of Employment and Labor, 2011–13 [Internet]; 2011. Available from: http://www.kosha.or.kr/www/. 16. Health and Safety Executive: Workplace exposure limits. London, UK: Health and Safety Executive; 2011. 16. Health and Safety Executive: Workplace exposure limits. London, UK: Health and Safety Executive; 2011. 17. International Agency for Research on Cancer: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 100F. Lyon: IARC Press; 2012:544. 17. International Agency for Research on Cancer: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 100F. Lyon: IARC Press; 2012:544. 18. Barnes L, Eveson JW, Reichart P, Sidransky D: World Health Organization Classification of Tumours. Pathology and Genetics of Head and Neck Tumours. 3rd edition. Lyon: IARC Press; 2005:212–3. 221. 3rd edition. Lyon: IARC Press; 2005:212 3. 221. 19. Guzzo M, Locati LD, Prott FJ, Gatta G, McGurk M, Licitra L: Major and minor salivary gland tumors. Crit Rev Oncol Hematol 2010, 74(2):134–148. 20. Ho K, Lin H, Ann DK, Chu PG, Yen Y: An overview of the rare parotid gland cancer. Head Neck Oncol 2011, 3:40. y 19. Guzzo M, Locati LD, Prott FJ, Gatta G, McGurk M, Licitra L: Major and minor salivary gland tumors. Crit Rev Oncol Hematol 2010, 74(2):134–148. 20. Ho K, Lin H, Ann DK, Chu PG, Yen Y: An overview of the rare parotid gland cancer. Head Neck Oncol 2011, 3:40. 21. Mancuso TF, Brennan MJ: Epidemiological considerations of cancer of the gallbladder, bile ducts and salivary glands in the rubber industry. J Occup Med 1970, 12(9):333–341. Received: 21 May 2013 Accepted: 4 October 2013 Published: 17 October 2013 Received: 21 May 2013 Accepted: 4 October 2013 Published: 17 October 2013 Authors’ contributions KDH and KEA conceived and designed the study. KDH, CSY and HYI were involved in conduction of the study. LNR and HYI performed the analysis and estimation of the environmental assessment. PCY and KEA interpretation of data. KEA, KDH, PCY, LNR and HYI were involved in writing the manuscript. KBG and YSD performed the revision the manuscript. All authors read and approved the final manuscript. References was not available. This forced us to rely on estimations based on statements from the patient and measurements from a tire manufacturing company in a previous study. Second, we presumed that the risk factors for head and neck salivary gland tumors and tracheal ACC are similar, which needs more thoughtful consideration. Third, it is known that the chemical composition analysis of rubber fumes is difficult and that data about their harmful effects are scanty [22], so we cannot but explain a biological plausibility for their carcinogenesis insufficiently. Fourth, some information about other possible risk factors such as EBV was not available, so we could not evaluate its effect on this case. 1. Urdaneta AI, Yu JB, Wilson LD: Population based cancer registry analysis of primary tracheal carcinoma. Am J Clin Oncol 2011, 34(1):32–37. 1. Urdaneta AI, Yu JB, Wilson LD: Population based cancer registry analysis of primary tracheal carcinoma. Am J Clin Oncol 2011, 34(1):32–37. 2. Honings J, van Dijck JAAM, Verhagen AFTM, van der Heijden HFM, Marres HAM: Incidence and treatment of tracheal cancer: a nationwide study in the Netherlands. Ann Surg Oncol 2007, 14(2):968–976. 2. Honings J, van Dijck JAAM, Verhagen AFTM, van der Heijden HFM, Marres HAM: Incidence and treatment of tracheal cancer: a nationwide study in the Netherlands. Ann Surg Oncol 2007, 14(2):968–976. g 3. Manninen MP, Antila PJ, Pukander JS, Karma PH: Occurrence of tracheal carcinoma in Finland. Acta Otolaryngol (Stockh.) 1991, 111(6):1162–1169. 4. Licht PB, Friis S, Pettersson G: Tracheal cancer in Denmark: a nationwide study. Eur J Cardio-Thorac Surg 2001, 19(3):339–345. g 3. Manninen MP, Antila PJ, Pukander JS, Karma PH: Occurrence of tracheal carcinoma in Finland. Acta Otolaryngol (Stockh.) 1991, 111(6):1162–1169. 3. Manninen MP, Antila PJ, Pukander JS, Karma PH: Occurrence of tracheal carcinoma in Finland. Acta Otolaryngol (Stockh.) 1991, 111(6):1162–1169. 4. Licht PB, Friis S, Pettersson G: Tracheal cancer in Denmark: a nationwide study. Eur J Cardio-Thorac Surg 2001, 19(3):339–345. y g 4. Licht PB, Friis S, Pettersson G: Tracheal cancer in Denmark: a nationwide study. Eur J Cardio-Thorac Surg 2001, 19(3):339–345. 4. Licht PB, Friis S, Pettersson G: Tracheal cancer in Denmark: a nationwide study. Eur J Cardio-Thorac Surg 2001, 19(3):339–345. 5. Webb BD, Walsh GL, Roberts DB, Sturgis EM: Primary tracheal malignant neoplasms: the university of Texas MD Anderson cancer center experience. J Am Coll Surg 2006, 202(2):237–246. g 6. Macchiarini P: Primary tracheal tumours. Lancet Oncol 2006, 7(1):83–91. Conclusion These include rubber manufacturing, hairdressing, and industries with exposure to nickel compounds [18-20]. This study has some limitations. We aimed to describe a relationship between rubber fume exposure and the development of tracheal ACC. First, for the 10-year ex- posure period, information regarding the first 6 years Mancuso et al. [21] suggested that nitroso compounds caused salivary gland tumors in laboratory mice, and the presence of nitroso compounds in rubbers could be an Page 4 of 5 Page 4 of 5 Kim et al. Annals of Occupational and Environmental Medicine 2013, 25:22 http://www.aoemj.com/content/25/1/22 Kim et al. Annals of Occupational and Environmental Medicine 2013, 25:22 http://www.aoemj.com/content/25/1/22 Table 2 Maximal concentrations of rubber fumes during the rubber curing process as estimated by a regression equation* Table 2 Maximal concentrations of rubber fumes during the rubber curing process as estimated by a regression equation* 1996 1997 1998 1999 2000 2001 Total dust† NA NA 0.64 NA 0.91 0.31 Rubber fumes‡ NA NA 0.57 NA 0.79 0.30 NA: Not available. *y = 0.81x + 0.049 (x = total dust, y = concentration of rubber fumes) adapted from Lee et al. [13]. †Measured concentration in mg/m3. ‡Estimated concentration in mg/m3. Consent Written informed consent was obtained from the pa- tient’s guardian/parent/next of kin for the publication of this report and any accompanying images. 22. Kim B, Lee JS, Choi BS, Park SY, Yoon JH, Kim H: Ultrafine particle characteristics in a rubber manufacturing factory. Occup Hyg 2013, 57(6):728–739. 22. Kim B, Lee JS, Choi BS, Park SY, Yoon JH, Kim H: Ultrafine particle characteristics in a rubber manufacturing factory. Occup Hyg 2013, 57(6):728–739. Page 5 of 5 Page 5 of 5 Kim et al. Annals of Occupational and Environmental Medicine 2013, 25:22 http://www.aoemj.com/content/25/1/22 23. Straif K, Baan R, Grosse Y, Secretan B, El Ghissassi F, Cogliano V: WHO international agency for research on cancer monograph working group. Carcinogenicity of polycyclic aromatic hydrocarbons. Lancet Oncol 2005, 6(12):931–932. 24. Hecht SS: Approaches to cancer prevention based on an understanding of N-nitrosamine carcinogenesis. Proc Soc Exp Biol Med 1997, 216(2):181–191. doi:10.1186/2052-4374-25-22 Cite this article as: Kim et al.: A Case of Tracheal Adenoid Cystic Carcinoma in a Worker Exposed to Rubber Fumes. Annals of Occupational and Environmental Medicine 2013 25:22. doi:10.1186/2052-4374-25-22 Cite this article as: Kim et al.: A Case of Tracheal Adenoid Cystic Carcinoma in a Worker Exposed to Rubber Fumes. Annals of Occupational and Environmental Medicine 2013 25:22. 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https://openalex.org/W137979270	https://hal.inria.fr/hal-01457456/file/978-3-642-41151-9_25_Chapter.pdf	English	null	Development of a Catchment Management Tool to Assess Environmental Risk from Nutrient Loadings Using Open Source GIS	IFIP advances in information and communication technology	2,013	cc-by	3,336	Development of a Catchment Management Tool to Assess Environmental Risk from Nutrient Loadings using Open Source GIS Ian Packham, Eva Mockler, and Michael Bruen UCD Dooge Centre for Water Resource Research, School of Civil, Structural and Environmental Engineering, University College Dublin, Belfield, Dublin 4, Ireland {i.packham, eva.mockler, michael.bruen}@ucd.ie Abstract. A Catchment Management Tool (CMT) is being developed for the Irish Environmental Protection Agency (EPA) that will allow River Basin Man- agers to evaluate the environmental risk from organic and inorganic nutrient loadings due to various land uses and human activities. The CMT was built us- ing Open Source GIS (Geographical Information System) software, to facilitate development and widen the research user-base of the tool. The user interface of the CMT is designed to be flexible to allow local knowledge to be included in the system as well as the possibility to try what-if scenarios in relation to envi- ronmental assessment. Contaminant loadings are calculated from publicly available data and hydrogeologically susceptible areas (HSAs) are formed by combining soil and geological GIS layers. Further combinations of Loadings and HSAs allow Critical Source Areas to be delineated that identify areas con- tributing significant amounts of a given contaminant to any selected water body. Keywords: Catchment Management, Critical Source Areas, Open Source GIS Software, Contaminant Loadings, Hydrogeology, Susceptibility Keywords: Catchment Management, Critical Source Areas, Open Source GIS Software, Contaminant Loadings, Hydrogeology, Susceptibility 1 Introduction 1 In response to the European Union’s Water Framework Directive (WFD) require- ments that River Basin Districts meet good quality status by 2015, the Irish Environ- mental Protection Agency (EPA) has commissioned a Catchment Management Tool (CMT) to assist in planning its activities. The CMT is one of the outputs of the Path- ways Project, a multidisciplinary collaborative project that aims to improve under- standing of hydrological and hydrogeological pathways of water-borne nutrient transport. The knowledge gained from this and other recent projects will inform the CMT and in particular the evaluation of environmental risk due to nutrient loadings. This paper begins by describing the development of the Catchment Management Tool using Open Source Geographical Information System (GIS) software. Open Source software was chosen during the design and development stage of the CMT for fast, accessible ongoing software development and to widen the potential research user-base of the tool. Then the paper describes the data sources and methods used to compute the nutrient loadings for a chosen catchment and thus how the various in- formation sources are combined to produce Critical Source Areas of environmental risk. The paper concludes with potential further applications of the CMT, such as varying loadings due to land-use change and other what-if scenarios. The flexible nature of the CMT is designed to help river basin managers select appropriate measures to achieve good WFD status. 1.1 Critical Source Areas Critical Source Areas are defined as those areas that contribute a disproportionally high pollutant loading to the receiving river, and occur where high loadings coincide with hydrogeologically susceptibility areas (HSAs). HSAs are those areas that are sensitive to a given contaminant (such as the nutrients nitrogen or phosphorous) in relation to a given receptor (such as groundwater or stream/lake). The coincidence of a sensitive area with the likelihood of high loading of that contaminant indicates a possible environmental risk. When defined in this way, critical source areas can be delineated using GIS layers and appropriate factors. 2.1 Free and Open Source Software for Geospatial Open Source software often allows for a flexible development environment because the software in question is often designed to be easy to set up and interact with other software [1]; in contrast to proprietary software that is often designed as a stand-alone system. The Open Source Geospatial Foundation (OSGeo [2]) is the umbrella organi- zation that supports collaborative projects and organizes the annual international Free and Open Source Software for Geospatial (FOSS4G) conference. As well as the pro- jects overseen by OSGeo, there are a large number of other GIS projects listed at [3] and [4] and reviewed, for example, in [5]. The GIS desktop viewer chosen for the Pathways CMT was Quantum GIS (QGIS) [6] which can view multiple sources of GIS data, such as ESRI shapefiles, Raster files and databases. The principal data source is the PostgreSQL [7] database which can be spatially enabled using PostGIS [8]. The advantage of using such a database is that the available data can be processed and stored for viewing, in this case via the QGIS Python plugin architecture. Working copies of data can be saved as individual pro- jects and updated to simulate separately different scenarios. Other Open Source soft- ware combinations are available, but the QGIS / PostGIS pairing was seen as the most flexible option for the Pathways project. 2.2 Three Levels of Interaction To allow for different levels of prior knowledge and GIS experience for the users, a range of levels of interaction is provided in the user interface, see Fig. 1. Level 1: Source Layers. At the first level of interaction, the user can choose the basic source data to view as GIS-layers for the catchment of interest, such as soil properties, geology and land-use as well as potential point sources of contaminants. This data is pre-processed so the user can easily view data of interest with minimal interaction; summary reports and statistics are readily available. Level 2: Static Analysis Layers. At the next level of interaction, users can choose a specific catchment and view in detail the loadings and static critical source areas formed by combining the Level 1 layers. Initially the data is created automatically from default data and parameters, but the user can choose to update this data, if they have more local knowledge or experience of the actual processes involved. Level 3: Dynamic Analysis Layers. The final level of interaction requires the user to input hydrological data specific to the catchment. The more complicated lateral flows are modeled using a rainfall-runoff model, producing dynamic critical source areas. Fig. 1. CMT user interaction diagram Fig. 1. CMT user interaction diagram 2.3 Flexible Software Design The software was designed as a loosely coupled system so that individual modules can be developed independently and replaced if necessary in the future. The QGIS software is used to view the GIS data; Fig. 2 shows an example, on the left hand side is the layer legend. A plugin written in the Python language lets the user choose which data to load from the PostGIS database (right hand side of Fig. 2). The Python code uses the QGIS application programming interface (API) to define different types of data needed for the CMT plugin and loads this via the database API into QGIS. Summary data and statistics of the relevant layers are easily computed in the plugin. The data has been preprocessed to speed up access for Level 1 Source Layers but also to allow combinations of data sources to be intersected “on the fly” (created dynami- cally) for Level 2 Static Analysis Layers. New data tables are created in the database using SQL (structured query language) and PostGIS spatial functions. Fig. 2. Screenshot of the CMT – note usual GIS tools are provided by QGIS, the plugin to the right allows the user to choose data layers and view summary statistics of those layers below Fig. 2. Screenshot of the CMT – note usual GIS tools are provided by QGIS, the plugin to the right allows the user to choose data layers and view summary statistics of those layers below Some tables in Level 2 are copies of Level 1 source layers and can be edited by the user. Functionality to change the underlying data is available in QGIS and many other desktop GIS systems. The user might modify the data because, for example, they might know that the in-built default loading values are not appropriate and should be changed or want to simulate changes in, for instance, land-use. After updating the data source, all the other related data tables can be updated from one button click and the outcome of those changes will be immediately seen. For Level 3, the Pathways Computational Engine (PACE) has been written as a standalone FORTRAN package [9]. Communication with the CMT and database is undertaken only in the form of reading and writing text files. 2.3 Flexible Software Design This loose coupling means that the Computational Engine can be developed in parallel with the GIS com- ponent and can be amended without affecting the rest of the CMT. 3.1 Source Layers Geological and soil mapping information were supplied by the EPA and GSI (Geo- graphical Survey of Ireland) in digital form. Derivatives of the EPA / Teagasc (Irish Agriculture and Food Development Authority) soil maps [10] include soil drainage and subsoil permeability. Other relevant maps include aquifer type, groundwater vul- nerability [11], Corine land-cover, slope and lakes, a number of these were combined to make the national recharge map [12]. Point sources such as landfills, water treat- ment plants and registered IPPC (integrated pollution prevention and control) facili- ties are also included as well as possible hazards such as abstractions and quarries. These maps are all available to view in Level 1 of the CMT (Fig. 2). 3.2 Calculating Nutrient Loadings In the CMT, contaminant loadings to land surfaces are calculated from publicly avail- able data from the Irish Central Statistics Office (CSO). Population figures and the number of houses and type of sewage system that serves each house [13] are availa- ble. Land use and the number and type of animals in an Electoral District is provided by the Census of Agriculture [14] which will soon be updated from 2000 to 2010 data. This data is then used to calculate nutrient (Nitrogen and Phosphorous) loading to different hydrological pathways using the Groundwater Task Team (GTT) [15] methodology, modified for Irish settings by the EPA. Fig. 3. Example of total input Nitrate loading to the Mattock_mid catchment, on the border of Co Louth and Co Meath in Ireland; inset shows averages broken down by land-use Fig. 3. Example of total input Nitrate loading to the Mattock_mid catchment, on the border of Co Louth and Co Meath in Ireland; inset shows averages broken down by land-use Fig. 3. Example of total input Nitrate loading to the Mattock_mid catchment, on the border of Co Louth and Co Meath in Ireland; inset shows averages broken down by land-use The hydrological pathways are modeled at river basin sub-catchment level (aver- age area 13 km2), so intersection of these with the Irish Electoral District (average area 20 km2) gives a coarse variation in loadings across the chosen catchment. An example output of the loadings calculated for nitrate by the CMT is shown in Fig. 3, including a statistical summary. The GTT methodology also models, in an empirical way, the processes of the nu- trient going through the soil and to the particular receptor that is required to model attenuation processes. For example leaching in Grassland Pastures is calculated using NCycle_IRL [16]. Leaching of Nitrogen is related to soil drainage or texture, so the combination of the loadings map (Fig. 3) and the soil drainage map (Fig. 2) are used to create a loading map of N from soil drainage (not shown). 3.3 Creating Hydrogeologically Susceptible Areas (HSAs) Hydrogeologically susceptible areas are those areas that are sensitive to a given nutri- ent reaching a given water body receptor. Creating these layers requires identifying the soil or geological properties that are relevant through literature review, expert opinion or empirical analysis. For example, for nitrogen to groundwater, the combina- tion of subsoil permeability and depth to bedrock is used to determine ranges of sus- ceptibility. The EPA has published a risk-based methodology for single house treat- ment system that includes a number of susceptibility factors [17]; they also consider denitrification in bedrock for the Groundwater pathway. These factors are being re- viewed and modified for use in the CMT. Fig. 4. Hydrogeologically Susceptible Areas (HSAs) of Nitrogen to the Groundwater receptor for the Mattock_mid catchment Fig. 4. Hydrogeologically Susceptible Areas (HSAs) of Nitrogen to the Groundwater receptor for the Mattock_mid catchment Fig. 4 shows an example of the HSAs derived for the Mattock catchment, in the North East of Ireland, for Nitrogen to groundwater. Very High and High susceptibility relate to areas of extreme vulnerability (subsoil less than 3m subsoil thick) or highly permeable subsoils. These categories can be given quantitative risk values which in effect are the attenuation rates of nitrogen to groundwater. Susceptibility maps for phosphorous to groundwater can be created using the same procedure. Susceptibility to surface water receptors is more problematic as no tem- poral variation is considered when using the GIS layers. Multiple receptors might also exist in the catchment (in terms of lakes and streams), so only the loading, mobiliza- tion and delivery to the local surface water receptor is being considered. The compu- tational engine in Level 3 of the CMT has the capabilities to model the lateral flow and temporal dynamics required for generic surface water receptors. 3.4 Critical Source Areas (CSAs) By combining the Loadings information with the Hydrogeological Susceptibility in- formation it is possible to create Critical Source Area (CSA) maps for each contami- nant. When intersecting the maps, the loadings rates from soil drainage can be multi- plied by the attenuation rates or other factors derived from the susceptibility maps. The amount of loading that reaches groundwater also needs to take into account as well as the proportion of effective rainfall involved. Fig. 5. Critical Source Areas for Nitrogen to Groundwater receptor from diffuse sources; this is final loading after attenuation for Mattock_mid catchment; inset shows averages Fig. 5. Critical Source Areas for Nitrogen to Groundwater receptor from diffuse sources; this is final loading after attenuation for Mattock mid catchment; inset shows averages Fig. 5. Critical Source Areas for Nitrogen to Groundwater receptor from diffuse sources; this is final loading after attenuation for Mattock_mid catchment; inset shows averages Fig. 5. Critical Source Areas for Nitrogen to Groundwater receptor from diffuse sources; this is final loading after attenuation for Mattock_mid catchment; inset shows averages The national recharge map [12] is available within the CMT which includes effec- tive rainfall and recharge coefficient. The latter coefficient determines the amount of effective rainfall that will reach the groundwater depending on the hydrogeological category of the area. Most of the Mattock catchment shown in the figures is underlain by a “non-productive” aquifer, that is, it does not have the storage capacity or trans- mission capability to supply more than a few houses. This is simulated in the Re- charge Map by a “cap” that limits the amount of water that the aquifer will accept. The recharge cap has an additional effect on the model output as it is assumed that excess rainfall is effectively rejected by the aquifer. In the CMT, a modified recharge coefficient is used that takes into account the recharge cap for non-productive aqui- fers. Fig. 5 shows the Critical Source Areas to groundwater after combining loadings with soil drainage, the attenuation factors and modified recharge coefficients. 4 Further Applications and What-if Scenarios The CMT is designed to be user friendly by pre-processing as much of the available data as possible so an initial overview can be evaluated by the user or River Basin District Manager very quickly. If the Manager is aware, from local knowledge, of inaccuracies in the underlying loadings data or subsequent output, then changes to the input data and parameters are allowed because copies of the original data can be saved under a different project name. This flexibility also allows for what-if scenarios to be generated as the user can change the loadings data to simulate an increase in, for instance, inorganic fertilizer or leakage at a waste water treatment facility, for exam- ple. Also a change in land-use could be simulated by allowing changes to the copy of the Corine land-cover layer. So a change in arable land to urban could be simulated or vice versa. It might also be possible to simulate the effects of climate change by ad- justing meteorological inputs or changing the properties of the soil or geology layers and analyze the outcome in the Critical Source Areas. 5 Conclusions A Catchment Management Tool (CMT) is under development for the Irish EPA. The user interface of the CMT is designed to be flexible to allow local knowledge to be included in the system as well as the possibility to try what-if scenarios in relation to environmental assessment. Contaminant loadings are calculated from publicly availa- ble data and hydrogeologically susceptible areas (HSAs) are formed by combining soil and geological GIS layers. Further combination of Loadings and HSAs allow Critical Source Areas to be delineated that identify high risk areas from a given con- taminant to a receptor of interest. This paper has described the software architecture of the CMT and the data sources required for the loadings and geological susceptibility layers. Examples of critical source area maps produced from the CMT were shown. Changes in those maps due to user interaction can be used to simulate the changes in environmental risk due to pos- sible future scenarios. Initial feedback suggests the tool will be useful for the EPA and River Basin District Managers; further user testing and validation of the loading and attenuation rates is required before final release. sible future scenarios. Initial feedback suggests the tool will be useful for the EPA and River Basin District Managers; further user testing and validation of the loading and attenuation rates is required before final release. Acknowledgments. The Pathways Project is an EPA STRIVE funded project (2007- W-CD-1-S1), the collaborative partners are Queens University Belfast, University College Dublin and Trinity College Dublin. The authors would also like to thank the project Steering Committee for invaluable discussion and advice. References 1. Sanz-Salinas, J.-G. & Montesinos-Lajara, M.: Current Panorama of the FOSS4G Ecosys- tem. Novatica, X(2), 43–51 (2009) 2. Open Source Geospatial Foundation, http://www.osgeo.org 2. Open Source Geospatial Foundation, http://www.osgeo.org 3. Open Source GIS, http://www.opensourcegis.org 3. Open Source GIS, http://www.opensourcegis.org 4. Free and Open Source GIS, http://www.freegis.org 5. Steiniger, S. and Bocher, E.: An Overview on Current Free and Open Source Desktop GIS Developments. Int. J. of Geographical Information Science 23 (10) 1345–1370 (2008) 5. Steiniger, S. and Bocher, E.: An Overview on Current Free and Open Source Desktop GIS Developments. Int. J. of Geographical Information Science 23 (10) 1345–1370 (2008) 6. Quantum GIS: http://www.qgis.org 7. PostgreSQL: http://www.postgresql.org 7. PostgreSQL: http://www.postgresql.org 8. PostGIS: http://www.postgis.org 9. Mockler, E. & Bruen, M.: A variable-structure catchment model as the engine for a water quality decision support tool facilitating scientific debate and collaboration. In Seppelt, R., Voinov, A.A., Lange, S., Bankamp, D. (eds) International Congress on Environmental Modelling and Software - iEMSs2012 (2012) 10. Fealy, R. M., Green, S., Loftus, M., Meehan, R., Radford, T., Cronin, C. and Bulfin, M.: Teagasc EPA Soil and Subsoils Mapping Project-Final Report. Volume I, Teagasc, Dublin (2009) 11. DELG/EPA/GSI: Groundwater Protection Schemes. Department of the Environment and Local Government, Environmental Protection Agency and Geological Survey of Ireland (1999) 12. Hunter Williams, N., Misstear, B., Daly D., Johnston, P., Lee, M., Cooney, P., Hickey, C.: A National Groundwater Recharge Map for Ireland, National Hydrology Conference (2011) 13. CSO, Small Area Population Statistics (SAPS): (2006): http://census.cso.ie/census/ReportFolders/ReportFolders.aspx 13. CSO, Small Area Population Statistics (SAPS): (2006): http://census.cso.ie/census/ReportFolders/ReportFolders.aspx 14. CSO, Census of Agriculture 2000 Detailed Results: (2000): http://www.cso.ie/px/pxcoa2000/database/census of agriculture 2000/census of agriculture 2000.asp http://www.cso.ie/px/pxcoa2000/database/census of agriculture 2000/census of agriculture 2000.asp 15. Groundwater Task Team: Cumulative Nitrogen and Phosphorus Loadings to Groundwa- ter, produced by ENTEC on behalf of the Scottish Environment Protection Agency, the Environment Agency (England & Wales), the Environmental Protection Agency and the Northern Ireland Environment Agency (2010) g y 16. del Prado, A., Scholefield, D., Brown, L.: NCYCLE_IRL: A model to predict N fluxes in Irish grasslands. Environmental Protection Agency, Ireland (2005) 17. EPA: A risk-based methodology to assist in the regulation of Domestic Waste Water Treatment Systems, Environmental Protection Agency, Ireland (2013)
https://openalex.org/W2899648588	https://www.preprints.org/manuscript/201811.0251/v1/download	English	null	A Comparative Study of Advanced Stationary Phases for Fast Liquid Chromatography Separation of Synthetic Food Colorants	Molecules/Molecules online/Molecules annual	2,018	cc-by	4,358	Ivona Lhotská 1, Petr Solich 1 and Dalibor Šatínský 1,* Ivona Lhotská 1, Petr Solich 1 and Dalibor Šatínský 1,* Ivona Lhotská 1, Petr Solich 1 and Dalibor Šatínský 1,* 1 Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, Hradec Králové 500 05, Czech Republic; lhotski@faf.cuni.cz; solich@faf.cuni.cz; satinsky@faf.cuni.cz * Correspondence: satinsky@faf.cuni.cz; Tel.: +420-495-067-228 Abstract: Food analysis demands fast methods for routine control and high throughput of samples. Chromatographic separation enables simultaneous determination of numerous compounds in complex matrices, several approaches increasing separation efficiency and speed of analysis were involved. In this work, modern types of column with monolithic rod or superficially porous particles were employed and compared for determination of eight synthetic food dyes, their chromatographic performance was evaluated. During method optimization, cyano stationary phase Chromolith Performance CN 100 × 4.6 mm and Ascentis Express ES-CN 100 × 4.6 mm, 5 µm were selected for the separation of polar colorants. The separation was performed by gradient elution of acetonitrile/methanol and 2% water solution of ammonium acetate at flow rate 2.0 ml min-1. Mobile phase composition and the gradients were optimized in order to enable efficient separation on both columns. The method using fused-core particle column provided higher separation efficiency, narrow peaks of analytes resulted in increased peak capacity and shortening of analysis time. After the validation, the method was applied for analysis of colored beers, soft drinks and candies. Keywords: monolithic column; porous shell column; food additive; dye; food colorant; chromatography; fast chromatography. org) \| NOT PEER-REVIEWED \| Posted: 9 November 2018 org) \| NOT PEER-REVIEWED \| Posted: 9 November 2018 doi:10.20944/preprints201811.0251.v1 Article Article A comparative study of modern stationary phases for fast liquid chromatography separation of synthetic food colorants Ivona Lhotská 1, Petr Solich 1 and Dalibor Šatínský 1,* 1 Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, Hradec Králové 500 05, Czech Republic; lhotski@faf.cuni.cz; solich@faf.cuni.cz; satinsky@faf.cuni.cz * Correspondence: satinsky@faf.cuni.cz; Tel.: +420-495-067-228 Ivona Lhotská 1, Petr Solich 1 and Dalibor Šatínský 1,* 1 Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, Hradec Králové 500 05, Czech Republic; lhotski@faf.cuni.cz; solich@faf.cuni.cz; satinsky@faf.cuni.cz * Correspondence: satinsky@faf.cuni.cz; Tel.: +420-495-067-228 1. Introduction Food colorants are one of the food additives used extendedly, especially in beverages and candies. There are various natural colors, however their tint is dependent on pH and the color can be lost during time and storage conditions [1]. Hence, the artificial dyes obtained by chemical synthesis from highly purified oil products are used with advantage as those colorants are more stable and in advance cheaper. Recently, there is a trend to return to natural origin of colorants in high-class products or intended for children consumption as the customers demand according to the latest findings [2]. Increased attention was paid to adverse effects of synthetic food colorants and their long-term toxicity. Several food dyes demonstrated positive results in genotoxicity tests [3, 4], but usually the tested concentrations overtop levels present in food. Some azo dyes have already been banned due to their carcinogenic degradation products [5], azo dyes tartrazine and sunset yellow are suspected from xenoestrogenic behavior [6]. Although remaining controversial, using of synthetic coloration seems to be safe, meeting the recommended acceptable daily intake (ADI) values [1], which are exposed to legislative limits [7]. However even with low consumption of colorings, hypersensitive reactions may occur in susceptible individuals. Many allergic and other immune reactive disorders have been reported [8-10]. Moreover, behavioral changes depending on diet are observed in sensitive children [9]. Ingestion of great amount of foreign antigenic load, such as sugar, synthetic food colorants and other additives has been connected with development of attention and hyperactivity disorders in children. Reasonably, the synthetic colors application and used concentration in food doi:10.20944/preprints201811.0251.v1 should be controlled. The food additives contained in particular foodstuff have to be listed on label and there are specified concentrations for each product [7]. There are plenty of possibilities for dyes determination as they have varying physico-chemical properties. Water-soluble food colorants such as azo-dyes, triarylmethane dyes, quinopthalone, or indigoid dyes differ in lipophilicity and pKa values. Oxidation of some dyes can be used for electrochemical determination [11]. The sensitive simultaneous determination of colorants has been achieved by capillary electrophoresis [12], and even miniaturized electrophoresis on a chip [13]. Spectrophotometry could be method of choice for analyzing colorful substances. However, their simultaneous determination is very limited due to the similar spectral characteristics [14, 15]. On the other hand, spectrophotometric detection is extensively utilized mainly in combination with chromatographic separation of analytes. 2.1. Method development Most of the parameters were optimized on the monolithic column first. The middle polar cyano stationary phase was chosen as it showed good selectivity for mostly polar colorants. In advance, a mobile phase with high ionic strength containing ammonium acetate showed great influence of ‘salting out effect’ enabling increased interaction of compounds with stationary phase and thus good retention of the more polar colorants. The resolution of critical pairs of peaks (Tartrazine – Indigo carmine; Green S – Patent blue; Brilliant blue – Fast green) was finally solved with raising the concentration of ammonium acetate from 0.1% up to 2% and gradient elution program with methanol. Low back pressure due to lower mass transfer resistance of monolith enabled to use a flow rate 2 mL min-1. Afterwards, fused-core particle column was taken of similar parameters (cyano stationary phase, 10 cm length, and 4.6 mm diameter), the same buffer, flow rate and injection volume 10 µL. However, original gradient was optimized and it was taken advantage of acetonitrile in mobile phase for its lower viscosity, higher elution power and better resolution of peaks on this stationary phase. 1. Introduction After appropriate pre-extraction and clean-up, it is a very common and versatile method for simultaneous determination of numerous colorings in complex food matrices. Despite of some recent interesting applications [16], thin-layer chromatography has been almost superseded by liquid chromatography (HPLC-DAD) [17-23], similarly very fast and simple method of sequential injection chromatography has been reported [24]. Nowadays, in many cases mass spectrometry detection with mass confirmation has been involved [25, 26]. In this study, two methods using different technology of stationary phase for determination of food synthetic colors in drinks and candies were developed and validated. The chromatography columns packed with monolithic sorbent and superficially porous particles as the two important approaches in current trends in fast chromatography were chosen for comparison of their chromatographic performance. Those types of packing materials represent development in liquid chromatography providing higher separation efficiency and the speed of analysis [27]. 2.2. Method validation and comparison Two methods for determination of food synthetic colors employing either monolithic sorbent or superficially porous particles columns were developed, their chromatographic performances were evaluated. Comparison was performed in terms of repeatability, peak symmetry, peak capacity and linear range (column loading capacity). Peak capacity representing separation efficiency parameter is calculated from peak width. As can be seen in Figures 1 and 2 and according the values in Table 1, fused-core column in combination with acetonitrile in mobile phase provided very narrow peaks, in shorter gradient time, leading to higher peak capacity. The analytes were concentrated in narrow zones represented by narrow peaks with higher response. That lowered limit of detection to 0.2 mg L-1; but evidentially the detector was overloaded soon (upper limit of detection 100 mg L-1), although in case of higher concentrations, simple dilution of sample would solve that. Figure 1. Chromatogram of 8 food synthetic dyes separation on monolithic column Chromolith Performance CN 100 × 4.6 mm with guard column Chromolith CN 5 × 4.6 mm. Gradient elution with mobile phase comprising of (A) methanol and (B) water solution of 2% acetic acid (pH 7.0). Detection of individual dyes using the wavelength according to their maximal absorption. reprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 November 2018 doi:10.20944/preprints201811.0251.v1 Peer-reviewed version available at Molecules 2018, 23, 3335; doi:10.3390/molecules23123335 doi:10.20944/preprints201811.0251.v1 Figure 1. Chromatogram of 8 food synthetic dyes separation on monolithic column Chromolith Performance CN 100 × 4.6 mm with guard column Chromolith CN 5 × 4.6 mm. Gradient elution with mobile phase comprising of (A) methanol and (B) water solution of 2% acetic acid (pH 7.0). Detection of individual dyes using the wavelength according to their maximal absorption. Figure 1. Chromatogram of 8 food synthetic dyes separation on monolithic column Chromolith Performance CN 100 × 4.6 mm with guard column Chromolith CN 5 × 4.6 mm. Gradient elution with mobile phase comprising of (A) methanol and (B) water solution of 2% acetic acid (pH 7.0). Detection of individual dyes using the wavelength according to their maximal absorption. Figure 2. Chromatogram of 8 food synthetic dyes separation on fused-core column Ascentis Express ES-CN 100 × 4.6 mm, 5 µm. Gradient elution with mobile phase comprising of (A) acetonitrile and (B) water solution of 2% acetic acid (pH 7.0). Detection of individual dyes using the wavelength according to their maximal absorption. Table 1. 2.2. Method validation and comparison System suitability test results Analyte λ (nm) tR (min)a Repeatability whb Figure 2. Chromatogram of 8 food synthetic dyes separation on fused-core column Ascentis Express ES-CN 100 × 4.6 mm, 5 µm. Gradient elution with mobile phase comprising of (A) acetonitrile and (B) water solution of 2% acetic acid (pH 7.0). Detection of individual dyes using the wavelength according to their maximal absorption. Pcc doi:10.20944/preprints201811.0251.v1 tR , RSD (%)d Peak area, RSD (%)e Peak symmetry Monolithic column Tartrazine 420 0.95 0.3 0.4; 0.1; 0.9 2.47 0.12 22.37 Indigo carmine 625 1.18 0.3 1.9; 0.4; 1.1 1.38 0.12 21.83 Sunset yellow 482 1.99 0.2 0.4; 0.1; 0.8 1.32 0.12 21.49 Fast green 625 6.53 0.1 0.3; 0.6; 0.7 1.19 0.12 22.37 Brilliant blue 625 6.91 0.1 0.3; 0.4; 0.5 1.09 0.12 21.66 Green S 625 7.67 0.1 1.6; 0.9; 1.0 1.53 0.16 16.72 Patent blue 625 7.96 0.1 0.7; 2.1; 0.6 2.32 0.18 15.12 Quinoline yellow 420 9.44 0.1 1.3; 0.4; 4.5 1.29 0.11 22.93 Fused-core column Tartrazine 420 1.51 0.2 1.5; 1.8; 1.0 1.60 0.02 60.38 Indigo carmine 625 1.74 0.2 1.7; 1.8; 1.5 1.82 0.03 48.50 Sunset yellow 482 2.05 0.1 1.6; 1.8; 1.3 1.77 0.03 50.14 Green S 625 3.26 0.1 3.7; 1.8; 1.1 1.33 0.05 29.50 Fast green 625 3.45 0.1 1.9; 2.0; 1.5 1.53 0.04 34.14 Brilliant blue 625 3.67 0.1 1.8; 1.8; 1.2 1.45 0.05 31.98 Patent blue 625 4.60 0.1 2.1; 2.0; 1.2 1.71 0.04 41.71 Quinoline yellow 420 5.67 0.1 1.8; 1.5; 0.9 1.38 0.04 39.51 a Retention time b Peak width at 50% of the peak height c Peak capacity (counting with time of gradient elution without final equilibration, 10 min and 5.7 min for monolithic and fused-core column, respectively) d RSD was calculated from eight injections of standard mixture at concentration level c=50 mg L-1 e RSD was calculated from six injections of standard mixture at concentration levels: c1=200 mg L-1, c2=50 mg L-1, c3=5 mg L-1 on monolithic column and c1=100 mg L-1, c2=50 mg L-1, c3=5 mg L-1 on fused-core column b Peak width at 50% of the peak height On the monolithic column, the resolution of all colorants was achieved only with longer methanol gradient. Probably worsened by the solvent used, the peaks were not so narrow, high and symmetric. Table 2. The analytical characteristics of the developed method in comparison of two types of columns 2.2. Method validation and comparison a Standard solutions at 0.2 mg L-1, 0.5 mg L-1, 2 mg L-1, 5 mg L-1, 10 mg L-1, 25 mg L-1, 50 mg L-1, 75 mg L-1, 100 mg L-1, 200 mg L-1 concentration level were measured, each in triplicate. rd solutions at 0.2 mg L 1, 0.5 mg L 1, 2 mg L 1, 5 mg L 1, 10 mg L 1, 25 mg L 1, 50 mg L 1 mg L-1, 200 mg L-1 concentration level were measured, each in triplicate. 2.2. Method validation and comparison As is mentioned in Table 2, the linear range is than shifted to higher concentrations (0.5- 200 mg L-1). In addition, the compounds order change was observed (retention times in Table 1). It was caused by using acetonitrile instead of methanol and better solubility of Green S in acetonitrile mobile phase used for fused-core particle column. The separation on monolithic column showed to be more complicated due to a strong peak tailing of Patent blue. This negative effect was not observed on fused core particle column. Moreover, three critical pairs Tartrazine – Indigo carmine; Green S – Patent blue; Brilliant blue – Fast green, depicted in Figure 2 showed peak resolution factor lower than 1.5 which complicated reliable validation of the method. The peaks of Tartrazine and Indigo carmine were not well separated on monolithic column and their quantification was possible only due to a different spectral characteristics. On the other hand, the advantages of the monolithic column method were lower back pressure and less interferences on blank chromatogram. Repeatability of both methods and correlation coefficients of regression equations were similar. Table 2. The analytical characteristics of the developed method in comparison of two types of columns doi:10.20944/preprints201811.0251.v1 Analyte Linear range (mg L-1)a Regression equation R2 LOD (mg L-1) LOQ (mg L-1) Monolith Tartrazine 0.5-200 y=11,250x+21,652 0.9973 0.03 0.09 Quinoline yellow 0.5-200 y=20,143x+106,689 0.9934 0.03 0.11 Sunset yellow 2-200 y=2,149x+7,683 0.9967 0.26 0.86 Indigo carmine 0.5-200 y=6,834x+23,541 0.9945 0.05 0.17 Fast green 0.5-200 y=28,509x+96,884 0.9958 0.02 0.08 Brilliant blue 0.5-200 y=31,272x+116,370 0.9954 0.02 0.06 Green S 0.5-200 y=23,582x+89,043 0.9961 0.03 0.10 Patent blue 0.5-200 y=39,910x-16,514 0.9997 0.02 0.06 Fused-core Tartrazine 0.2-100 y=12,770x-3,835 0.9999 0.05 0.17 Quinoline yellow 0.2-100 y=30,986x-13,046 0.9999 0.05 0.17 Sunset yellow 0.5-100 y=2,042x-42 0.9999 0.20 0.67 Indigo carmine 0.5-100 y=11,438x-6,214 0.9999 0.08 0.25 Green S 0.2-100 y=45,731x-78,955 0.9955 0.02 0.05 Fast green 0.2-100 y=34,452x-23,308 0.9998 0.02 0.06 Brilliant blue 0.2-100 y=33,215x-22,166 0.9998 0.02 0.07 Patent blue 0.2-100 y=47,507x-145,350 0.9908 0.01 0.04 a Standard solutions at 0.2 mg L-1, 0.5 mg L-1, 2 mg L-1, 5 mg L-1, 10 mg L-1, 25 mg L-1, 50 mg L-1, 75 mg L-1, 100 mg L-1, 200 mg L-1 concentration level were measured, each in triplicate. 3.1. Chemicals and materials Standards of synthetic colorants Quinoline yellow (95%), Tartrazine (99%), Sunset yellow (95%), Fast green FCF (98%), Green S (98%), Indigo carmine (98%), and Patent blue (97%) were purchased from Fluka (Germany). Brilliant blue FCF was supplied by Sigma-Aldrich (Czech Republic). Glacial acetic acid was produced by Fluka (Germany), ammonium hydroxide 26% and HPLC-grade solvents acetonitrile and methanol were obtained from Sigma-Aldrich (Czech Republic). The ultra-pure water purification for mobile phase preparation was carried out through a Milli-Q (Millipore, USA). Other chemicals and used materials were of analytical grade. Chromatographic column Chromolith Performance CN 100 × 4.6 mm with guard column Chromolith CN 5 × 4.6 mm was ordered from Merck (Czech Republic) and Ascentis Express ES-CN 100 × 4.6 mm, 5 µm from Sigma-Aldrich (Supelco, Czech Republic). Seasonal beers on tap were collected from local bars and just filtered to vial (0.45 µm PTFE). Other samples of drinks and candies were bought in local shop. Soft drinks were prepared by dissolving of powder or syrup in water at first, keeping the manufacturer instructions. Candies were dissolved in mixture of MeOH and water 50:50 (v/v) using ultrasound. Candy extracts were five times diluted before HPLC analysis due to high viscosity. All samples were filtered before analysis. 3.2. Chromatographic system Chromatographic system Shimadzu Prominence consisted of a SIL-20AC autosampler, LC- 20AD solvent delivery modules with a DGU-AS on-line degasser, a CTO-20AC column oven, an SPD- M20A detector and a CBM-20A communication module. The system control, data acquisition and data evaluation were performed by Shimadzu “LC Lab-Solution” software (Shimadzu Corporation, Japan). 2.3. Analysis of real samples After previous evaluation, the method showing efficient and fast separation on fused-core column was chosen for application on real samples analysis. Soft drinks (including syrups and powders for syrup preparation), candies and seasonal Easter green beers were tested. Liquid samples were just filtrated and 10 µL of sample solution was directly injected. Syrups and powders were diluted and candies extracted in mixture of MeOH and water 50:50 (v/v). Samples were filtered before analysis through PTFE filter, nylon filters were excluded due to partial adsorption of analytes. Detected synthetic colorants were enlisted in the product label as legislative demands; their concentrations were in reasonable limits. Sample chromatograms of colored drinks and candies are shown in Figure 3. doi:10.20944/preprints201811.0251.v1 Figure 3. Chromatograms of sample matrices: seasonal colored beer, candies and soft drinks prepared from powder or syrup (analyzed by Method II) Figure 3. Chromatograms of sample matrices: seasonal colored beer, candies and soft drinks prepared from powder or syrup (analyzed by Method II) 3.5. Method II: Chromatographic separation on Fused-Core particle column For the second method, Ascentis Express ES-CN 100 × 4.6 mm, 5 µm particle size column with superficially porous particles was utilized for separation. The buffer (solvent B) remained water solution of 2% acetic acid adjusted at pH 7.0 with addition of ammonia; organic part of the mobile phase was acetonitrile (solvent A). A gradient elution started after conditioning with water mobile phase by raising solvent A to 20% in 1.5 min, the ratio was held to 2nd min, then slowly increased to 25% in 3.5 min and finally to the 50% in 5th min. The system was equilibrated back to 100% of solvent B for 2 min. Total analysis time for separation of eight colorants was 7 min. All the other conditions were kept the same for both methods. 3.3. Preparation of standard solutions Standard stock solutions of individual colorants in concentration 4000 mg L-1 were prepared, 5 mg of Green S, Brilliant blue FCF, Patent blue, Fast green FCF were dissolved in methanol, Sunset yellow and Indigo carmine in water, Quinoline yellow in tetrahydrofuran and Tartrazine in doi:10.20944/preprints201811.0251.v1 methanol-water mixture (4:1, (v/v)). The standard stock solutions were stored in freezer. The mixed standard working solution in concentration 500 mg L-1 of each dye was prepared by mixing aliquot amount of all eight standard solutions. The calibration standard solutions were dissolved in the mixture of acetonitrile with water (1:1) over the concentration range from 0.2 mg L-1 to 200 mg L-1. 3.4. Method I: Chromatographic separation on monolithic column In the first method, eight synthetic food colorants of varying physico-chemical properties were separated on monolithic column Chromolith Performance CN 100 × 4.6 mm with guard column Chromolith CN 5 × 4.6 mm by gradient elution, tempered at 30°C. The separation was initiated with 100% water solution of 2% acetic acid adjusted at pH 7.0 with ammonia (solvent B) at a flow rate of 2.0 mL min-1 for 2 min, followed by linear gradient of methanol (solvent A) to 40% in 10th min. Total analysis last 12 min, including 2 min of equilibrating with initial conditions after separation. Injection volume was 10 µL. Colorants were detected according to their absorption maximum at wavelengths 625 nm for the blue colors (Indigo carmine, Brilliant blue FCF, Paten blue, Fast green FCF and Green S), 482 nm for Sunset yellow and 420 nm for the rest of yellow colors (Quinoline yellow, Tartrazine). Conflicts of Interest: The authors declare no conflict of interest. Acknowledgments: I. Lhotská would like to acknowledge financial support of the project of specific researc Charles University, no. SVV 260 412. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 November 2018 doi:10.20944/preprints201811.0251.v1 References 1. Amchova, P.; Kotolova, H.; Ruda-Kucerova, J. Health safety issues of synthetic food colorants. Regul. Toxicol. Pharmacol. 2015, 73, 914-922, https://doi.org/10.1016/j.yrtph.2015.09.026 2. Martins, N.; Roriz, C.L.; Morales, P.; Barros, L.; Ferreira, I.C.F.R. Food colorants: Challenges, opportunities and current desires of agroindustries to ensure expectations and regulatory practices. Trends Food Sci. Technol. 2016, 52, 1-15, https://doi.org/10.1016/j.tifs.2016.03.009 3. Kus, E.; Eroglu, H.E. Genotoxic and cytotoxic effects of sunset yellow and brilliant blue, colorant food additives, on human blood lymphocytes. Pak. J. Pharm. 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Tartrazine and sunset yellow are xenoestrogens in a new screening assay to identify modulators of human oestrogen receptor transcriptional activity. Toxicology. 2012, 298, 40-51, https://doi.org/10.1016/j.tox.2012.04.014 7. Regulation EC No 1333/2008 on food additives. Off. J. Eur. Union. 2008, L354, 16-33. g ff 8. Leleu, C.; Boulitrop, C.; Bel, B.; Jeudy, G.; Vabres, P.; Collet, E. Quinoline Yellow dye-induced fixed food- 8. Leleu, C.; Boulitrop, C.; Bel, B.; Jeudy, G.; Vabres, P.; Collet, E. Quinoline Yellow dye-induced fixed food- and-drug eruption. Contact Dermat. 2013, 68, 187-188, https://doi.org/10.1111/cod.12019 9. Stevens, L.J.; Kuczek, T.; Burgess, J.R.; Stochelski, M.A.; Arnold, L.E.; Galland, L. Mechanisms of behavioral, atopic and other reactions to artificial food colors in children. Nutr. Rev. 2013, 71, 268-281, https://doi.org/10.1111/nure.12023 p g 10. Vojdani, A.; Vojdani, C. Immune reactivity to food coloring. Altern. Ther. Health Med. 2015, 21, 52-62. 11. 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Their peaks were symmetric, very narrow and with high detector response; hence the linear range was limited and the detector was overloaded in concentration higher than 100 mg L-1. Both columns with cyano stationary phase exhibited extremely low back pressure and higher flow rates accelerating analysis were enabled. Author Contributions: Methodology, D.Š.; resources, D.Š. and P.S.; investigation, I.L.; validation, I.L., formal analysis, I.L. and D.Š.; data curation, I.L. and D.Š.; visualization, I.L.; writing—original draft preparation, I.L.; writing—review and editing, D.Š.; supervision, D.Š.; funding acquisition, D.Š. and P.S. Funding: This research was funded by the project EEFSA-CDN (No. CZ.02.1.01/0.0/0.0/16_019/0000841) co- funded by the ERDF. Acknowledgments: I. Lhotská would like to acknowledge financial support of the project of specific research of Charles University, no. SVV 260 412. Conflicts of Interest: The authors declare no conflict of interest. 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https://openalex.org/W2808061162	https://www.frontiersin.org/articles/10.3389/fmicb.2018.01481/pdf	English	null	A Novel PLP-Dependent Alanine/Serine Racemase From the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3	Frontiers in microbiology	2,018	cc-by	5,921	Abbreviations: AlaR, alanine racemase; ASR, Ala/Ser racemase; BAR, broad substrate speciﬁcity amino acid racemase; GABA-AT, γ-aminobutyric acid aminotransferase; IPTG, isopropyl-β-D-thiogalactopyranoside; NBC, N-tert- butyloxycarbonyl-L-cystein; OPA, o-phthalaldehyde; SerR, serine racemase. Edited by: Tohru Yoshimura, Nagoya University, Japan Nagoya University, Japan Reviewed by: Kouhei Ohnishi, Kochi University, Japan Tadao Oikawa, Kansai University, Japan Correspondence: Ryushi Kawakami kawakami@tokushima-u.ac.jp Reviewed by: Kouhei Ohnishi, Kochi University, Japan Tadao Oikawa, Kansai University, Japan Correspondence: Ryushi Kawakami kawakami@tokushima-u.ac.jp Correspondence: Ryushi Kawakami kawakami@tokushima-u.ac.jp Specialty section: This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology A Novel PLP-Dependent Alanine/Serine Racemase From the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3 Ryushi Kawakami1, Tatsuya Ohshida2, Haruhiko Sakuraba2 and Toshihisa Ohshima3 1 Division of Bioscience and Bioindustry, Graduate School of Technology, Industrial and Social Sciences, Tokushima University, Tokushima, Japan, 2 Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Kagawa, Japan, 3 Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology, Osaka, Japan 1 Division of Bioscience and Bioindustry, Graduate School of Technology, Industrial and Social Sciences, Tokushima University, Tokushima, Japan, 2 Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Kagawa, Japan, 3 Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology, Osaka, Japan We recently identiﬁed and characterized a novel broad substrate speciﬁcity amino acid racemase (BAR) from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. Three genes, PH0782, PH1423, and PH1501, encoding homologs exhibiting about 45% sequence identity with BAR were present in the P. horikoshii genome. In this study, we detected pyridoxal 5′-phosphate (PLP)-dependent amino acid racemase activity in the protein encoded by PH0782. The enzyme showed activity toward Ala, Ser, Thr, and Val, but the catalytic efﬁciency with Thr or Val was much lower than with Ala or Ser. The enzyme was therefore designated Ala/Ser racemase (ASR). Like BAR, ASR was highly stable at high temperatures and over a wide range of pHs, though its hexameric structure differed from the dimeric structure of BAR. No activity was detected in K291A or D234A ASR mutants. This suggests that, as in Ile 2-epimerase (ILEP) from Lactobacillus buchneri JCM1115, these residues are involved in Schiff base formation and substrate interaction, respectively. Unlike BAR, enhanced ASR activity was not detected in P. horikoshii cells cultivated in the presence of D-Ala or D-Ser. This is the ﬁrst description of a PLP-dependent fold type I ASR in archaea. Keywords: Ala racemase, Ser racemase, PLP-dependent enzyme, hyperthermophilic archaea, Pyrococcus horikoshii OT-3, D-amino acid INTRODUCTION D-Amino acids play a variety of important roles in many organisms. In mammals, for example, D-Ser and D-Asp are known to be N-methyl-D-Asp (NMDA) receptor co-agonists (Errico et al., 2015), while D-Ala is a major osmolyte responsible for intracellular isosmotic regulation in the tissues of bivalve and kuruma prawn (Abe et al., 2005; Yoshikawa and Yokoyama, 2015). In bacteria, it is well known that D-Ala and D-Glu are components of the peptidoglycan cell wall (Hancock, 1960; Ghuysen, 1961), as are D-Ser and D-Asp (Reynolds and Courvalin, 2005; Veiga et al., 2006). Although free D-amino acids such as D-Asp, D-Ala, and D-Ser have also been Received: 12 January 2018 Accepted: 13 June 2018 Published: 09 July 2018 ORIGINAL RESEARCH published: 09 July 2018 doi: 10.3389/fmicb.2018.01481 Citation: Kawakami R, Ohshida T, Sakuraba H and Ohshima T (2018) A Novel PLP-Dependent Alanine/Serine Racemase From the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3. Front. Microbiol. 9:1481. doi: 10.3389/fmicb.2018.01481 July 2018 \| Volume 9 \| Article 1481 1 Frontiers in Microbiology \| www.frontiersin.org Ala/Ser Racemase From P. horikoshii Kawakami et al. The three genes were ampliﬁed using PCR with P. horikoshii genomic DNA as the template. The primer sets used in the PCR are listed in Supplementary Table S1. Forward and reverse primers introduced an NdeI site overlapping the 5′ initiation codon and a BglII (for PH0782) or BamHI (for PH1423 and PH1501) site proximal to the 3′ end of the termination codon. PCR reactions were run using PrimeStar Max DNA polymerase (Takara, Tokyo) according to the manufacturer’s instructions. Ampliﬁed fragments were puriﬁed, introduced into pCR4- TOPO (Invitrogen, Tokyo) and sequenced. The resultant TOPO/PH0782, TOPO/PH1423 and TOPO/PH1501 were digested with NdeI and BamHI (BglII for PH0782) and introduced into pET11a (Novagen, Tokyo) to generate pET11a/PH0782, pET11a/PH1423, and pET11a/PH1501, respectively. detected in hyperthermophilic archaea (Matsumoto et al., 1999; Nagata et al., 1999; Long et al., 2001), the cell walls of archaea such as the Pyrococcus and Thermococcus species are S-layers, composed of hexagonally or tetragonally arranged proteins or glycoproteins and do not contain D-amino acids. The physiological function of D-amino acids in archaeal cells thus remains unclear. detected in hyperthermophilic archaea (Matsumoto et al., 1999; Nagata et al., 1999; Long et al., 2001), the cell walls of archaea such as the Pyrococcus and Thermococcus species are S-layers, composed of hexagonally or tetragonally arranged proteins or glycoproteins and do not contain D-amino acids. The physiological function of D-amino acids in archaeal cells thus remains unclear. Free D-amino acids in cells are generally produced by amino acid racemases, which catalyze pyridoxal 5′-phosphate (PLP)-dependent or PLP-independent racemization of amino acids. PLP-independent Asp racemases (AspRs) and PLP- dependent AlaR and SerR have been found in archaeal cells and characterized (Matsumoto et al., 1999; Long et al., 2001; Moore and Leigh, 2005; Ohnishi et al., 2008; Aihara et al., 2016; Washio et al., 2016). This includes detailed structural and functional characterization of a PLP-independent AspR from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 (Liu et al., 2001, 2002a,b; Yoshida et al., 2006; Ohtaki et al., 2008; Kita et al., 2009). Citation: We recently detected novel PLP-dependent amino acid racemase activity toward Met, Leu, and Phe in the crude extract of P. horikoshii, identiﬁed the enzyme gene (ORF ID: PH0138), and determined the properties of the recombinant protein (Kawakami et al., 2015, 2017). That enzyme showed activity toward 10 amino acids and was therefore named BAR. For construction of the K291A and D234A mutants, two sets of primers were designed (Supplementary Table S1) and pET11a/PH0782 was used as the template. The non-PCR reaction was run with PrimeStar Max DNA polymerase (Takara, Tokyo) using the standard protocol supplied by the manufacturer. The restriction enzyme DpnI was added to the reaction mixture to digest the template DNA. An aliquot of the reaction mixture was then used to transform TOP 10 cells (Stratagene, Tokyo). To screen for the correct mutation, the plasmids were extracted from the transformants and whole gene sequencing was conducted using a genetic analyzer (Model 3130, Applied Biosystems, Tokyo). This enzyme was originally annotated as a GABA-AT in the genome database, and its primary structure is consistent with a fold-type I PLP-dependent enzyme. Three other genes within the P. horikoshii genome, PH0782, PH1423, and PH1501, encode proteins that are also annotated as GABA-ATs and exhibit high similarity to BAR. This suggests these homologs also function as amino acid racemases. We previously reported that BAR activity is markedly increased in P. horikoshii cells grown on medium supplemented with D-allo-Ile, which suggests BAR mediates utilization of D-amino acids for growth (Kawakami et al., 2015). In the present study, we constructed expression systems for BAR homologs using pET vector and detected amino acid racemase activity toward Ala and Ser in the recombinant PH0782 enzyme. We then characterized the enzyme and identiﬁed the residues responsible for its catalytic activity. To better understand the physiological function of this enzyme, we examined the level of AlaR activity in P. horikoshii cells under various growth conditions. Expression and Puriﬁcation of Recombinant Enzymes The procedures used to express and purify PH0782 were similar to those used for BAR (Kawakami et al., 2017), except that Escherichia coli BL21 (DE3) cells (Stratagene) were used as the competent cells. For analysis of the mutant enzymes and substrate screening, enzymes partially puriﬁed through heat treatment at 90◦C for 20 min were used. Protein concentrations were determined using the Bradford method (Bradford, 1976). Bovine serum albumin served as the standard. Materials o-Phthalaldehyde and N-tert-butyloxycarbonyl-L-cysteine were from Wako (Osaka, Japan) and Sigma–Aldrich (Tokyo, Japan), respectively. D- and L-amino acids were from Wako and Tokyo Chemical Industry (Tokyo). All other chemicals were of reagent grade. MATERIALS AND METHODS SDS–PAGE (Laemmli, 1970) was used to determine the subunit molecular mass of the enzymes. Myosin (200 kDa), β-galactosidase (116.3 kDa), phosphorylase B (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa), and aprotinin (6.5 kDa) were used as molecular mass standards (Bio-Rad, Tokyo). The native molecular mass of PH0782 was determined using gel ﬁltration chromatography with a HiLoad 26/60 Superdex 200 column (GE Healthcare, Tokyo). Thyroglobulin (670 kDa), γ-globulin (158 kDa), Ovalbumin (44 kDa), and Myoglobin (17 kDa) were used as molecular mass standards (Bio-Rad). Characterization of Enzymes y g Escherichia coli BL21 (DE3) cells harboring expression plasmids encoding BAR homologs (pET11a/PH0782, pET11a/PH1423, and pET11a/PH1501) were grown in LB medium, and gene expression was induced using IPTG. The cells were then disrupted by sonication, and the resultant supernatants were heated at 90◦C for 20 min in the presence of 0.1M citrate (pH 5.5) buﬀer. The heat-treated supernatants were then subjected to SDS–PAGE, and bands derived from BAR homologs were clearly detected at about 50 kDa (data not shown). To screen for amino acid racemase activity, L-forms of Ala, Val, Leu, Ile, Phe, Met, Trp, Tyr, Ser, Thr, Asn, Gln, and Arg were used as substrates with the BAR homologs, and the reaction mixtures were analyzed using UPLC. Peaks for the D-forms of Ala and Ser were clearly detected in the reaction mixture containing PH0782. Weaker peaks for D-Val and D-allo-Thr were also detected with PH0782 (data not shown). Slight peaks for the D- forms of Met, Phe, and Leu were detected in the reaction mixture containing PH1501. No peak corresponding to a D-amino acid was detected in the reaction mixture containing PH1423 (data not shown). In addition, spectrophotometric assay revealed no racemase activity toward Pro in any of the enzymes (data not shown). For substrate screening, 10 µg of each enzyme were added to the reaction mixture and incubated for 60 min. Ala, Val, Leu, Ile, Phe, Met, Trp, Tyr, Ser, Thr, Asn, Gln, and Arg were used as substrates. To determine pH dependency, L-Ala was used as the substrate and the activity was assayed under various buﬀer conditions (acetate [pH 5.5–6.5], MES [pH 5.5–7.0], phosphate [pH 6.0–8.0], HEPES [pH 6.5–8.5], and CHES [pH 8.5–9.5]; buﬀer pH was adjusted at 25◦C). To determine the kinetic parameters, racemase activity was assayed in the presence of 1– 20 mM Ala or Ser (n = 3). Reaction rates were independently calculated, and apparent Vmax and Km values were analyzed with Prism 5.0 (GraphPad Software) using a non-linear regression model. To assess the pH stability of PH0782, the enzyme (0.1 mg/ml) was incubated at 80◦C for 2 h in several buﬀers (100 mM, acetate [pH 4.0–6.0], phosphate [pH 7.0–8.0], glycine [pH 9.0–10.0], and phosphate [pH 11.0–12.0]), and the residual activity was assayed. The thermostability of PH0782 was assessed by determining the residual activity after incubation at selected temperatures for 30 min and at 80◦C for several diﬀerent incubation times. Characterization of Enzymes The residual activities in the temperature and pH stability assays were determined spectrophotometrically using L-Ala as the substrate, as described previously (Kawakami et al., 2015). Construction of Expression Plasmids for BAR Homologs In this study, we constructed expression plasmids for PH0782, PH1423, and PH1501 genes using a pET system. July 2018 \| Volume 9 \| Article 1481 Frontiers in Microbiology \| www.frontiersin.org 2 Ala/Ser Racemase From P. horikoshii Kawakami et al. Separation and Determination of DL-Amino Acids Using UPLC a D-amino acid was previously described by Kawakami et al. (2015). In the kuruma prawn, D-Ala produced from L-Ala by AlaR reportedly functions as an osmolyte responsible for intracellular isosmotic regulation (Yoshikawa and Yokoyama, 2015). To determine whether ASR is responsible for increasing the NaCl concentration in the medium, P. horikoshii was cultivated for 3 h at several NaCl concentrations (1.5, 2.5, 3.5, and 4.5%) in standard medium after cultivation for 18 h at 90◦C in standard medium (2.5% NaCl). The P. horikoshii cells collected by centrifugation were disrupted by sonication, after which the extracts were cleared through another centrifugation, and the supernatants were used for determination of racemase activity. Spectrophotometric assays were performed as described previously (Kawakami et al., 2015), and L-Met and L-Ala were used as substrates for BAR and ASR, respectively. g UPLC analyses were performed for quantitative determination of L- or D-amino acids produced in the enzyme assays used for substrate screening, assessment of pH dependency, and kinetic analysis. For the enzyme assays, the standard reaction mixture included 100 mM HEPES (pH 7.0), 10 mM L-amino acid, 0.04 mM PLP, and 1 µg of enzyme in a 100-µL volume, which was incubated at 80◦C for 30 min. After incubation, the reaction mixture was immediately cooled, 6% trichloroacetic acid was added, and the precipitate was removed by centrifugation. The supernatant was then neutralized using NaOH, and the amino acids in the mixture were derivatized with OPA and NBC. In the screening assays, the derivatized amino acids were analyzed using an X-pressPak V-C18 column (2.0 mm by 50 mm, Jasco, Tokyo) as described previously (Kawakami et al., 2017). For simultaneous kinetics analysis using Ala, Ser, Val, and Thr as substrates, citrate solutions (5 mM, pH5.8) in 15 and 60% ethanol were used as buﬀers A and B, respectively, and the gradient program was operated as follows: 0–20% B for 4.5 min, 20–40% B for 2.5 min, 40–70% B for 1.0 min, 70% B for 0.5 min, and 70–0% B for 0.5 min. Cultivation of P. horikoshii Under Various Conditions and Determination of Racemase Activities PH0782 was successfully puriﬁed using the same procedure used for BAR (Figure 1A). About 4 mg of puriﬁed enzyme were obtained from 3.2 L of LB medium. SDS–PAGE showed the subunit molecular mass of puriﬁed PH0782 to be about 54.9 kDa, which approximates the molecular mass (50.5 kDa) calculated from the primary structure. Using gel ﬁltration chromatography, the native molecular mass of PH0782 was estimated to be about 300 kDa, which suggests the enzyme exists as a hexamer. The activity of BAR was greatly increased in P. horikoshii cells grown on medium supplemented with D-allo-Ile (Kawakami et al., 2015). We therefore investigated ASR activity in P. horikoshii cells grown on medium supplemented with D- Ala, D-Ser or D-allo-Ile. Cultivation of P. horikoshii OT-3 with July 2018 \| Volume 9 \| Article 1481 Frontiers in Microbiology \| www.frontiersin.org 3 Ala/Ser Racemase From P. horikoshii Kawakami et al. (31.4 ± 0.80 µmol/min/mg), respectively. The Km values for L- Ala (3.3 ± 0.22 mM) and L-Ser (4.5 ± 0.32 mM) were lower than those for D-Ala (5.5 ± 0.59 mM) and D-Ser (5.9 ± 0.40 mM), respectively. The kinetic parameters for Val and Thr were not determined because of the high Km and/or low Vmax predicted from the preliminary analysis. FIGURE 1 \| SDS–PAGE analysis of the puriﬁcation of PH0782 (A) and its mutants (B). (A) 15% polyacrylamide gel was used for the analysis. Lanes: M, markers; 1, crude extract (30 µg); 2, heat treatment (6.5 µg); 3, butyl-Toyopearl (1.4 µg); and 4, DEAE-celluroﬁne (1.4 µg). (B) Crude extracts of cells expressing wild type and mutant PH0782 were heated at 90◦C for 20 min with 0.1M citrate (pH 5.5), and the supernatants (each of about 7 µg) were applied to 12% polyacrylamide gel. Lanes: M, markers; 1, wild type; 2, K291A mutant; and 3, D234A mutant. Mutation Analysis of the Catalytic Residues Two PH0782 mutants (K291A and D234A) were constructed to conﬁrm that these residues are important for the enzyme’s catalytic activity. Figure 3 shows an alignment of the primary structures of BAR homologs and ILEP from Lactobacillus buchneri JCM1115 (Mutaguchi et al., 2013). The crystal structure of ILEP revealed that K280 and D222 are responsible for the interaction with PLP (Hayashi et al., 2017). These residues corresponded to residues K291 and D234 in PH0782 (Figure 3). After expression in E. coli, the mutant proteins were clearly observed on SDS–PAGE after heat treatment at 90◦C (Figure 1B). Spectrophotometric assay using L-Ala as a substrate detected no activity with either the K291A or D234A mutant. FIGURE 1 \| SDS–PAGE analysis of the puriﬁcation of PH0782 (A) and its mutants (B). (A) 15% polyacrylamide gel was used for the analysis. Lanes: M, markers; 1, crude extract (30 µg); 2, heat treatment (6.5 µg); 3, butyl-Toyopearl (1.4 µg); and 4, DEAE-celluroﬁne (1.4 µg). (B) Crude extracts of cells expressing wild type and mutant PH0782 were heated at 90◦C for 20 min with 0.1M citrate (pH 5.5), and the supernatants (each of about 7 µg) were applied to 12% polyacrylamide gel. Lanes: M, markers; 1, wild type; 2, K291A mutant; and 3, D234A mutant. AlaR Activity in P. horikoshii Cells Grown Under Various Conditions To determine whether ASR mediates utilization of D-amino acids for growth, P. horikoshii was cultivated in medium containing several D-amino acids. The activity toward Met derived from BAR was detected in all P. horikoshii cells tested. As previously described, the highest activity was detected in cells grown on medium containing D-allo-Ile (Kawakami et al., 2015). By contrast, no AlaR activity was detected in any of the tested cells. Characterization of PH0782 The activity of the puriﬁed enzyme was doubled by the addition of PLP and completely inhibited by the addition of 1 mM hydroxylamine hydrochloride, a known inhibitor of PLP- dependent enzymes, suggesting PLP is a cofactor. To prepare an apo-protein of PH0782, the enzyme was incubated at 80◦C for 18 h in the assay mixture. The apo-enzyme exhibited no activity in the absence of PLP and recovered its activity by addition of PLP. Alanine racemase activity in P. horikoshii cells cultivated at several NaCl concentrations (1.5, 2.5, 3.5, and 4.5%) was determined to test whether ASR is responsible for increasing the NaCl concentration in the medium, but no activity was detected in any of the tested cells. To assess the thermostability of PH0782, its enzyme activity was determined after incubation at diﬀerent temperatures for various times. When the enzyme was incubated for 30 min at temperatures between 50 and 100◦C, full enzyme activity was retained until 90◦C, but about 30% was lost at 100◦C (Figure 2A). When the enzyme was incubated at 80◦C, full activity was retained for at least 5 h (Figure 2B). When the enzyme was incubated at 80◦C for 2 h at various pHs, full enzyme activity was retained at pHs ranging from 6 to 10 (Figure 2C). To determine the optimal pH for enzyme activity, the assay was performed at pHs ranging from 5.5 to 9.5. Highest activity was detected at around pH 6.5–7.0 in MES and HEPES buﬀers (Figure 2D). Frontiers in Microbiology \| www.frontiersin.org DISCUSSION This indicates that apo- protein is probably prepared by incubation in the presence of substrate at 80◦C. But further studies might be needed to clarify these observations. The enzyme activity of PH0782 produced equimolar amounts of L-Ala and D-Ala and of L-Ser and D-Ser, and the equilibrium constant for racemization (Keq) was determined to be nearly 1 (Supplementary Figure S1). These results indicate that PH0782 catalyzes the racemization reaction. Kinetic analysis revealed that the catalytic eﬃciency with Ala (7.19 ± 0.52 /s/mM for L-Ala and 7.58 ± 0.87 /s/mM for D-Ala) is comparable to that with Ser (5.87 ± 0.44 /s/mM for L-Ser and 5.88 ± 0.43 /s/mM for D-Ser), but the eﬃciency was much lower with Val or Thr. We therefore conclude is known to be thermolabile. On the other hand, after the enzyme was incubated at 80◦C for 18 h in the assay mixture containing L-Ala as substrate, it showed no activity but recovered its activity by the addition of PLP. This indicates that apo- protein is probably prepared by incubation in the presence of substrate at 80◦C. But further studies might be needed to clarify these observations. The enzyme activity of PH0782 produced equimolar amounts of L-Ala and D-Ala and of L-Ser and D-Ser, and the equilibrium constant for racemization (Keq) was determined to be nearly 1 (Supplementary Figure S1). These results indicate that PH0782 catalyzes the racemization reaction. Kinetic analysis revealed that the catalytic eﬃciency with Ala (7.19 ± 0.52 /s/mM for L-Ala and 7.58 ± 0.87 /s/mM for D-Ala) is comparable to that with Ser (5.87 ± 0.44 /s/mM for L-Ser and 5.88 ± 0.43 /s/mM for D-Ser), but the eﬃciency was much lower with Val or Thr. We therefore conclude that PH0782 is an ASR, not a BAR. Further analyses will be necessary to identify the substrates and activities of PH1423 and PH1501, as they exhibited little or no activity in the present study. Alanine racemases and serine racemases are well characterized as PLP-dependent racemases (Yoshimura and Esaki, 2003; Radkov and Moe, 2014; Hernández and Cava, 2016) that TABLE 1 \| Kinetic analysis of the amino acid racemase activity of PH0782. July 2018 \| Volume 9 \| Article 1481 DISCUSSION In an earlier study, we revealed that PH0138, which was originally annotated as a putative GABA-AT, catalyzed the racemization of a number of hydrophobic and aromatic amino acids. We therefore designated the enzyme a BAR (Kawakami et al., 2015). In addition, we found that three other genes, PH0782, PH1423, and PH1501, were also annotated as putative GABA-AT genes and had high sequence similarity with BAR (about 45%). In the present study, we succeeded in constructing expression plasmids encoding these genes and found that PH0782 has amino acid racemase activity toward Ala, Ser, Val, and Thr. The racemase activity was completely inhibited by hydroxylamine hydrochloride, but strangely, we did not succeed to prepare apo-protein of PH0782 by dialysis against the buﬀer containing hydroxylamine hydrochloride. In addition, apo-protein was not obtained even after incubation at 80◦C for 18 h with or without hydroxylamine. This suggests that PLP binds tightly to PH0782 and is stabilized in the enzyme complex, although free PLP To determine the kinetic parameters of PH0782 enzyme activity toward Ala and Ser, the initial velocities of both D-amino acid formation and L-amino acid formation were measured at concentrations ranging from 1 to 20 mM. The relation between substrate concentration and initial velocity ﬁtted well into a non-linear regression model. The calculated Vmax and Km values for PH0782 are shown in Table 1. The Vmax values toward D-Ala (49.5 ± 2.09 µmol/min/mg) and D-Ser (41.2 ± 1.10 µmol/min/mg) were 1.7 and 1.3 times higher than those toward L-Ala (28.2 ± 0.76 µmol/min/mg) and L-Ser July 2018 \| Volume 9 \| Article 1481 Frontiers in Microbiology \| www.frontiersin.org 4 Ala/Ser Racemase From P. horikoshii Kawakami et al. FIGURE 2 \| Enzyme stability against temperature (A), time (B), and pH (C), and the pH dependency (D) of PH0782. In (D), acetate (squares), MES (diamonds), phosphate (closed circles), HEPES (triangles), and CHES (open circles) were used as buffers. FIGURE 2 \| Enzyme stability against temperature (A), time (B), and pH (C), and the pH dependency (D) of PH0782. In (D), acetate (squares), MES (diamonds), phosphate (closed circles), HEPES (triangles), and CHES (open circles) were used as buffers. is known to be thermolabile. On the other hand, after the enzyme was incubated at 80◦C for 18 h in the assay mixture containing L-Ala as substrate, it showed no activity but recovered its activity by the addition of PLP. DISCUSSION Substrate Vmax (µmol/min/mg) Km (mM) kcat/Km (/s/mM) L-Ala 28.2 ± 0.76 3.3 ± 0.22 7.19 ± 0.52 L-Ser 31.4 ± 0.80 4.5 ± 0.32 5.87 ± 0.44 D-Ala 49.5 ± 2.09 5.5 ± 0.59 7.58 ± 0.87 D-Ser 41.2 ± 1.10 5.9 ± 0.40 5.88 ± 0.43 TABLE 1 \| Kinetic analysis of the amino acid racemase activity of PH0782. Frontiers in Microbiology \| www.frontiersin.org 5 Ala/Ser Racemase From P. horikoshii Kawakami et al. FIGURE 3 \| Sequence alignment of BAR homologs and ILEP from L. buchneri. Conserved residues are indicated by asterisks. Residues interacting with PLP, whic were determined from the structure of ILEP (Hayashi et al., 2017), are boxed. ASR in P. horikoshii suggests that D-Ala and/or D-Ser serve some function in these cells. We previously observed that cellular BAR activity in P. horikoshii is dramatically enhanced when the cells are grown on medium supplemented with D-allo-Ile (Kawakami et al., 2015). This was the ﬁrst clue that D-amino acids are involved with expression of enzyme genes in hyperthermophilic archaea. We therefore investigated ASR activity in P. horikoshii grown on medium supplemented with D-amino acids; however, no activity was detected in any of the cells tested. It is also known that D-Ala acts as an osmolyte to protect tissues in crustaceans from changes in osmotic pressure (Yoshikawa and Yokoyama, 2015). We therefore investigated ASR activity in P. horikoshii cells grown in the presence of high and low salt concentrations, but again no activity was detected. Although the physiological function of ASR remains unclear, the ASR-encoding PH0782 gene forms a cluster with PH0781 gene, which encodes a putative alanine transport protein, and this gene cluster is widely distributed in other Pyrococcus and Thermococcus strains. This suggests the enzyme may function in D-Ala production from L-Ala in these strains. generally show strict substrate speciﬁcity for Ala and Ser, and are classiﬁed as fold type III and type II PLP-dependent enzymes, respectively. By contrast, ASR shows equivalent substrate speciﬁcity for Ala and Ser and is classiﬁed as fold type I. Although racemase activities toward Ala and Ser have been detected in extract from the hyperthermophilic archaeon Pyrobaculum islandicum, the details remain unclear (Nagata et al., 2002). Recently, ILEP from L. DISCUSSION In addition, the D222A and D222N ILEP mutants exhibit almost no activity (Hayashi et al., 2017). Likewise, in the present study, the K291A and D234A ASR mutants exhibited no activity. These results suggest that, as in ILEP, these residues are involved in Schiﬀbase formation and substrate interaction, and that the monomer structures of ASR and BAR are similar to that of ILEP, though the quaternary structures diﬀer among the three enzymes: BAR is dimeric, ILEP is tetrameric, and ASR is hexameric. Frontiers in Microbiology \| www.frontiersin.org DISCUSSION buchneri was found to share 37 and 40% sequence identity with ASR and BAR, respectively, and to be a fold type I enzyme that catalyzes the epimerization and/or racemization of multiple hydrophobic amino acids, including Ile and Val (Mutaguchi et al., 2013). More recently, the crystal structures of ILEP in its apo-form and in complex with PLP, PLP-L-Ile, and PLP-D-allo-Ile were determined at resolutions of 1.94–2.77 Å (Hayashi et al., 2017). The residues in ILEP that interact with PLP are well conserved in both ASR and BAR (Figure 3). Among these residues, K280 (K291 in ASR) forms an internal aldimine (Schiﬀ-base) linkage with PLP, and D222 (D234) is the most proximate to the Cα atom of D-allo- Ile. In addition, the D222A and D222N ILEP mutants exhibit almost no activity (Hayashi et al., 2017). Likewise, in the present study, the K291A and D234A ASR mutants exhibited no activity. These results suggest that, as in ILEP, these residues are involved in Schiﬀbase formation and substrate interaction, and that the monomer structures of ASR and BAR are similar to that of ILEP, though the quaternary structures diﬀer among the three enzymes: BAR is dimeric, ILEP is tetrameric, and ASR is hexameric. generally show strict substrate speciﬁcity for Ala and Ser, and are classiﬁed as fold type III and type II PLP-dependent enzymes, respectively. By contrast, ASR shows equivalent substrate speciﬁcity for Ala and Ser and is classiﬁed as fold type I. Although racemase activities toward Ala and Ser have been detected in extract from the hyperthermophilic archaeon Pyrobaculum islandicum, the details remain unclear (Nagata et al., 2002). Recently, ILEP from L. buchneri was found to share 37 and 40% sequence identity with ASR and BAR, respectively, and to be a fold type I enzyme that catalyzes the epimerization and/or racemization of multiple hydrophobic amino acids, including Ile and Val (Mutaguchi et al., 2013). More recently, the crystal structures of ILEP in its apo-form and in complex with PLP, PLP-L-Ile, and PLP-D-allo-Ile were determined at resolutions of 1.94–2.77 Å (Hayashi et al., 2017). The residues in ILEP that interact with PLP are well conserved in both ASR and BAR (Figure 3). 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https://openalex.org/W3004955663	https://research-information.bris.ac.uk/ws/files/223943879/journal.pone.0226311.pdf	English	null	Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina	PloS one	2,020	cc-by	8,387	Bell, O. H., Carreño, E., Williams, E. L., Wu, J., Copland, D. A., Bora, M., Kobayter, L., Fruttiger, M., Sim, D. A., Lee, R. W. J., Dick, A. D., & Chu, C. J. (2020). Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina. PLoS ONE, 15(2). https://doi.org/10.1371/journal.pone.0226311 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1371/journal.pone.0226311 Link to publication record on the Bristol Research Po PDF-document Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1371/journal.pone.0226311 Link to publication record on the Bristol Research Portal PDF-document This is the final published version of the article (version of record). It first appeared online via Elsevier at https://www.sciencedirect.com/science/article/abs/pii/S0360319920302779 . Please refer to any applicable terms of use of the publisher. Bell, O. H., Carreño, E., Williams, E. L., Wu, J., Copland, D. A., Bora, M., Kobayter, L., Fruttiger, M., Sim, D. A., Lee, R. W. J., Dick, A. D., & Chu, C. J. (2020). Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina. PLoS ONE, 15(2). https://doi.org/10.1371/journal.pone.0226311 Bell, O. H., Carreño, E., Williams, E. L., Wu, J., Copland, D. A., Bora, M., Kobayter, L., Fruttiger, M., Sim, D. A., Lee, R. W. J., Dick, A. D., & Chu, C. J. (2020). Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina. PLoS ONE, 15(2). https://doi.org/10.1371/journal.pone.0226311 University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ RESEARCH ARTICLE Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina Oliver H. BellID1, Ester Carreño2, Emily L. Williams1, Jiahui Wu1, David A. Copland1, Monalisa Bora2, Lina Kobayter2, Marcus Fruttiger3,4, Dawn A. Sim4, Richard W. J. Lee1,2,3,4, Andrew D. Dick1,2,3,4, Colin J. Chu1,2* Oliver H. BellID1, Ester Carreño2, Emily L. Williams1, Jiahui Wu1, David A. Copland1, Monalisa Bora2, Lina Kobayter2, Marcus Fruttiger3,4, Dawn A. Sim4, Richard W. J. Lee1,2,3,4, Andrew D. Dick1,2,3,4, Colin J. Chu1,2* 1 Academic Unit of Ophthalmology, Department of Translational Health Sciences, Faculty of Health Sciences, University of Bristol, Bristol, United Kingdom, 2 Bristol Eye Hospital, University Hospitals Bristol NHS Foundation Trust, Bristol, United Kingdom, 3 UCL Institute of Ophthalmology, London, United Kingdom, 4 Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * colin.chu@bristol.ac.uk Editor: Olaf Strauß, Eye Hospital, Charite´, GERMANY Received: November 20, 2019 Accepted: January 29, 2020 Published: February 13, 2020 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0226311 Copyright: © 2020 Bell et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS Citation: Bell OH, Carreño E, Williams EL, Wu J, Copland DA, Bora M, et al. (2020) Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina. PLoS ONE 15(2): e0226311. https://doi.org/10.1371/ journal.pone.0226311 Abstract It is not currently possible to reliably visualise and track immune cells in the human central nervous system or eye. Previous work demonstrated that indocyanine green (ICG) dye could label immune cells and be imaged after a delay during disease in the mouse retina. We report a pilot study investigating if ICG can similarly label immune cells within the human retina. Twelve adult participants receiving ICG angiography as part of routine standard of care were recruited. Baseline retinal images were obtained prior to ICG administration then repeated over a period ranging from 2 hours to 9 days. Matched peripheral blood samples were obtained to examine systemic immune cell labelling and activation from ICG by flow cytometry with human macrophage cultures as positive controls. Differences between the delayed near infrared ICG imaging and 488 nm autofluorescence was observed across pathologies, likely arising from the retinal pigment epithelium (RPE). Only one subject dem- onstrated ICG signal on peripheral blood myeloid cells and only three distinct cell-sized sig- nals appeared over time within the retina of three participants. No significant increase in immune cell activation markers were detected after ICG administration. ICG accumulated in the endosomes of macrophage cultures and was detectable above a minimum concentra- tion, suggesting cell labelling is possible. ICG can label RPE and may be used as an addi- tional biomarker for RPE health across a range of retinal disorders. Standard clinical doses of intravenous ICG do not lead to robust immune cell labelling in human blood or retina and further optimisation in dose and route are required. is stored in the Flow Repository (http:// flowrepository.org/) under the ID: FR-FCM-Z2EV. angiographic dye employed in routine ophthalmic practice for the diagnosis and monitoring of retinal disease [1]. Previously published work identified that ICG administered as a systemic depot injection in the mouse could label myeloid immune cells in the inflamed retina after 48 hours which could be imaged using commercially-available platforms [2]. This approach could advance our understanding of ocular disease and responses to treatment if similar find- ings can be demonstrated in man. Funding: This work was supported by a grant from the David Telling Charitable Trust, Bristol. CJC is supported as an NIHR Academic Clinical Lecturer. OHB is funded by the National Eye Research Centre. ELW, DAC, RWJL, ADD, and CJC are supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. The views expressed are those of the Author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Equally, it has been shown in patients with rheumatoid arthritis and psoriatic arthritis that joints with active arthritis selectively accumulate more ICG than joints from healthy patients, when measured in the hands of patients and controls [3]. Furthermore, ICG colocalised with macrophages in the lipid-rich atheromas within 20 minutes of intravenous injection in an experimental model in rabbits [4]. This suggests that ICG could selectively accumulate within macrophages and granulocytes. Following intravenous ICG administration, the main peak of absorption is at a wavelength of 805 nm and the peak of fluorescence emission ranges from 820 to 835 nm, within the near infrared spectrum [5]. It is however well-recognised that the ICG absorption spectra and to a lesser extent the emission spectra, can shift depending on concentration and the other constit- uents in solution [6]. ICG is injected as an intravenous bolus for ocular angiography up to a maximum of 25 mg in usual practice and retinal images are typically only captured over the first 30-minutes. This means that labelling of cells may not have been observed previously in the human eye, as it is unlikely that cells will have had enough time to be labelled with ICG or to infiltrate the eye if stained peripherally. Ethics The study was prospectively registered with the ISRCTN registry under study number 30128134 (https://doi.org/10.1186/ISRCTN30128134). Ethical approval was obtained from the United Kingdom Health Research Authority and local NHS Research Ethics Committee (17/ SW/0030). Human blood samples were handled in accordance with the Human Tissue Act 2004 and the research followed the tenets of the Declaration of Helsinki. Written informed consent was obtained from all participants. Introduction It is not currently possible to reliably visualise and track immune cells in human central ner- vous system tissues. The eye is unique as the only fully-accessible and optically-transparent neural organ in the human body. Indocyanine green (ICG) is a safe and widely-used Data Availability Statement: All relevant data are within the manuscript, its Supporting Information files, and in repositories. The flow cytometric data PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 1 / 14 is stored in the Flow Repository (http:// flowrepository.org/) under the ID: FR-FCM-Z2EV. Competing interests: I have read the journal’s policy and the authors of the manuscript have the following competing interests: M. Fruttiger and D. A. Sim: Patent (expired). The remaining authors have declared that no competing interests exist. ICG is known to enhance the visibility of the retinal pigment epithelium (RPE) mosaic and has been used in adaptive optics imaging [7, 8]. This staining has also been seen late after administration in the normal rat retina [9] in an approach termed delayed near-infra red anal- ysis (DNIRA). It has been suggested this may provide a marker of RPE status in addition to 488 nm autofluorescence but has not been extensively investigated in a clinical setting. With diffuse background staining of RPE by ICG, any signal from immune cell ICG labelling would need to exceed the RPE signal intensity to be detected. This was achieved during retinal inflammation after depot ICG administration in the mouse [2]. Here we report a pilot human study that begins the translation of findings from rodent sys- tems starting by determining if ICG could permit the imaging of immune cells within the human retina using standard intravenous ocular angiographic doses. By extending the imaging period and analysing matched peripheral blood samples we aimed to determine if the currently employed doses and route are sufficient for wider application. Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina is stored in the Flow Repository (http:// flowrepository.org/) under the ID: FR-FCM-Z2EV. PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 Recruitment criteria Twelve adult participants (18 years) receiving ICG angiography as part of routine standard of care were recruited. Participants required a suspected diagnosis of neovascular age-related macular degeneration (nAMD), central serous retinopathy (CSR) or intermediate, posterior PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 2 / 14 Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina and panuveitis. The rationale for combining these diverse patient groups was based on the hypothesis that myeloid immune cells will be present and intraocular leakage of fluid might accumulate ICG at a sufficient concentration within the retina itself. Ocular inclusion criteria for nAMD or CSR were: 1) likely or suspected choroidal neovascu- lar membrane or central serous retinopathy that clinically required ICG angiography (with or without combined fluorescein angiography); 2) macular sub-retinal fluid present and at least 500 μm in diameter on spectral-domain (SD) optical coherence tomography (OCT) scan; 3) any obscuring haemorrhage should not exceed more than 50% of the area of the sub-retinal fluid; 4) not due for intravitreal injection or photodynamic therapy within the first 48 hours of the study period. Ocular inclusion criteria for uveitis included: 1) likely or suspected intermediate or poste- rior uveitis or panuveitis that clinically requires ICG angiography (with or without combined fluorescein angiography); 2) clinically-suspected vasculitis; 3) no intravitreal therapy adminis- tered within 3 months prior or will be administered during the study period; 4) mild vitritis only (with a SUN haze score 2) to not obscure retinal imaging [10]. The following general exclusion criteria applied: 1) known fluorescein, ICG, iodine, or shellfish allergy; 2) any known contraindication to topical tropicamide and phenylephrine dilating drops; 3) known renal (eGFR 80 mL/min/1.73 m2) or hepatic dysfunction or active diseases that in the opinion of the investigator will contraindicate the administration of ICG; 4) inability to be easily imaged on the Spectralis, Optos or Topcon retinal imaging machines (e.g. marked kyphosis or physical impairment); 5) significant media opacity leading to poor image quality (e.g. vitreous haemorrhage or cataract); 6) inability to donate a peripheral blood sample or known HIV, Hepatitis B/C; 7) pregnant or lactating women; 8) unable to consent to study participation or 9) already enrolled in another research trial. As a pilot study, an exact sample size was not calculated in advance. Participant blood sam- ples were anonymised, and laboratory staff and imaging technicians were masked to clinical details. Recruitment criteria Image analysis was performed by CJC and EC with full clinical details available. PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 Cell preparation Human blood-derived macrophages: (hBDMs). One mM of EDTA and 50 μL/mL RosetteSep human monocyte enrichment cocktail (STEMCELL Technologies, Vancouver, Canada) were added to blood and incubated for 20 minutes at room temperature before dilu- tion with an equal volume of PBS enriched with 2% FCS. Cells were isolated by Ficoll gradient and resuspended at 0.5106 cells/mL in RPMI complete media (RPMI supplemented with 10% FCS, 2 mM L-glutamine, and 100 U/mL penicillin streptomycin). Fifty ng/mL M-CSF was added and cells were seeded at 2 million CD14+ cells per well of a 6-well plate. Media exchange was performed on days 4, 7 and 10. PBMCs. Whole blood was prepared for ficoll as described above, with the exception that the RosetteSep human monocyte enrichment cocktail was not used. After centrifugation, cells were re-suspended to 0.5106 cells/mL in freezing media (15% FCS and 10% v/v DMSO in RPMI complete media) and frozen at -80˚C. Cells were thawed by adding pre-heated RPMI complete media prior to use. PBMCs. Whole blood was prepared for ficoll as described above, with the exception that the RosetteSep human monocyte enrichment cocktail was not used. After centrifugation, cells were re-suspended to 0.5106 cells/mL in freezing media (15% FCS and 10% v/v DMSO in RPMI complete media) and frozen at -80˚C. Cells were thawed by adding pre-heated RPMI complete media prior to use. ICG (in vitro). ICG (25 mg powder for solution for injection; PL 44791/0001, Diagnostic Green, Aschheim-Dornach, Germany) was dissolved to a stock solution of 5 mg/mL in pure water before dilution into cell culture media as required. ICG (in vitro). ICG (25 mg powder for solution for injection; PL 44791/0001, Diagnostic Green, Aschheim-Dornach, Germany) was dissolved to a stock solution of 5 mg/mL in pure water before dilution into cell culture media as required. Study visits and interventions Following consent, participants underwent collection of clinical and medication history, fol- lowed by ocular and systemic examination with recording of weight, blood pressure, pulse, and temperature. Retinal imaging by colour photography, SD-OCT, infrared reflectance, and 488 nm and 780 nm (standard) and 532 nm and 802 nm fundus autofluorescence (ultra-wide- field) was performed at baseline, prior to ICG and fluorescein angiography (where indicated as part of standard of care). The same imaging set was performed after 2, 4, 6, and 8 hours post- ICG injection. Follow-up imaging was performed at a minimum of a further two timepoints of either 24 hours, 48 hours, 7 days, and 9 days post-ICG injection (S1 Table). Each subject received a single injection of ICG at baseline only. No adverse events occurred during the study. A peripheral blood sample of up to 10 mL was taken from the contralateral arm of that injected with ICG at either 2 or 4 hours post-administration and at a later, broad range of timepoints (24 or 48 hours, or 7 days post-administration) to maximise the chance of detecting a positive signal, given the expected time to labelling was unknown. Where possible following a study amendment, a pre-injection blood sample from the same participant was also obtained for the first flow cytometric run for direct comparison pre- and post-administration–for more robust ICG gating but to also assess potential changes in activation markers of the immune cells to a patient’s own baseline. For the second run (days later) fresh healthy donor blood was used as a control, rather than storage and reuse of the participant’s own blood, due to potential PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 3 / 14 Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina depletion of subsets of immune cells as ICG-free blood was required to enable preparation of a fluorescence-minus-one (FMO) control to assists with gating. Blood samples were processed within 30 minutes of venepuncture for flow cytometry. Retinal imaging platforms Retinal angiography and imaging were performed using two different devices: a Spectralis HRA+OCT system (Heidelberg Engineering, Heidelberg, Germany) and an Optos California (Optomap, Optos plc, Scotland, UK). Colour fundus photography used a TRC-50DX camera (Topcon Medical, Tokyo, Japan). SD-OCT imaging was obtained on the Spectralis system. PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 Flow cytometry hBDMs and whole blood. One-hundred μL of samples were added to each well of a V- well bottomed plate, 100 μL LIVE/DEAD Fixable Violet Dead Cell Stain (L34955; Thermo Fisher Scientific) was added (to a final dilution of 1:1000 in PBS), and incubated at 4˚C for 15 minutes. Each well was re-suspended with 50 μL of an antibody cocktail and incubated at 4˚C for 30 minutes; two antibody cocktails were used: 1) FITC-conjugated anti-human CD3 mAb (Clone UCHT1; BD Biosciences), PE-conjugated anti-human CD4 mAb (Clone RPA-T4; BD Biosciences), PE-dazzle594-conjugated anti-human CD66b mAb (Clone G10F5; Biolegend, San Diego, CA), PerCP-Cy5.5-conjugated anti-human CD62L mAb (Clone DREG-56; Biole- gend), BV605-conjugated anti-human CD25 mAb (Clone BC96; Biolegend), APC-conjugated anti-human CD69 mAb (Clone FN50; Biolegend); 2) PE-dazzle594-conjugated anti-human CD66b mAb, eVolve 605-conjugated anti-human CD14 mAb (Clone 61D3; eBioscience (Thermo Fisher Scientific)), BV711-conjugated anti-human HLA-DR mAb (Clone L243; Bio- legend), APC-conjugated anti-human CD80 mAb (Clone 2D10; Biolegend) (all antibodies were diluted in FWB (2% v/v FCS and 1 mM EDTA in PBS); residual volume of blood was considered to be 20 μL). Two-hundred μL of BD FACS lysing solution (349202) was then added and incubated at room temperature for 10 minutes, washed and prepared for flow cytometry acquisition. For antibody cocktail 1, cells were gated-for on size and granularity (FSC-A vs. SSC-A) and singlets (FSC-A vs. FSC-H), then live cells (LIVE/DEAD Fixable Violet; gate-drawing was PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 4 / 14 Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina assisted by heat-killed cells), then for CD3+CD4+ (CD4+ T cells), CD3+CD4- (CD4- T cells), and CD3- cells, and (via the CD3- gate) for CD66b+ (granulocytes), and CD4int cells (mono- cytes). Cell populations were subsequently gated-for on activation markers using FMO con- trols (CD25 [11], CD62L [12–14], and CD69 [11]; S1 Fig). For antibody cocktail 2, live cell singlets were gated-for as described above, and then CD14+ CD66b- cells (monocytes) were gated-for (CD14 gate-drawing was assisted by FMO controls). The monocytes were subsequently gated-for on activation markers using FMO controls (HLA-DR [15] and CD80 [16]). For all flow cytometry experiments, 1 μL of mAbs were also added to one drop of compen- sation beads (Anti-Rat Ig, κ/Negative Control Compensation Particles Set (BD Biosciences)) to prepare single-stain controls for compensation; for live-dead compensation, either heat- killed cells or the ArC Amine Reactive Compensation Bead Kit (Invitrogen) were used. Results It has been identified that systemic ICG administration in the mouse could label myeloid immune cells which were then visualised in the retina [2]. To determine if it could be used for immune cell labelling in man an experimental pilot study was initiated. As ICG is already rou- tinely used for ocular angiography at accepted doses, twelve adults with an existing ocular indi- cation for ICG administration (as part of their standard of care) were enrolled and imaged for up to nine days following angiography. Following a study amendment, from participant 3 onwards the maximum dose of 25mg was always injected. Demographics and the weight- adjusted dose of ICG administered are summarised in Table 1. Delayed retinal imaging after ICG administration reveals disparities between standard autofluorescence Near-infrared (790 nm excitation) retinal autofluorescence signals were minimally detectable or absent at baseline in most of the patients, but clearly visible in all following ICG administra- tion, even nine-days later (S1 Fig). Differences in signal patterns between this DNIRA and standard 488 nm autofluorescence could be seen across a range of ocular pathology (Fig 1) and may provide an additional method for visualising RPE and cell health. Light microscopy Cells were washed 5 times (for 5 minutes each) in PBS. Images were acquired on an EVOS FL Color Imaging System (Thermo Fisher Scientific). Data analysis Flow cytometry data was analysed and presented using FlowJo v10 (FlowJo LLC, Ashland, OR). Statistical analysis was performed using Prism 7 (GraphPad Software Inc., San Diego, CA). Participant data (percentage positive cells or MFI) was analysed using the Kruskal-Wallis test (one-way ANOVA [17]), with FDR step-up correction of p-values [18]. Flow cytometry Sam- ples were acquired using a BD LSRFortessa X-20 using BD FACSDIVA software (BD Biosciences). ICG labelling of Human immune cells in vivo is not robust using standard intravenous doses Only one subject demonstrated ICG signal on peripheral blood immune cells using flow cytometry. Angiography confirmed an acute choroidal neovascular membrane (CNV) which 5 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina Table 1. Demographics of participants enrolled in the study with a clinical indication for routine ICG angiography. ID Age (years) Gender (M/F) ICG indication and eye Weight (kg) Dose of ICG (mg/ kg) Lens Status Visual Acuity RE (Letter score) Visual Acuity LE (Letter score) Past Medical History Current systemic medication Recent ocular medication ICG+ cells detected in blood 1 76 M nAMD LE 96 0.08 C 73 62 Treated Bladder and Prostate Carcinoma Atorvastatin Aflibercept 2 days prior NO 2 78 F nAMD BE 58 0.30 IOL 72 78 None Multivitamin supplements Lucentis 6 months prior, previous PDT NO 3 79 F nAMD RE 112 0.22 C 63 81 Hysterectomy and stoma from bowel injury. TIA Lutein None YES 4 22 F Uveitis BE (MFC) 125 0.20 N 75 2 None Tacrolimus, Mycophenolate mofetil None NO 5 79 F nAMD RE 75 0.33 C 42 79 None None Aflibercept one month prior NO 6 64 F nAMD LE 65 0.38 N 78 34 Depression, hypertension Paroxetine None NO 7 76 F Uveitis BE (Panuveitis) 60 0.42 IOL 83 65 Ocular sarcoidosis None None NO 8 30 M Uveitis BE (Panuveitis) 84 0.30 IOL 83 85 Arthritis of the knee None Ozurdex one month prior & topical prednisolone NO 9 86 F nAMD LE 87 0.29 IOL 76 6 Hiatus hernia, osteoporosis, hypertension, hypercholesterolaemia Lansoprazole, Adcal D3, Lisinopril, Cefalexin Aflibercept six months prior NO 10 78 M CSR BE 72 0.35 N 37 60 Polymyalgia rheumatica, hypertension, Diabetes Mellitus, Myeloma Prednisolone, Amlodipine, Atorvastatin, Metformin, Cholecalciferol, Ramipril None NO 11 34 F Uveitis BE (Panuveitis) 56 0.45 IOL 79 60 Tuberculosis, Sarcoidosis Rifater, Moxifloxacin, Pyridoxime Dexamethasone & Dorzolamide drops NO 12 74 F nAMD LE 90 0.28 N 78 15 None Atorvastatin, Clopidogrel, Indapamide MR None NO ID = identification number, nAMD = neovascular age-related macular degeneration, CSR = Central serous retinopathy, MFC = Multifocal Choroiditis with Panuveitis, LE = Left eye, RE = Right eye, BE = Both eyes, N = No cataract, C = cataract, IOL = intraocular lens, TIA = transient ischaemic attack. PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 LE = left eye, RE = right eye. Scale bars = 600 μm. https://doi.org/10.1371/journal.pone.0226311.g001 https://doi.org/10.1371/journal.pone.0226311.g001 https://doi.org/10.1371/journal.pone.0226311.g001 speed of change is unknown. There was no evidence of systemic infection or inflammation in this participant to explain why ICG signal was present in the blood. Compensating for the potential confounding factor of signal arising from RPE dysfunction and the expected rare occurrence of labelled immune cells using the intravenous ICG route, it was possible to identify distinct cell-sized signals (Fig 3) in only three locations of three sub- jects. In each, the DNIRA signals newly-appear over time in a region where there is no alter- ation in 488 nm autofluorescence. No ICG signal was observed in the retinal vasculature at late timepoints, in agreement with published data, showing it has a relatively short half-life of min- utes in the circulation [19, 20]. ID = identification number, nAMD = neovascular age-related macular degeneration, CSR = Central serous retinopathy, MFC = Multifocal Choroiditis with Panuveitis, LE = Left eye, RE = Right eye, BE = Both eyes, N = No cataract, C = cataract, IOL = intraocular lens, TIA = transient ischaemic attack. Note total ICG dose injected was increased to a fixed maximal 25mg from participant 3 onwards. ID = identification number, nAMD = neovascular age-related macular degeneration, CSR = Central serous retinopathy, MFC = Multifocal Choroiditis with Panuveitis, LE = Left eye, RE = Right eye, BE = Both eyes, N = No cataract, C = cataract, IOL = intraocular lens, TIA = transient ischaemic attack. Note total ICG dose injected was increased to a fixed maximal 25mg from participant 3 onwards. had been symptomatic for five days prior to ICG injection (Fig 2A–2D). Discrete signals around the CNV lesion were visible only in the near-infrared channel and were approximately 30 μm in diameter compatible with myeloid cells (Fig 2E and 2F). Longitudinal observation demonstrated alteration in position of these signals between the 24 hour and 7 day imaging intervals without corresponding changes in 488 nm autofluorescence, indicating the changes are unlikely to arise from the RPE. It is possible these may represent labelled cells, but the only characteristic possible to confirm this would be movement in position across time, though the PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 6 / 14 Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina Fig 1. Differences in retinal images between late near infrared imaging following ICG (DNIRA) and standard 488 nm autofluorescence. Four examples from the eyes of different subjects illustrate different appearances that provide complementary information on RPE health. Retinal ICG fluorescence 790 nm excitation channel images are shown alongside matched 488 nm excitation autofluorescence images. CNV = Choroidal neovascular membrane, CSR = Central serous retinopathy. LE = left eye, RE = right eye. Scale bars = 600 μm. Fig 1. Differences in retinal images between late near infrared imaging following ICG (DNIRA) and standard 488 nm autofluorescence. Four examples from the eyes of different subjects illustrate different appearances that provide complementary information on RPE health. Retinal ICG fluorescence 790 nm excitation channel images are shown alongside matched 488 nm excitation autofluorescence images. CNV = Choroidal neovascular membrane, CSR = Central serous retinopathy. PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 Discussion Whilst immune cell labelling was the central objective, we illustrate a range of retinal pathology can be imaged late after ICG administration, with distinctive signals arising from the RPE. DNIRA has been described before, but here we demonstrate findings across a further diverse range of human ocular pathology using a commercially available SLO platform [9] that com- plements recent findings using adaptive optics imaging of the RPE in inherited retinal degen- erations [8]. Several examples of unique patterns only seen by DNIRA identify additional or complementary information for monitoring RPE health versus traditional 488 nm autofluores- cence imaging. These data highlight an opportunity for superior stratification and earlier detection of pathology such as reversible RPE dysfunction, critical to identifying therapeutic response signals, as has remained problematic for example in studies of geographic atrophy [21]. In terms of immune cell labelling, using the intravenous route with standard angiographic doses does not achieve robust labelling within the retina. ICG-positive myeloid cells were only detected in the peripheral blood of subject 3, in which distinct near infrared punctate signals appeared without corresponding 488 nm signal alterations. These new signals of the appropri- ate size for a leukocyte changed over time and might represent immune cell trafficking of an ICG-labelled cell. Two similar signals were only seen in two other eyes of two different partici- pants. The only feasible non-invasive method of characterising them as immune cells in man would be to identify clear movement over a period of minutes to hours. Co-labelling with Fig 3. Cell-sized hyperfluorescent near-infrared signals appearing in regions with no prior autofluorescence or RPE signal change during the study. Retinal ICG fluorescence 790 nm excitation channel images are shown alongside matched 488 nm excitation autofluorescence images. Subject 3 had detectable ICG cell staining in peripheral blood. By size and location these could represent weakly ICG labelled cells infiltrating the retina, rather than arising from RPE staining alone. LE = left eye, RE = right eye. Scale bars = 200 μm. htt //d i /10 1371/j l 0226311 003 Fig 3. Cell-sized hyperfluorescent near-infrared signals appearing in regions with no prior autofluorescence or RPE signal change during the study. Retinal ICG fluorescence 790 nm excitation channel images are shown alongside matched 488 nm excitation autofluorescence images. Subject 3 had detectable ICG cell staining in peripheral blood. Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina Fig 2. Only one subject demonstrated ICG-positive cells in peripheral blood and retinal signals that altered over time. (A) Subject 3 presented a week following new onset visual loss and distortion in the right eye. Existing RPE pigment abnormalities were visible on colour fundus images, (B) with a large pigment epithelial detachment and subretinal fluid visible on spectral-domain OCT. (C) A choroidal neovascular membrane was confirmed by fluorescein and ICG angiography, at 90 and 40 seconds post-injection respectively. (D) Optos ultra-widefield ICG imaging (802 nm excitation) confirmed regions of leakage or high signal were present only at the macula. (E) At different points timed from ICG administration, Spectralis HRA imaging demonstrated punctate signals in the ICG channel only (790 nm excitation) that altered markedly between 24 hours and day 7. (F) Enlarged region illustrating changes in signal intensity and location. (G) Flow cytometry histogram of the peripheral blood sample taken at 24 hours demonstrating ICG+ signal (green) on monocyte and granulocyte populations compared to a no-ICG control (grey). Scale bars = 200 μm unless stated. https://doi.org/10.1371/journal.pone.0226311.g002 No peripheral blood immune cell activation was observed following ICG administration For a subset of participant blood samples, it was possible to measure activation markers using flow cytometry (Fig 4, S2 and S3 Figs). Aggregate data did not demonstrate evidence of ICG resulting in an inflammatory or activated immunophenotype. As only one subject had detect- able ICG signal in peripheral blood, in vitro human macrophage cell cultures were used to examine their ability to develop ICG-positive signals on flow cytometry. Microscopy demon- strated endosomal accumulation of ICG in both cell cultures at an in vitro dose of 0.2 mg/ml, implying a sufficient concentration or protracted exposure may be required to achieve label- ling where cells would be clearly visible if they gain entry into the retina. PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 7 / 14 Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 8 / 14 https://doi.org/10.1371/journal.pone.0226311.g002 Discussion Each point is a single blood sample from 7 subjects. All activation markers underwent validation with positive control stimuli. Human macrophage cell cultures developed visible signal within endosomes following 24 hours of lt ith 0 2 / L ICG b i hi h ld b d t t d b fl t t i th i f d h l (D) R t ti i h f tivate immune cells in peripheral blood but can be observed in endosomes of macrophage cell cultures. (A) Fig 4. ICG administration did not activate immune cells in peripheral blood but can be observed in endosomes of macrophage cell cultures. (A) Aggregated flow cytometry activation marker changes in participant peripheral blood samples taken across the study are shown measuring CD62L on T-cells (p = 0.914), (B) CD80 on monocytes (p = 0.914), and (C) CD62L on granulocytes (p = 0.698). Each point is a single blood sample from 7 subjects. All activation markers underwent validation with positive control stimuli. Human macrophage cell cultures developed visible signal within endosomes following 24 hours of culture with 0.2 mg/mL ICG by microscopy, which could be detected by flow cytometry in the near infrared channel. (D) Representative images shown for human cell cultures, with (green histogram) and without (grey histogram) ICG administration. Scale bars = 10 μm. https://doi.org/10.1371/journal.pone.0226311.g004 cellular dyes or antibodies are not currently established. No rapid alterations were seen during imaging sessions, potential cell labelling could only be identified in post-processing and the expected speed of migration within retinal tissue remains unknown. Evidence from 2-photon imaging of the inflamed mouse eye however demonstrates myeloid cell movement may be much slower than expected, even compared to other tissues, at around 1 μm per minute [22]. This could explain the larger time intervals required to observe signal position shifts that could represent labelled immune cells. Complementing our data and drug safety, no evidence of immune cell activation could be detected in peripheral blood following intravenous ICG injection. To better understand the pharmacodynamics of ICG in relation to cellular accumulation, we tested human-derived macrophage cell cultures which confirmed the localisation of ICG within extranuclear vesicular structures. Whilst not directly comparable to the in vivo situa- tion, this suggests if the correct delivery and concentration can be achieved, labelling is possi- ble. Discussion By size and location these could represent weakly ICG labelled cells infiltrating the retina, rather than arising from RPE staining alone. LE = left eye, RE = right eye. Scale bars = 200 μm. https://doi.org/10.1371/journal.pone.0226311.g003 https://doi.org/10.1371/journal.pone.0226311.g003 9 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina Fig 4. ICG administration did not activate immune cells in peripheral blood but can be observed in endosomes of macrophage cell cultures. (A) Aggregated flow cytometry activation marker changes in participant peripheral blood samples taken across the study are shown measuring CD62L on T-cells (p = 0.914), (B) CD80 on monocytes (p = 0.914), and (C) CD62L on granulocytes (p = 0.698). Each point is a single blood sample from 7 subjects. All activation markers underwent validation with positive control stimuli. Human macrophage cell cultures developed visible signal within endosomes following 24 hours of culture with 0.2 mg/mL ICG by microscopy, which could be detected by flow cytometry in the near infrared channel. (D) Representative images shown for human cell cultures, with (green histogram) and without (grey histogram) ICG administration. Scale bars = 10 μm. Fig 4. ICG administration did not activate immune cells in peripheral blood but can be observed in endosomes of macrophage cell cultures. (A) Aggregated flow cytometry activation marker changes in participant peripheral blood samples taken across the study are shown measuring CD62L on T-cells (p = 0.914), (B) CD80 on monocytes (p = 0.914), and (C) CD62L on granulocytes (p = 0.698). Each point is a single blood sample from 7 subjects. All activation markers underwent validation with positive control stimuli. Human macrophage cell cultures developed visible signal within endosomes following 24 hours of culture with 0.2 mg/mL ICG by microscopy, which could be detected by flow cytometry in the near infrared channel. (D) Representative images shown for human cell cultures, with (green histogram) and without (grey histogram) ICG administration. Scale bars = 10 μm. https://doi org/10 1371/journal pone 0226311 g004 Fig 4. ICG administration did not activate immune cells in peripheral blood but can be observed in endosomes of macrophage cell cultures. (A) Aggregated flow cytometry activation marker changes in participant peripheral blood samples taken across the study are shown measuring CD62L on T-cells (p = 0.914), (B) CD80 on monocytes (p = 0.914), and (C) CD62L on granulocytes (p = 0.698). PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 Discussion The intracellular localisation is in contrast with other cells such as hepatocytes, where nuclear localisation has been observed, suggesting differential uptake or processing routes [23]. The uptake of ICG has been shown, in primary human RPE cultures, to be dependent on the Na+/K+-ATPase pump and localised within extranuclear compartments [24]. It has also been observed to bind the RPE of human, primate, and mouse subjects in vivo [9, 25]. In 10 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina Subject 3, several cell types were weakly-positive for ICG and this is likely due to membrane binding as ICG is well-characterised as able to bind to amphiphilic structures [26]; however the phagocytic populations (monocytes and granulocytes) were far more strongly-positive for ICG than other immune cells. This suggests that ICG may be internalised and accumulated by these cells, comparable to that observed in our macrophage cell cultures and may therefore be the only visible population brighter than the RPE if they enter the retina. Our findings highlight the potential of ICG, though considerably more refinement is needed prior to further clinical application because we identified ICG-positive blood in only one subject at 24 hours post-administration. It was not possible to test peripheral blood at every imaging visit, so positive findings may have been missed. Preclinical mouse data sug- gested the intravenous route of ICG administration would not provide the most efficient cell labelling, but it was prudent from an ethical and practical perspective to show the established administration route was insufficient [2]. The results of this study provide justification to pur- sue alternative methods of administration, reformulation or higher doses. In clinical hepatic function studies, approximately ten-fold higher systemic ICG doses than used here have been given successfully without clear evidence of toxicity [27]. This dose has typically only been given as a bolus and given the endosomal accumulation and preferential staining of myeloid cells with phagocytic potential, it is likely more sustained exposure of cells to a minimal concentration of ICG would be needed for efficient labelling. The superior intra- peritoneal or subcutaneous routes identified in mouse are not readily applicable in man given the safety, dose, and side-effect considerations for ocular application [2]. Discussion However, administra- tion by slow intravenous infusion or reformulation to an oral preparation may be preferable and our in vitro modelling suggests a sustained blood level of even 6 μg/ml of ICG for two hours might enable strong cell marking to be reliably detectable using flow cytometry (S2 Fig). With improvement in the efficiency of cell labelling, the relative amplitude of signal from immune cells against that of the RPE could result in clear separation of the two, as achieved in the mouse [2]. Furthermore, engaging methods of superior imaging resolution such as adap- tive optics scanning laser ophthalmoscopy will allow single immune cells to be clearly distin- guished from the RPE, identified and tracked. This study provides further support towards the translational goal of imaging the cellular components of the immune system in the human eye and with considerable refinement ICG might be a viable method to achieve this. PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 Supporting information S1 Table. Imaging session and blood sample collection timings for each participant. Mea- sured from time of ICG injection. Timings were determined by participant availability and fol- lowing an amendment, extension to cover a range of longer intervals to maximise the chance of signal detection. S1 Fig. Baseline 488 and 790 nm fundal images. A representative selection of baseline images acquired from patients in the study illustrating weak to minimal infrared autofluorescence prior to ICG administration. Scale bars = 300 μm. (TIF) S2 Fig. Representative gating strategy used to analyse human blood. (A) The representative gating strategy used for antibody cocktail #1 (“lymphocyte panel”) is shown. For antibody cocktail #2 (“monocyte panel”), live cells were gated into granulocytes (CD66b+) or monocytes (CD14+ CD66b-, a CD14 FMO was used to assist with drawing the CD14+ gate)). For the histo- grams (activation markers: CD25, CD62L, and CD69), FMOs are indicated in red, whilst a S2 Fig. Representative gating strategy used to analyse human blood. (A) The representative gating strategy used for antibody cocktail #1 (“lymphocyte panel”) is shown. For antibody cocktail #2 (“monocyte panel”), live cells were gated into granulocytes (CD66b+) or monocytes (CD14+ CD66b-, a CD14 FMO was used to assist with drawing the CD14+ gate)). For the histo- grams (activation markers: CD25, CD62L, and CD69), FMOs are indicated in red, whilst a 11 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina representative participant sample is indicated in blue. Validation of the activation marker anti- bodies was performed via whole-blood staining (individually for 24 hours) with phytohaemag- glutin P (PHA; at a final concentration of 20 μg/mL), CD3/CD28 activating beads (Dynabeads; 0.63 μL of beads (stock concentration 4.0 107 beads/mL) were used), and li popolysaccharide (LPS; at a final concentration of 0.1 ng/mL). (B) Representative histograms from an experi- ment where whole human blood was stained ex vivo with 6 ug/mL indocyanine green (ICG) for 2 hours (green), as compared to an ICG FMO sample (grey). The staining is less bright than has been observed with cell lines and the participant data, possibly due to the abundance of erythrocytes (as compared-to cell cultures), lower ICG concentration, and/or shorter stain- ing time. S3 Fig. Scatterplots showing changes in activation after ICG administration. Acknowledgments The Authors wish to thank Andrew Herman and Lorena Ballesteros (Flow Cytometry Facility, University of Bristol) for technical assistance; Eleanor Hiscott, Julie Cloake and the staff of the Bristol Eye Hospital Clinical Research Unit, and Optos Plc who kindly supplied the Optos Cal- ifornia machine for the duration of this study. Supporting information (A–B) Scatter- plots for CD3+CD4+ cells show changes in CD25 and CD69, (C–E) for CD3+CD4- cells show changes in CD25, CD62L, and CD69, and (F) for CD14+ cells (monocytes) show changes in HLA-DR. The y-axis is expressed as fold-change in the percentage of positive cells (CD25, CD62L, CD69) or the fold-change of the MFI (CD80, HLA-DR). Loss of CD62L is associated with activation, whilst expression of CD25 and CD69, and up-regulation of CD80 and HLA-DR associate with activation; statistical tests were not significant (p 0.05). The data was acquired using flow cytometry (n = 7). (TIF) References 1. Obana A, Miki T, Hayashi K, Takeda M, Kawamura A, Mutoh T, et al. Survey of complications of indo- cyanine green angiography in Japan. American journal of ophthalmology. 1994; 118(6):749–53. Epub 1994/12/15. https://doi.org/10.1016/s0002-9394(14)72554-1 PMID: 7977601. 2. Sim DA, Chu CJ, Selvam S, Powner MB, Liyanage S, Copland DA, et al. A simple method for in vivo labelling of infiltrating leukocytes in the mouse retina using indocyanine green dye. Disease Models & Mechanisms. 2015; 8(11):1479–87. https://doi.org/10.1242/dmm.019018 PMID: 26398933; PubMed Central PMCID: PMC4631782. 3. 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Funding acquisition: Ester Carreño, Colin J. Chu. Funding acquisition: Ester Carreño, Colin J. Chu. Investigation: Oliver H. Bell, Ester Carreño, Emily L. Williams, Jiahui Wu, David A. Copland, Monalisa Bora, Colin J. Chu. Methodology: Emily L. Williams. Project administration: Oliver H. Bell, Ester Carreño, Monalisa Bora, Colin J. Chu. Resources: Emily L. Williams, David A. Copland, Monalisa Bora, Richard W. J. Lee, Andrew D. Dick. Supervision: David A. Copland, Richard W. J. Lee, Andrew D. Dick, Colin J. Chu. Validation: Oliver H. Bell, Emily L. Williams. Validation: Oliver H. Bell, Emily L. Williams. Visualization: Oliver H. Bell, Ester Carreño, Lina Kobayter, Colin J. Chu. Writing – original draft: Oliver H. Bell, Ester Carreño, Colin J. Chu. 12 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0226311 February 13, 2020 Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina Writing – review & editing: Oliver H. 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https://openalex.org/W4298015057	http://istoria.unbari.ac.id/index.php/OJSISTORIA/article/download/137/113	Indonesian	null	PERKEMBANGAN TENGKULUK DI KOTA JAMBI TAHUN 1946 - 2017	Istoria : jurnal ilmiah pendidikan sejarah Universitas Batanghari	2,022	cc-by-sa	5,346	Abstrak Tujuan penelitian ini untuk mengetahui perkembangan Tengkuluk Di Kota Jambi Tahun 1946 – 2017. Metode penelitian yang digunakan adalah metode historis karena untuk mengumpulkan sumber-sumber sejarah secara efektif, menilainya secara kritis dan menyajikan suatu sintesis tertulis atas hasil yang dicapai. Berdasarkan hasil penelitian bahwa tengkuluk menjadi suatu indentitas tersendiri bagi kaum perempuan di Jambi. Menurut sejarah, kain tengkuluk sudah ada sejak abad ke 7, yang pada saat itu digunakan perempuan melayu untuk menghadiri suatu acara adat ataupun saat kegiatan bercocok tanam seperti di sawah dan ladang. Bukan hanya sekedar sebagai penutup kepala, sejatinya kain tengkuluk memiliki makna yang lebih mendalam.Jambi kaya akan budaya yang mana belum banyak diketahui masyarakatluas. Kota Jambi yang terletak di pesisir timur di bagian tengah Pulau Sumatera inimempunyai budaya Melayu sejak dahulu. Salah satu bukti adat budaya BangsaMelayu di Jambi ialah warisan tradisi penutup kepala yang disebut tengkulukbagikaum wanita. Tengkuluk merupakan identitas jatidiri perempuan melayu. Tradisi penutup kepala atau tengkuluk sudah ada sebelum masyarakat Jambi mengenal agama Islam. Pada masa itu tengkuluk hanya sebagai aksesoris atau hiasan kepala sebagai suatu keindahan perempuan-perempuan melayu Jambi, yang digunakan sebagai penutup kepala pada saat pesta, atau hari-hari besar. Tengkuluk juga dijadikan sebagai penahan kepala dari beban-beban ketikan meletakan barang bawaan di atas kepala atau sebagai pelindung kepala dari teriknya sengat matahari ketika perempuan melayu pergi berladang atau berumo pada saat itu.Sebagai bentuk kebudayaan tengkuluk mengalami berbagai perubahan hingga saat ini seperti perkembangan persepsi masyarakat tentang tengkuluk. Bagi wanita yang berhijab, menggunakan tengkuluk adalah dengan cara menggunakan dahulu hijab untuk menutupi rambut, telinga dan leher. Lalu baru di pasangkan tengkuluk di atas kepala. Perubahan juga terjadi dari segi bahan-bahan yang digunakan dalam unsur-unsur yang terdapat pada tengkuluk. K t K i T k l k K t J bi PERKEMBANGAN TENGKULUK DI KOTA JAMBI TAHUN 1946 - 2017 Ikrima Yaziva,* Siti Heidi Karmela** Alumni Prodi Pendidikan Sejarah FKIP Unbari Dosen Prodi Pendidikan Sejarah FKIP Unbari Jurnal IstoriaProdi Pendidikan Sejarah FKIP Universitas Batanghari Jambi Jurnal IstoriaProdi Pendidikan Sejarah FKIP Universitas Batanghari Jambi E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 Abstract Keywords: Tengkuluk, Jambi City Abstract The purpose of this study was to determine the development of Tengkuluk in Jambi City in 1946 – 2017. The research method used is the historical method because it collects historical sources effectively, evaluates them critically and presents a written synthesis of the results achieved. Based on the results of the study, the tengkuluk became a separate identity for women in Jambi. According to history, the tengkuluk cloth has existed since the 7th century, which at that time was used by Malay women to attend a traditional event or during farming activities such as in the fields and fields. Not just as a head covering, in fact the tengkuluk cloth has a deeper meaning. Jambi is rich in culture which is not widely known by the public. The city of Jambi, which is located on the east coast in the central part of Sumatra Island, has a Malay culture since ancient times. One proof of the Malay cultural customs in Jambi is the legacy of the traditional head covering called tengkuluk for women. Tengkuluk is the identity of Malay women. The tradition of covering the head or tengkuluk existed before the Jambi community knew Islam. At that time, the tengkuluk was only used as an accessory or a headdress as a beauty for Jambi Malay women, which was used as a head covering at parties, or on big days. The Tengkuluk is also used as a head restraint from the burdens of typing to put luggage on the head or as a head protector from the scorching heat of the sun when Malay women go to farming or berumo at that time. As a form of culture, the Tengkuluk has undergone various changes to date such as the development of public perception. about the neck. For women who wear the hijab, using the nape of the neck is to first use the hijab to cover the hair, 12 E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 Jurnal IstoriaProdi Pendidikan Sejarah FKIP Universitas Batanghari Jambi ears and neck. Then just put the neck on the top of the head. Changes also occur in terms of the materials used in the elements found in the tengkuluk. Keywords: Tengkuluk, Jambi City ears and neck. Then just put the neck on the top of the head. Changes also occur in terms of the materials used in the elements found in the tengkuluk. A. PENDAHULUAN umo dan tudung lingkup yang terispirasi dari anak-anak pesantren yang ada di seberang Kota Jambi yaitu sorban dan cadar yang digunakan oleh para santriwati. Indonesia dikenal sebagai bangsa dan negara yang memiliki kekayaan budaya luar biasa, dari segi keindahaannya, juga nilai falsafah yang terkandung di dalamnya. Di Indonesia sendiri terdapat banyak suku bangsa yang mendiami sepanjang kepulauan ini. Setiap suku bangsa memiliki unsur kebudayaan yang berbeda-beda dengan suku lainnya. Salah satunya yang sudah diakui dunia yaitu tengkuluk. g p Tengkuluk (tutup kepala) sebagai bagian penting dalam pakaian yang dipakai oleh perempuan Jambi terdiri dari etnik dengan ragam budaya penduduk asli dan pendatang tampak indah dan anggun, merupakan kebanggaan masyarakat Jambi sebagai masyarakat yang berkebudayaan tinggi. Menampilkan kembali tutup kepala (tengkuluk) dalam kesehariannya ditengah-tengah masyarakat sama dengan membangkitkan batang yang terendam dan mengait barang yang teranyut (Nurlaini. 2017). Tengkuluk merupakan sebuah peninggalan budaya melayu di Kota Jambi. Kota Jambi merupakan sebuah Provinsi di Indonesia yang terletak di pesisir timur dibagian tengah Pulau Sumatra. Kota Jambi berbatasan langsung dengan Provinsi Riau dan Sumatra Selatan. Wilayahnya dikenal dengan sebutan“Sepucuk Jambi, Sembilan Lurah betanggo alam barajo”. Jambi kaya akan budaya yang mana belum banyak diketahui masyarakatluas. Propinsi yang terletak di pesisir timur di bagian tengah Pulau Sumatera inimempunyai budaya Melayu sejak dahulu. Salah satu bukti adat budaya BangsaMelayu di Jambi ialah warisan tradisi penutup kepala yang disebut tengkulukbagikaum wanita. Tutup kepala atau dalam bahasa Jambi lebih dikenal tengkulukadalah salah satu pelengkap adat tradisi berbusana, yang sering digunakan baikuntuk sehari-hari maupun untuk acara khusus. Dalam setiap model tengkulukterkandung falsafah yang memiliki nilai/norma yang menentukan bagaimana kitabersikap, bertindak, dan berperilaku, juga memberikan kita aturan untuk hidup.Tengkulukadalah produk adat j Tengkuluk menjadi suatu indentitas tersendiri bagi kaum perempuan di Jambi. Menurut sejarah, kain Tengkuluk sudah ada sejak abad ke 7, yang pada saat itu digunakan perempuan melayu untuk menghadiri suatu acara adat ataupun saat kegiatan bercocok tanam seperti di sawah dan ladang. Bukan hanya sekedar sebagai penutup kepala, sejatinya kain tengkuluk memiliki makna yang lebih mendalam. Kain tengkuluk yang berarti sebagai lambang kesahajaan dan budi pekerti luhur perempuan Jambi. Pada tahun 1946 tengkuluk pertama kali masuk ke Kota Jambi yaitu di Seberang Kota Jambi digunakan masyarakat seberang sebagai tengkuluk untuk ke 13 Jurnal IstoriaProdi Pendidikan Sejarah FKIP Universitas Batanghari Jambi E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 E-ISSN 2597-8845 Vol. 6 No. 1. A. PENDAHULUAN April 2022 Permasalahan pokok penelitian ini didasarkan pada latar belakang masalahnya adalah “bagaimana latar belakang dan masuknya tengkuluk di Kota Jambi”. Oleh karenanya rumusan masalah yang dapat diajukan sesuai dengan pokok permasalahannya adalah Bagaimana perkembangan dan penggunaan tengkuluk di Kota Jambi ?” dan budaya yang mengungkapkan aspek kehidupan bermasyarakat (Nurdin.2010). Tengkuluk berupa sebuah selendang denganberbagai jenis bahan yang dilipat dan dililitkan di kepala. Sebagaimana mengenakkan jilbab/kerudung untuk para muslimah, bedanya tengkuluk hanya dilipat dan dililitkan saja tanpa menggunakan alat jarum, peniti, dan lain-lain. Bagi non muslim pun juga bisa mengenakkan tengkuluk sebagai penambah kecantikan penampilan. Lebih jelasnya rumusan masalah ini menjelaskan bagaimana perkembangan tengkuluk dari 1946- 2017. Melihat sejarah budaya masyarakat Jambi yang begitu beragam, maka pengetahuan tentang tutup kepala/tengkuluk daerah Jambi, dan teknik-teknik pemakaiannya, sangat memegang peranan di dalam kehidupan sejarah budaya masyarakat Jambi. Disaat telah berkembang berbagai model penutup kepala, tetapi tengkuluk masih dipakai oleh masyarakat Kota Jambi khususnya seberang Kota Jambi. Banyak anak mudayang tidak tahu dan tidak mengenali tengkuluk sebagai warisan leluhur Jambi. Padahal, penggunaan tengkuluk sejatinya membuat wanita lebih bersahaja dan sekaligus melestarikan salah satu kekayaan budaya yang dimiliki Indonesia. B. METODE Metode penelitian yang digunakan adalah metode sejarah karena jenis penelitian yang dilakukan termasuk jenis penelitian historis, yang merekonstruksi ulang peristiwa sejarah penting di masa lampau dalam kehidupan masyarakat melalui sumber-sumber yang menjadi bukti atau jejak sejarah untuk mengungkapkan fakta-fakta sehingga dapat diambil kesimpulan secara kronologis. Oleh karena itu metode sejarah yang dimaksud adalah suatu sistem berdasarkan prosedur yang benar untuk mencapai kebenaran sejarah. Keseluruhan prosedur metode sejarah dapat dicapai melalui beberapa tahapan, yaitu pengumpulan sumber (heuristik), kritik dan seleksi sumber, interpretasi, dan penulisan (historiografi). y y g Tengkuluk menjadi kebudayaan Masyarakat Jambi yang telah dikenal oleh seluruh Indonesia, menjadi kebudayaan khas perempuan Jambi pada upacara-upacara adat tertentu menggunakan tengkuluk sebagai pelengkap busana perempuan di Kota Jambi, bahkan pernah digunakan oleh beberapa tokoh pemerintahan dalam agenda penting mereka. Berbagai macam bentuk fashion penutup kepala masa kini seperti hijab, turban, pashmina dll Sehingga sangat perlu dilakukan penelitian ini untuk mengetahui bagaimana sejarah tengkuluk di Kota Jambi, sehingga penulis tertarik untuk melakukan penelitian tentang “Perkembangan Tengkuluk Di Kota Jambi Tahun 1946 - 2017”. C. HASIL DAN PEMBAHASAN C. HASIL DAN PEMBAHASAN PENELITIAN 1. Hasil Penelitian C. HASIL DAN PEMB PENELITIAN 1. Hasil Penelitian PENELITIAN 1. Hasil Penelitian 14 E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 Jurnal IstoriaProdi Pendidikan Sejarah FKIP Universitas Batanghari Jambi Jurnal IstoriaProdi Pendidikan Sejarah FKIP Universitas Batanghari Jambi Secara teoritis penamaan Jambi ada dugaan kuat untuk menamakan sebuah ibu kota pemerintahan atau negara. Kalau dikaitkan dengan seorang tokoh seorang putri yang bernama Putri Selaras Pinang Masak (lara Pinang Masak) yang bersuamikan Paduka Datuk Berhala yang Islam masaih terlalu muda, dikarenakan Islam secara catatan historis diterima masyarakat Sumatra berkisar abad ke XIII atau abad XIV, padahal cerita Cina menyatakan nama Jambi sudah ada sejak abad ke IX. Sebelum abad itu tidak ada catatan tentang nama Jambi. Nama Jambisangat dekat dengan nama Melayu, yang berkaitan erat dengan peristiwa expedisi pemalayu tahun tahun 1275 oleh Kartanegara dari Singosari. Pemalayu artinya berangkat dari Malayu. a) Karakteristik Melayu di Kota Jambi Daerah Jambi tidak dapat dipisahkan dari sungai. Sungai bagi masyarakat Jambi adalah urat nadi dan sumber pendapatan mereka. Sungai di Provinsi Jambi dimanfaatkan untuk kegiatan mengelola sarana transportasi sungai, seperti perahu dan kapal. Sungai juga menjadi jalur transportasi utama ke arah pedalaman. Hal ini disebabkan karena sarana transportasi darat (jalan raya) yang ada belum dapat menjangkau semua daerah pedalaman. Sungai yang membentang dari hulu sampai hilir Jambi adalah sungai Batanghari. Sungai ini merupakan jalur penting bagi pelayaran perdagangan dan bukti mengetahui peradaban yang ada di pedalaman Jambi. Lain daripada itu, sungai Batanghari merupakan jalur pelayaran dan perdagangan terpenting bagi masyarakat Jambi. Ia memiliki peranan penting dalam budaya, ekonomi, dan politik Jambi dengan banyaknya pendatang yang menggunakannya sebagai jalur untuk bisa keluar-masuk ke pedalaman Jambi (Nurdin.2010: 26). Satu satunya sumber tertulis tertua yang menyebutkan nama Jambi adalah berita dan catatan Cina dengan sebutan Chan- pei. I-tsing, pendeta Budha dari Cina menuliskan negara-negara di laut selatan yang memeluk agama Budha termasuk Chan-pei dalam pelayaran ke Nalanda, terletak di Sungai Batang Hari yang sebelumnya pada abad ke VII bernama Malayu. Jambi baru muncul tahun 853 M. Melayu Jambi memiliki suatu karakteristik tersendiri, untuk mengetahui karakteristiknya penulis akan membahas nama Jambi. Secara historis, yang dikemukakan oleh Budiharjo nama Jambi berasal dari nama Jambi. Sebuah nama tumbuhan yang diberi nama lain pinang. Jadi antara nama Jambi dan pinang setali tiga uang. Dari pengertian tersebut timbul suatu pertanyaan kapan, siapa, mengapa ada nama Jambi untuk menyebutkan suatu perkampungan (Anonim. 1997:6). Pencapaian kehidupan Melayu di nusantara, tidak hanya lekat pada Islam saja, melainkan sebuah pencapaian yang sangat komplek, melaui perdagangan yang membentuk dan menyebarkan budaya, hingga menjadi bangsa penakluk daereh- daerah lain. Pencapaian ini tentu 15 Jurnal IstoriaProdi Pendidikan Sejarah FKIP Universitas Batanghari Jambi E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 sampai di Alam Melayu beberapa sebelum kelahiran Isalm. Gugusan kepulauan Melayu telah terkenal dengan kawasan yang kaya dengan hasil bumi yang sangat diperlukan oeleh negara-negara asing (Yahaya. 1998:3). saja tidak hanya di maknai sebagai sebuah peristiwa sejarah yang hanya untuk dikenang dalam memori kolektif kita di dalam seminar ataupun di dalam akademisi, melainkan kita posisikan sebagai sejarah bangsa yang dapat menginspirasi masyarakat sekarang untuk membangun masa depan bangsa. Didalam pemikiran, terbentuknya suatu kebudayaan masyarakat tidak terlepas dari proses sejarah itu sendiri. Melayu Jambi menjadi objekkebudayaan, tidak terlepas dari proses sejarah menjadi kebudayaan. Sejarawan Leonard Y. b) Sejarah b) Sejarah Perkembangan Tengkuluk Masa Kuno Atau Seberang Tempo Dulu a) Karakteristik Melayu di Kota Jambi Andaya menyatakan, dalam konteks regional maupun internasional di masa lampau, Jambi merupakan salah satu sumber peradaban dan asal-usul orangorang Melayu di Sumatera (Andaya. 2001: 315-330). Penafsiran sebuah peristiwa sejarah merupakan sebuah keharusan, sehingga intisari dari peristiwa sejarah dapat kita resapi dan maknai dalam kehidupan, dapat membangun peradaban yang lebih tinggi dan agung untuk kesejahteraan umat manusia. Dalam sudut pandang agama, sikap mengambil pelajaran terhadap peristiwa sejarah menjadi salah satu bagian penting dalam kehidupan, dalam sudut pandang Islam sesunggugnya Allah SWT memberikan sinyal hukum kesejarahan (historicl law atau sunnah tarikhiyah) yang yang berlaku di alam atau dalam masyarakat. y Masyarakat Melayu Jambi dilihat dari tradisi yang mana ungkapkan bagian terdahulu, bahwa tradisi suatu konsep agama tradisional. Konsep yang di maksud adalah Islam tradisonal seperti kebiasaan, adat keagamaan, dan praktek keagamaan. Secara harfiah adat mempunyai arti suatu kebiasaan yang terjadi berulang kali tetapi tidak mengalami perubahan pada sifat dan zatnya. Menurut istilah, adat adalah suatu aturan yang dibuat manusia yang berasal dari kebiasaan-kebiasaan yang dipandang baik untuk mengatur cara hidup, berpikir, berbuat, dan bertindak dalam kehidupan bermasyarakat (Pemkot & Lembaga Adat Kota Jambi. 2004:16). Penyebaran agama Islam di Alam Melayu melalui dua tahap ; tahap kedatangan atau ketibaan dan tahan perkembangan. Berbagai-berbagai pendapat tentang tarihk kedatangan Islam ke Alam Melayu ; ada yang mengatakan abad kesembilan Masihi, ada yang menyatakan pada abad kedelapan Masehi dan ada pula mengatakan lebih awal lagi yaitu sejak zaman Nabi Muhammad SAW yaitu abad ketujuh Masehi Islam sudah tiba di Alam Melayu . Satu masalahyang tidak dapat bisa dinafikan bahwa kapa-kapal perniagaan Arab telah Masyarakat Melayu Jambi dilihat dari tradisi yang mana ungkapkan bagian terdahulu, bahwa tradisi suatu konsep agama 16 Jurnal IstoriaProdi Pendidikan Sejarah FKIP Universitas Batanghari Jambi E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 tradisional. Konsep yang di maksud adalah Islam tradisonal seperti kebiasaan, adat keagamaan, dan praktek keagamaan. Secara harfiah adat mempunyai arti suatu kebiasaan yang terjadi berulang kali tetapi tidak mengalami perubahan pada sifat dan zatnya. Menurut istilah, adat adalah suatu aturan yang dibuat manusia yang berasal dari kebiasaan-kebiasaan yang dipandang baik untuk mengatur cara hidup, berpikir, berbuat, dan bertindak dalam kehidupan bermasyarakat (Pemkot & Lembaga Adat Kota Jambi. 2004:16). tradisional. Konsep yang di maksud adalah Islam tradisonal seperti kebiasaan, adat keagamaan, dan praktek keagamaan. Secara harfiah adat mempunyai arti suatu kebiasaan yang terjadi berulang kali tetapi tidak mengalami perubahan pada sifat dan zatnya. a) Karakteristik Melayu di Kota Jambi Menurut istilah, adat adalah suatu aturan yang dibuat manusia yang berasal dari kebiasaan-kebiasaan yang dipandang baik untuk mengatur cara hidup, berpikir, berbuat, dan bertindak dalam kehidupan bermasyarakat (Pemkot & Lembaga Adat Kota Jambi. 2004:16). Tengkuluk adalah salah satu pelengkap adat dalam tradisi berbusana, yang sering digunakan baik untuk sehari-hari maupun untuk saat khusus, Tutup kepala adalah produk adat dan budaya yang menggunakan aspek kehidupan bermasyarakat. Penutup kepala merupakan lambang yang memiliki makna, simbol dan wibawah serta mencerminkan kepribadian masyarakat serta alam pikiran masyarakat setempat. masyarakat serta alam pikiran masyarakat setempat. Gambar 1. Tengkuluk Tudung Lingkup Pada Tahun 1942 Gambar 1. Tengkuluk Tudung Lingkup Pada Tahun 1942 Gambar 1. Tengkuluk Tudung Lingku Pada Tahun 1942 b) Sejarah Perkembangan Tengkuluk Masa Kuno Atau Seberang Tempo Dulu Indonesia dikenal sebagai bangsa dan Negara yang memiliki kekayaan budaya luar biasa. Keindahannya, keragamannya, juga nilai falsafah yang terkandung di dalamnya. Dalam keanekaragaman budaya tersebut, satu diantaranya adalah yang dimiliki Propinsi Jambi, Sumatera. Jambi dikenal sebagai daerah yang unik dan menyimpan banyak kekayaan alam dan budaya. Dibelah oleh Sungai Batanghari, membentuk kehidupan masyarakat yang bersendi pada adat budaya dan religi yang kuat sejak masa yang sangat lama. Pada tahun 1452 mulai tampak pengaruh Islam yang dibawa oleh Datuk Paduko Berhalo seorang Ulama Asia Panglima Tentara Turki yang menetap di Muara Sabak, konon ceritanya menikah dengan putri raja Jambi yang bernama Putri Selaras Pinang Masak Putri yang memegang teguh pada prinsip”Tiga Tungku Sajarangan” yang dimaksud dengan hal tersebut yaitu “Adat Bersendi Syara’, Syara’ Bersendi Kitabullah.” Makna yang terkandung di dalamnya adalah Syara’ (ayat) mengatakan Habluminallah. Adat memakai Habluminannas. Prinsip dalam falafah kehidupan ini memperlihatkan dan menjelaskan, tidak adanya pertentangan antara agama dengan adat pada sejak saat Salah satu kekayaan Jambi adalah kebiasaan kaum wanita mengenakan penutup kepala atau yang disebut tengkuluk. Setiap daerah di Jambi memiliki ragam tengkuluk yang unik, cantik sekaligus penuh makna. Meski arus moderenisasi tak terhindarkan, namun tengkuluk masih bisa ditemukan dalam kehidupan sehari- hari maupun pada acara-acara istimewah. 17 Jurnal IstoriaProdi Pendidikan Sejarah FKIP Universitas Batanghari Jambi E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 tengkuluk dan makna dari beberapa daerah”. agama Islam masuk (Aswar.2010). Pendapat lain, mengatakan bahwa keberadaan tengkuluk diketahui sudah ada sejak jaman kerajaan Melayu, yakni pada sebuah kerajaan yang terletak di pulau Sumatra sekitar abad ke-7. Pada masa tersebut, tengkuluk digunakan oleh kaum ibu- ibu dalam berbagai kegiatan dan kesempatan (Putu Elmira, 2018, https://www. Berdasarkan dari hasil wawancara diatas dapat dilihat bahwa masuknya tengkuluk di Kota Jambi kurang lebih pada abad ke 7 dan ke 14, namun demikian informasi ini belum dapat dibuktikan secara valid. Hanya berdasarkan keterangan dari beliau saat ini, kemudian berdasarkan wawancara yang dilakukan dengan Ujang Haryadi selaku pamong budaya museum siginjai dan dosen sejarah FKIPUnbari adalah sebagai berikut (Wawancara. Hariadi: 2021). p Liputan6.com/lifestyle/mengenal- Liputan6.com/lifestyle/mengenal- tengkulukpenutup-kepala-khas-perempuan- Jambi. (diakses tanggal 23 Juli 2019). tengkulukpenutup-kepala-khas-perempuan- Jambi. (diakses tanggal 23 Juli 2019). Gambar 2. Tengkuluk Umo Pada Tahun 1946 Gambar 2. Tengkuluk Umo Pada Tahun 1946 “Mengatakan bahwa mungkin sekitar tahun 19an tengkuluk masuk ke Kota Jambi, tapi memang belum ada tahun pastinya, akan tetapi pada abad ke 7 itu sudah ada tengkuluk Jambi yg terletak di daerah Kerinci, dan pada tahun 1452 tengkuluk itu mulai berkembang karena di bawa oleh datuk paduko berhalo karena pengaruh Islam.” Berdasarkan hasil wawancara yang dilakukan dengan Sri Purnama Syam selaku Kepala Dinas Pariwisata Kota Jambi, mengatakan bahwa : Tengkuluk atau kuluk merupakan penutup kepala warisan budaya tradisional perempuan melayu Jambi yang sudah mulai ditinggalkan, namun saat ini pemerintah bersama masyarakat melayu Jambi kembali mengeliatkan tradisi berbusana yang telah diwariskan oleh leluhurnya. Kemudian berdasarkan wawancara yang dilakukan dengan Nurlaini (2021) selaku kepala museum Jambi beliau mengatakan bahwa: , g “Beliau mengatakan bahwa tidak begitu memahami sejarah asal mulanya tengkuluk di Kota Jambi, karena beliau wisudawan seni tari, ia pun dari dulu memang penari, tetapi beliau mengatakan kurang lebih sekitar abad ke 7 dan 14 adanya tengkuluk, sedangkan di Kota Jambi sendiri kurang lebih masuk saat kemerdekaan Indonesia. Beliau juga mengatakan ada bnyak sekali jenis tengkuluk yg ada di seluruhProvinsi Jambi setiap daerah memilik jenis tengkuluknya masing-masing beliaupun menjelaskan jenis “Menjelaskan di Jambi ini yg terdeteksi ada sekitar 99 macam jenis tengkuluk itu sudah termasuk yg di modifikasi, tengkuluk ini sebenarnya sudah ada di Provinsi Jambi pada abad ke-7, tetapi blum masuk Kota 18 Jurnal IstoriaProdi Pendidikan Sejarah FKIP Universitas Batanghari Jambi E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 Jambi, saat itu tengkuluk ini hanya dipakai dalam acara ritual agama, dan kegiatan sakral lainnya, tidak hanya wanita tetapi zaman dulu laki-laki juga menggunakan tengkuluk, bentuknya pun tidak seperti tengkuluk zaman sekarang, bentuknya lebih seperti agak tidak beraturan, seiring berjalannya waktu tengkulukpun hanya di pakai oleh wanita dan menyebar ke seluruh daerah di Provinsi Jambi saat itu dipakai juga oleh wanita untuk bercocok tanam tidak hanya saat kegiatan resmi. Berselangnya waktu, pada tahun 1452 datuk paduko berhalopun datang membawa pengaruh islam dan disitu lah tengkuluk menjadi semakin sering di pakai oleh wanita di Daerah Jambi karen islam menganjurkan untuk wanita menutupi rambutnya. Sekitar tahun 1945 Indonesia mulailah tengkuluk masuk di Kota Jambi yaitu berada di Seberang Kota Jambi, tetapi mereka tetap memilik jenis tengkuluk sendiri walaupun sama seperti tengkuluk lain yg ada di Wilayah Provinsi Jambi. Han yaitu di zaman perunggu memberikan pengaruhnya ke Asia Tenggara sampai ke Indonesia. Gambar 2. Tengkuluk Umo Pada Tahun 1946 Pengaruh tersebut berawal dari kegiatan bertani, menangkap ikan di laut, berlayar dan memancing. Tradisi mereka berpakaian terutama pakaian wanita sangat mempengaruhi tradisi berpakaian suku-suku di Provinsi Jambi. Pengaruh ini tampak dari seratus macam tradisi penutup kepala atau tengkuluk yang tersebar diseluruh daerah yang ada di Jambi. Bentuk dan cara pemakaian yang beraneka ragam dari tengkuluk tersebut disesuaikan dengan penggunaannya dalam kegiatan sehari-hari. Namun pernyataan ini dibantah oleh kepala museum Jambi Nurlaini, selaku kepala museum Jambi beliau mengatakan bahwa (Wawancara. Nurlaili: 2021). “Tekuluk/tengkuluk sudah ada pada zaman melayu tua, sementara Vietnam masuk pada zaman melayu muda. Dimana ada kerajaan melayu maka di sana ada kebudayaan tengkuluk. Lebih lanjut Nurlaini menjelaskan bahwa pada awalnya tengkuluk yang dipakai oleh kaum wanita melayu Jambi merupakan jenis kain sarung berukuran (pendek) yang dipakai untuk kegiatan sehari- hari oleh para petani wanita melayu Jambi pergi ke umo/ladang/sawah. Berdasarkan wawancara tersebut dapat dilihat bahwa pada dasarnya tengkuluk sudah ada di Provinsi Jambi sejak dahulu, namun bukan di Kota Jambi, baru masuk ke Kota Jambi secara khusus semenjak masuk dan berkembangnya agama Islam di Kota Jambi yang dibawa oleh datuk Paduko Berhalo pada tahun 1542. Tradisi penutup kepala atau tengkuluk sudah ada sebelum masyarakat Jambi mengenal agama Islam. Pada masa itu tengkuluk hanya sebagai aksesoris atau hiasan kepala sebagai suatu keindahan perempuan- perempuan melayu Jambi, yang digunakan sebagai penutup kepala Selain itu, diduga bermula dari pengaruh kebudayaan Dongson. Kebudayaan Dongson merupakan kebudayaan yang berasal dari Vietnam yang ada pada masa dinasti 19 Jurnal IstoriaProdi Pendidikan Sejarah FKIP Universitas Batanghari Jambi E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 10% yang telah terpublikasi. Ragam tutup kepala berasal dari kata ragam berarti macam-macam, tutup berarti melindungi, kepala adalah bagian yang tertinggi dalam badan manusia berbagai bangsa di dunia, yang sakral yang terpening dari seluruh tubuh. Kepala juga merupakan tempat masuknya roh atau sukma manusia, oleh karena itu kepala perlu dihias dan dipelihara. Ragam tutup kepala berarti adalah bermacam-macam pelindung kepala yang bagian tertingginyadalam tubuh manusia. Lebih lanjut Ibu Nurlaini mengatakan, bahwa berbeda dengan budaya-budaya lain yang mengenal tutup kepala tekuluk ini. Tehnik mengikat dan menyisipkan tengkuluk/kuluk Melayu ini tidak dimiliki oleh suku dan budaya lain. pada saat pesta, atau hari-hari besar. Tengkuluk juga dijadikan sebagai penahan kepala dari beban ketika meletakan barang bawaan di atas kepala atau pun berfungsi sebagai pelindung kepala dari teriknya sengat matahari ketika perempuan melayu pergi berladang atau berumo pada saat itu. Gambar 2. Tengkuluk Umo Pada Tahun 1946 Tengkuluk dan baju kurung hanya sebagai penutup tubuh dan aksesoris. Namun setelah masuknya agama Islam di tanah melayu kebiasaan menggunakan baju kurung dan penutup kepala tengkuluk dapat diartikan sebagai bentuk ketaatan dalam menjalankan agama Islam. Adat dan agama bersatu padu membentuk suatu keharmonisan dalam kehidupan masyarakat. Sesuai dengan seloko adat melayu Jambi “adat bersendi syarak, syarak bersendi kitabullah. Syarak mengato, adat memakai.” Setelah masuknya agama Islam di tanah melayu Jambi, masyarakat melayu Jambi terkenal dengan budaya dan keyakinan yang kuat pada ajaran agama Islam. Sehingga antara adat dan ajaran Islam dalam kehidupan masyarakat Jambi selalu sejalan dengan pedoman hidupnya. Hal tersebut dalam seloko ada yang menjadi pedoman yaitu “adat bersendi syara’, syara’ bersendi kitabbullah” yang berarti yang semula berawal dari “adat bersendi layak, alur dan patut”. Kini layak dan patut adalah dalam ukuran simbolnya berdasarkan ketentuan agama Islam yaitu Syara’ dan Kitabbullah. Ketika agama Islam sudah berkembang di tanah melayu, baju kurung dan tengkuluk menjadi satu kepaduan yang berfungsi bukan hanya sekadar sebagai penutup kepala atau pembungkus tubuh, namun sudah keharusan sebagai penutup aurat perempuan sesuai dengan syariat Islam yang berkembang di tanah melayu. Dalam pemakaian ragam tengkuluk sebagai warisan budaya melayu tak terlepas dari estetika dan artistika. Jika estetika merupakan konsep yang dibentuk oleh masyarakat yang melahirkan bentuk-bentuk tengkuluk. Maka artistika merupakan suatu keindahan yang tampak atau yang terlihat. Dalam hal ini terlihat dalam berbagai macam tengkuluk. Pakaian adat perempuan melayu Jambi sesuai dengan agama yang mereka anut, umumnya perempuan dewasa melayu Jambi menutup aurat. Kepala dan rambut merupakan salah satu aurat kaum perempuan dan rambut sebagai mahkota perempuan tidak boleh terurai begitu saja. Oleh Menurut Ibu Nurlaini, dari estetika ragam koleksi tengkuluk di seluruh tanah melayu Jambi dan baru 20 Jurnal IstoriaProdi Pendidikan Sejarah FKIP Universitas Batanghari Jambi E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 Berdasarkan dari keseluruhan hasil wawancara dengan beberapa informan dari masyarakat sekitaran Kota Seberang penulis dapat menyimpulkan bahwa memang benar pada tahun 1946 masyarakat Seberang Kota Jambi telah mengenal penutup kepala yang pada masa itu belum memiliki nama dan jenis-jenis tertentu, hanya biasanya sebagai penutup kepala saat keluar rumah, mengaji, acar adat dan kegiatan keseharian lainnya. Seiring berjalannya waktu sekitar tahun 1960an mulai adanya beberapa jenis penutup kepala yang digunakan masyarakat (Wawancara, Yati:2021 ; Lia: 2021). karena itu, umumnya bagi masyarakat Jambi antara pakaian dan tutup kepala merupakan satu kesatuan yang saling berhubungan yang menjadikan simbol kepatutan dalam berbusana perempuan melayu Jambi. Gambar 2. Tengkuluk Umo Pada Tahun 1946 Tengkuluk merupakan salah satu pelengkap adat dalam tradisi berbusana yang harus digunakan baik untuk keperluan sehari-hari maupun untuk keperluan acara khusus. Pada setiap model tengkuluk/kuluk, terkandung falsafah yang memiliki makna nilai atau norma yang menentukan bagaimana kita harus bersikap, bertindak dan berperilaku, juga memberikan aturan untuk kita (Aswar. 2010). c) Fungsi Tengkuluk “Menurut Nurlaili, tutup kepala adalah produk adat dan budaya yang mengungkapkan aspek kehidupan masyarakat melayu. Penutup kepala dan pakaian merupakan simbol atau lambang yang memiliki makna, simbol maupun wibawa serta cerminan kepribadian masyarakat danalam pikiran masyarakat setempat tersebut. Lebih lanjut Nurlaili memberikan contoh, penutup kepala wanita suku Melayu Jambi yang penggunaan ujung kainnya terjuntai di sebelah kanan menandakan bahwa si wanita telah menikah dan jika ujung kainnya jatuh ke sebelah kiri berarti si wanita masih dapat dilamar yang berarti single”. Penutup kepala atau tengkuluk ini berfungsi sebagai pelindung kepala dari cuaca panas, mencerminkan status sosial dan budaya dalam bentuk identitas diri, dan simbol kehormatan, kebanggaan dan martabat seseorang. Tengkuluk sendiri mempunyai berbagai macam bentuk, jenis dan cara lipat sesuai dengan fungsi atau kegunaan. g Pada tahun 1946 tengkuluk hanya berfungsi sebagai kegiatan sehari-hari, untuk acara adat, dll akan tetapi hanya diletakkan diatas kepala sebagai penutup kepala melindungi para Wanita pada masa itu dari sinar matahari yang hanya dari kain sarung wanita, biasanya para Wanita juga memakai bedak beras sebagai alas mukanya. Kemudian tengkuluk pada masa itu juga sering digunakan dalam acara adat misalnya pernikahan dimana tengkuluk dari jenis kain tersebut dililitkan kekepala sebagai hiasan kepala untuk memperindah penampilan Wanita pada masa itu, biasanya juga ditambahkan daun- Berdasarkan penuturan tersebut dapat diketahui bahwa tengkuluk pada masa itu juga menjadi simbol dari seorang perempuan yang membedakan antara perempuan yang sudah menikah dan perempuan yang berlum menikah. 21 Jurnal IstoriaProdi Pendidikan Sejarah FKIP Universitas Batanghari Jambi E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 daun sebagai tambahan untuk memperindah, akan tetapi belum memiliki sebutan atau nama yang spesifik. masyarakat yang bersendi pada adat budaya dan religi yang kuat sejak masa yang sangat lama. Salah satu kekayaan Jambi adalah kebiasaan kaum wanita disana mengenakan penutup kepala atau yang disebut tengkuluk. Setiap daerah di Jambi memiliki ragam tengkuluk yang unik, cantik sekaligus penuh makna. Meski arus moderenisasi tak terhindarkan, namun tengkuluk masih bisa ditemukan dalam kehidupan sehari-hari maupun pada acara-acara istimewah. p Seiring berjalannya waktu pada tahun 1960 tengkuluk sudah memiliki beragam macam bentuk dan nama, nama yang disematkan pada setiap jenis tengkuluk dibuat oleh masyarakat berdasarkan jenis lilitan yang menyerupai suatu benda atau tumbuhan sekitar. Sehingga lahirlah macam-macam tengkuluk dengan nama-namanya tersendiri. Dari tahun 1960 kepopuleran tengkuluk semakin tidak diragukan lagi, sudah banyak dipakai oleh semua kalangan, termasuk kalangan pemerintahan dalam acara resmi Kota Jambi. c) Fungsi Tengkuluk Dan pada tahun 2017 tengkuluk semakin merambah kedunia fashion yaitu dengan adanya macam-macam event maupun kegiatan pemerintahan dimana tengkuluk hadir sebagai pakaian tradisional Kota Jambi, sehingga pada tahun 2020 Ketua DPR RI yaitu Puan Maharani menggunakan tengkuluk pada upacara kemendekaan RI, sehingga tengkuluk semakin memancanegara dengan adanya hal tersebut. Tengkuluk adalah salah satu pelengkap adat dalam tradisi berbusana, yang sering digunakan baik untuk sehari-hari maupun untuk saat khusus. Tutup kepala adalah produk adat dan budaya yang menggunakan aspek kehidupan bermasyarakat. Penutup kepala merupakan lambang yang memiliki makna, symbol, wibawa, dan kepribadian masyarakat serta alam pikiran masyarakat setempat. Sebagai contoh, penutup kepala yang ujung kainnya tergantung disebelah kanan, berarti wanita sudah menikah (Nurdin. 2010). 1. Penutup kepala yang ujung kainnya tergantung disebelah kanan berarti si wanita sudah menikah, lihat gambar di bawah ini. d) Eksistensinya Saat Ini Dan Nilai-Nilai Yang Relevan Didalamnya d) Eksistensinya Saat Ini Dan Nilai-Nilai Yang Relevan Didalamnya Indonesia dikenal sebagai bangsa dan Negara yang memiliki kekayaan budaya luar biasa. Keindahannya, keragamannya, juga nilai falsafah yang terkandung di dalamnya. Dalam keanekaragaman budaya tersebut, satu diantaranya adalah yang dimiliki Propinsi Jambi, Sumatera. Jambi dikenal sebagai daerah yang unik dan menyimpan banyak kekayaan alam dan budaya. Dibelah oleh Sungai Batanghari, membentuk kehidupan 2. Penutup kepala yang ujung kainnya tergantung disebelah kiri berarti wanita masih dapat di lamar atau masih gadis, gambar seperti yang di bawah ini. Tata hias tutup kepala ini menurut Jan Harold Brunvand merupakan bagian dari folklore. Folklore terdiri dari kata flok dan lore. Folk adalah sekelompok orang yang memiliki ciri-ciri pengenal kebudayaan yang 22 Jurnal IstoriaProdi Pendidikan Sejarah FKIP Universitas Batanghari Jambi E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 Kepala Khas Perempuan Jambi. Tanpa Penerbit. membedakan dari kelompok lain, dan lore adalah tradisi dari flok yang diwariskan turun temurun melalui tutur kata yang disertai dengan perbuatan. Melalui folklore inilah terwariskan nilai-nilai tradisi Klux dari generasi ke generasi, termasuk mengenai tutup hias kepala atau tengkuluk (Yusniana. 2013). Nurdin, Ratu. M. Z. 2010. Tengkuluk Penutup Kepala Warisan Luhur dari Jambi.Jakarta: Dian Rakyat Anonim. 1997. Sejarah Kota Jambi Pada Masa Lampau, Sekarang dan Yang Akan Datang, (Jambi: Lembaga Adat Tanah Pilih Kotamadya Jambi, 1997). Tanpa Penerbit. g D. KESIMPULAN Tengkuluk merupakan identitas jatidiri perempuan melayu. Tradisi penutup kepala atau tengkuluk sudah ada sebelum masyarakat Jambi mengenal agama Islam. Pada masa itu tengkuluk hanya sebagai aksesoris atau hiasan kepala sebagai suatu keindahan perempuan-perempuan melayu Jambi, yang digunakan sebagai penutup kepala pada saat pesta, atau hari-hari besar. Tengkuluk juga dijadikan sebagai penahan kepala dari beban beban ketikan meletakan barang bawaan di atas kepala atau sebagai pelindung kepala dari teriknya sengat matahari ketika perempuan melayu pergi berladang atau berumo pada saat itu. Yahaya. 1998. Islam di Alam Malayu. Kuala Lumpur: Dewan Bahasa dan Pustaka. Andayana. 2001. The Searh For The ‗Origins Of Melayu‘,Journal Of Southeast Asian Studies, 32. Pemerintah Kota Jambi Dan Lembaga Adat Tanah Pilih Pasko Batuah Kota Jambi. 2004. Ikhtisar Adat Melayu Kota Jambi. Aswar. 2010. Anta kesuma Suji. Jakarta: Djambatan. Putu Elmira, 2018, https://www. Liputan6.com/lifestyle/mengenal -tengkulukpenutup-kepala-khas- perempuan-Jambi. (diakses tanggal 23 Juli 2019) Sebagai bentuk kebudayaan tengkuluk mengalami berbagai perubahan hingga saat ini seperti perkembangan persepsi masyarakat tentang tengkuluk. Bagi wanita yang berhijab, menggunakan tengkuluk adalah dengan cara menggunakan dahulu hijab untuk menutupi rambut, telinga dan leher. Lalu baru di pasangkan tengkuluk di atas kepala. Perubahan juga terjadi dari segi bahan-bahan yang digunakan dalam unsur-unsur yang terdapat pada tengkuluk. Wawancara dengan Ema selaku Kepala Dinas Pariwisata Kota Jambi pada tanggal 28 September 2021 Wawancara dengan Ujang Haryadi Haryadi Hariadi selaku pamong budaya museum siginjai dan Wawancara dengan Ujang Haryadi Haryadi Hariadi selaku pamong budaya museum siginjai dan E. DAFTAR PUSTAKA Nurlaini, 2017. Mengenal Sejarah dan Filosofi Tengkuluk, Penutup Nurlaini, 2017. Mengenal Sejarah dan Filosofi Tengkuluk, Penutup 23 Jurnal IstoriaProdi Pendidikan Sejarah FKIP Universitas Batanghari Jambi E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 E-ISSN 2597-8845 Vol. 6 No. 1. April 2022 dosen sejarah fkip unbari pada tanggal 30 September 2021 Wawancara dengan Nurnaili selaku kepala museum Jambi pada 02 Oktober 2021 Usaha Kecil Menegah Riset & Penalaran EXIST, 2014 Wawancara dengan ibu Nurnaili selaku kepala museum Jambi pada 02 Oktober 2021 Wawancara Ndak Yati (Tetuo sebrang, tgl 10 Oktober 2021), Er (selaku pengrajin batik, tgl 10 Oktober 2021) dan Mis Lia (Tetuo, tgl 13 April 2021) Nurdin, Ratu. M. Z. 2010. Kuluk Penutup Kepala Warisan Luhur dari Jambi.Jakarta: Dian Rakyat. Yusniana, H. B. 2013. Tekuluk Penutup Kepala Warisan Luhur dari Jambi Edisi ke Dua. Jakarta: Dian Rakyat. 24
https://openalex.org/W4362738357	https://zenodo.org/record/7813632/files/A%20PR%C3%81TICA%20DO%20ALEITAMENTO%20MATERNO%20E%20FATORES%20RELACIONADOS%20AO%20DESMAME%20PRECOCE_%20UMA%20REVIS%C3%83O%20INTEGRATIVA%20%E2%80%93%20ISSN%201678-0817%20Qualis%20B2.pdf	Portuguese	null	A PRÁTICA DO ALEITAMENTO MATERNO E FATORES RELACIONADOS AO DESMAME PRECOCE: UMA REVISÃO INTEGRATIVA	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	5,573	A PRÁTICA DO ALEITAMENTO MATERNO E FATORES RELACIONADOS AO DESMAME PRECOCE: UMA REVISÃO INTEGRATIVA Ciências da Saúde, Edição 121 ABR/23 / 09/04/2023 THE PRACTICE OF BREASTFEEDING AND FACTORS RELATED TO EARLY WEANING: NA INTEGRATIVE REVIEW REGISTRO DOI: 10.5281/zenodo.7813632 Thais Novais da Silva Raphael Silva Nogueira Costa Joyce Vilarins Santos Soares Priscila Rodrigues e Silva Láisa Renata Souza Ascenso Ana Caruline de Sá Cavalcante Ingryd Eccel Kill Ana Luísa Melo Silva Juliana Maria Alves Moraes Giovana Jenifer Santana de Oliveira Giovanna Biângulo Lacerda Chaves Lucas Tavares Domingos Alexia Beatriz da Silva Maria Luísa do Couto Ribeiro Lopes Maria Eduarda De Sousa Matos 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ISSN 1678-0817 Qualis B2 Revista Científica de Alto Impacto. Thais Novais da Silva Raphael Silva Nogueira Costa Joyce Vilarins Santos Soares Priscila Rodrigues e Silva Láisa Renata Souza Ascenso Ana Caruline de Sá Cavalcante Ingryd Eccel Kill Ana Luísa Melo Silva Juliana Maria Alves Moraes Giovana Jenifer Santana de Oliveira Giovanna Biângulo Lacerda Chaves Lucas Tavares Domingos Alexia Beatriz da Silva Maria Luísa do Couto Ribeiro Lopes Maria Eduarda De Sousa Matos 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Resumo Introdução: Amamentar é muito mais que nutrir um bebê. É um ato que promove uma interação entre mãe e filho que repercute no estado nutricional da criança. Porém devido a vários fatores muitas mulheres optam pelo desmame precoce. Objetivo geral: analisar quais as evidências científicas sobre a prática do aleitamento materno e os fatores relacionados ao desmame precoce publicados no período de 2015 a 2020. Metodologia: Foi realizada uma revisão do tipo integrativa. Pesquisa foi feita em dados eletrônicos, disponíveis na Biblioteca Virtual de Saúde (BVS), como a Scientific Eletronic Library Online (SCIELO), Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS), Medical Literature Analysis and Retrieval System Online ( MEDLINE), Base de dados em Enfermagem (BDENF). Estudo qualitativo baseado na análise de conteúdo de Bardin (1977). Resultado e Discussão: O presente trabalho foi feito através de 11 artigos nacionais, segundo a pesquisa feita, os fatores de destaque relacionados ao desmame precoce encontrados foram: retorno ao mercado de trabalho, assistência prestada e saúde mental, foi verificado também que o pré-natal pode ser responsável pela decisão da mulher em amamentar, o acolhimento e esclarecimento das dúvidas podem evitar intercorrências responsáveis pelo desmame precoce. Considerações finais: Medidas simples podem ser tomadas para evitar o desmame precoce, buscando informar de forma clara que são vários motivos que podem levar ao fim do aleitamento, e que muitas causas podem ser resolvidas por meio do acesso à informação, criação de estratégias, acolhimento e escuta humanizada. Palavra-chave: Aleitamento; Desmame; Enfermagem. Abstract Introduction: Breastfeeding is much more than nurturing a baby. It is an act that promotes an interaction between mother and child that affects the child’s nutritional status. However, due to several factors, many women opt for early weaning. General objective: is to analyze the scientific evidence on the practice of breastfeeding and factors related to early weaning published from 2015 to 2020. Methodology: An integrative review was carried out. Research was carried out on electronic data, available in the Virtual Health Library (VHL), such as the Scientific Electronic Library Online (SCIELO), Latin American and Caribbean Literature on Health Sciences (LILACS), Medical Literature Analysis and Retrieval System Online ( MEDLINE), Database in Nursing (BDENF). Qualitative study based on Bardin’s (1977) content analysis. Result and Discussion: The present work was done through 11 national articles, according to the research done, the prominent factors related to early weaning found were: Return to the labor market, care provided and mental health, it was also verified that prenatal care can being responsible for the woman’s decision to breastfeed, welcoming and clarifying doubts can prevent complications responsible for early weaning. Final considerations: Simple measures can be taken to prevent early weaning, seeking to clearly inform that there are several reasons that can lead to the end of breastfeeding, and that many causes can be resolved through access to information, creation of strategies, reception and humanized listening. Keyword: Breastfeeding; Weaning; Nursing. 1. INTRODUÇÃO O aleitamento materno é a ingestão do leite materno independente de receber ou não outros alimentos é o vínculo natural, a proteção e nutrição da criança, consequentemente a mais econômica e eficaz intervenção para redução da morbimortalidade infantil. É uma prática importante para mãe e bebê e perdura de forma diferente de mulher para mulher. A Organização Mundial de Saúde (OMS) reconhece e utiliza definições do aleitamento materno, classificando em: Exclusivo, predominante, aleitamento materno, complementado, misto ou parcial (MINISTÉRIO DA SAÚDE, 2015). O aleitamento materno é recomendado como alimento exclusivo para o bebê até os 6 meses de vida, considerado o alimento ideal para a sobrevivência e crescimento do recém-nascido, possuindo propriedades nutricionais, imunológicas e emocionais para o bebê. A prática traz benefícios não só para o recém-nascido quanto para a mãe, como a perda de peso, involução uterina, menor incidência de câncer de mama e de ovário, sendo também uma opção econômica, por fim trazendo melhoria a saúde e qualidade de vida das mães e bebês (MOIMAZ et al.,2013). Pelo fato de trazer tantos benefícios a saúde do recém-nascido e da mãe, a Organização Mundial de Saúde (OMS) definiu que a amamentação deve ser realizada até os seis meses de forma exclusiva e a partir disso utilizar a complementação com outros tipos de alimentos até os dois anos, já que antes desse prazo não se deve fazer nenhuma introdução alimentar, pois pode causar prejuízos nutricionais a saúde da criança ( ARAÚJO et al.,2013). A maternidade e o aleitamento materno historicamente foram utilizados como meio para afastar a mulher do mercado de trabalho, definindo então a maternidade como papel social feminino, mas com o passar dos anos leis foram criadas em defesa da mãe trabalhadora, dando a proteção legal para garantir o seu direito, a licença maternidade (KALIL; AGUIAR, 2016). A lei que ampara possui algumas brechas, já que é preconizado aleitamento materno exclusivo até os seis meses de vida e a lei só cobre a extensão da licença para funcionárias públicas federais, deixando por decisão dos empregadores estaduais, municipais e empresas privadas a decisão não obrigatória da extensão da licença, favorecendo o desmame precoce (KALIL; AGUIAR, 2016). A assistência empregada pelo enfermeiro visa promover, proteger e recuperar a saúde da criança e família, uma das ações de maior relevância nas consultas é o incentivo ao aleitamento materno, fazendo o acolhimento precoce da gestante. Nordeste apresentou o percentual mais baixo comparado as outras regiões (37,0%) (LIMA; NASCIMENTO; MARTINS, 2018). Nordeste apresentou o percentual mais baixo comparado as outras regiões (37,0%) (LIMA; NASCIMENTO; MARTINS, 2018). Nordeste apresentou o percentual mais baixo comparado as outras regiões (37,0%) (LIMA; NASCIMENTO; MARTINS, 2018). Devido a influência que o profissional de saúde têm sobre a permanência do aleitamento materno, a educação em saúde foi uma forma encontrada para informar as pessoas sobre a importância dessa prática por meio de palestras, visitas domiciliares, panfletagem roda de conversa e oficinas com o intuito de promover a maior adesão de mães a prática de amamentar e a troca de conhecimento técnico e popular ( VIEIRA et al.,2016). As intervenções realizadas pelo Enfermeiro na atenção primária de saúde (APS) são responsáveis pela promoção e proteção da saúde da mãe e bebê, o acompanhamento desde o pré-natal conscientiza sobre a importância do aleitamento materno e desmistifica o conhecimento cultural que muitas mulheres possuem e que influenciam no aleitamento. A educação em saúde promovida pelo profissional faz com que as mulheres se sintam seguras no cuidado à criança ou caso apareça alguma intercorrência se sintam seguras para procurar a equipe (HIGASHI et al.,2021). Devido a grande importância do aleitamento materno e ao alto índice de desmame precoce, o objetivo geral da pesquisa é analisar quais as evidências científicas sobre a prática do aleitamento materno e os fatores relacionados ao desmame precoce publicados no período de 2015 a 2020. Tendo como objetivo específico: descrever o contexto e aspectos conceituais referente ao aleitamento materno e desmame precoce; identificar as barreiras e estratégias utilizadas pelos profissionais de saúde sobre essa prática. Dado o exposto, espera-se contribuir para a produção do conhecimento sobre a importância do aleitamento materno, e medidas simples que podem ser tomadas para evitar o desmame precoce, buscando informar de forma clara que são vários motivos que podem levar ao fim do aleitamento, e que muitas causas podem ser resolvidas por meio do acesso à informação. 1. INTRODUÇÃO Dentre as orientações a que mais se destaca é a proteção e incentivo ao aleitamento materno (MONTESCHIO; GAIVA; MOREIRA, 2015). Mesmo com o investimento dos programas, a prática no Brasil ainda está longe do preconizado pela Organização Mundial de Saúde (OMS). Uma pesquisa realizada pela Secretaria Estadual de São Paulo (SES/SP) mostrou que o aleitamento materno era 41% nas capitais brasileiras, a região Norte com 45,9%, seguida da Centro-Oeste com 45,0%, Sul (43,9%) e Sudeste (39,4%), a região 2. METODOLOGIA O presente estudo se trata de uma revisão bibliográfica do tipo integrativa. É um método que permite a junção do conhecimento e incorporação dos estudos de forma mais ampla e em etapas, para melhor entender o estudo na prática (SOUZA et al.,2017). Busca através de artigos originais esclarecer as dúvidas e problemas que serão observados durante o estudo. Para Realização de uma revisão integrativa é necessário seguir 6 etapas sendo elas: 1º – Identificação do tema e seleção da questão de pesquisa; 2º- Estabelecimento dos critérios de inclusão e exclusão; 3º- Identificação dos estudos pré-selecionados e selecionados, 4º- Categorização dos estudos selecionados; 5º- Análise e interpretação dos resultados; 6- Apresentação da revisão/síntese do conhecimento (MARINI;LOURENÇO;BARBA,2017). A pesquisa foi realizada através de busca bibliográfica com base de dados eletrônicos, disponíveis na Biblioteca Virtual de Saúde (BVS), como a Scientific Eletronic Library Online (SCIELO), Latino Literatura -Americana e do Caribe em Ciências da Saúde (LILACS), Medical Literature Analysis and Retrieval System Online ( MEDLINE), Base de dados em Enfermagem (BDENF).Foram utilizados artigos originais em português disponíveis na íntegra, que se encaixam no tema proposto, publicados nos anos de 2015 a 2020,e excluídos artigos que não se enquadraram nos critérios de inclusão, carta ao leitor e artigos duplicados e em outros idiomas. A seleção de artigos foi feita através do fluxograma de Prisma. Tabela 01- Fluxograma referente à busca eletrônica nas bases de dados LILACS, BDENF, MEDLINE E SCIELO. FONTE: Autoria própria FONTE: Autoria própria Após a coleta e seleção de artigos, foi realizada uma Análise de dados qualitativa baseado na análise de conteúdo de Bardin (1977). O objetivo foi apresentar uma análise crítica de conteúdo, que com o tempo se tornou mais rígida utilizando as inferências. A análise foi feita por um conjunto de critérios que se divide em três etapas: pré-análise, exploração e tratamento. Na pré análise é feita a escolha do material que será utilizado por meio da leitura flutuante, formulam-se hipóteses e elaborar indicadores que norteiam a interpretação final. Na fase de exploração se codifica os dados e são transformados e agregados em unidades utilizando inferências, dando início então ao tratamento onde as nferências são interpretadas, baseando-se na análise crítica do autor. inferências são interpretadas, baseando-se na análise crítica do autor. 3. RESULTADOS No quadro 02 foram incluídos 11 artigos originais, que tem como principal objetivo identificar fatores que levam ao desmame precoce e a influência dos profissionais de saúde sobre essa prática, o tipo de estudo dos seguintes artigos, variam entre 3 estudos da coorte, 1 descritivo exploratório, 1 empírica descritivo exploratório do tipo pesquisa de campo , 2 qualitativo descritivo exploratório,1 transversal retrospectivo , 1 relato comunicativo crítico, 1 qualitativo descritivo e 1 estudo transversal de análise bivariada e multivariada. Grande parte dos artigos selecionados foram publicados por revistas de Enfermagem, em variadas regiões do país, principalmente na Região Sudeste com destaque para a cidade de São Paulo, as demais regiões foram: Centro- Oeste; Sul; Nordeste. Logo abaixo segue o quadro construído para evidenciar e organizar os principais itens dos artigos utilizados no estudo. QUADRO 02- Informações referente aos 11 artigos selecionados para pesquisa Título Autor/Ano Objetivos Tipo de Estudo Resultado s Conclusão Intenção de amamenta r, duração do aleitament o Materno e motivos para o desmame: um estudo decoorte, AMARAL et al.,2019 Avaliar a intenção materna de amamenta r, duração do aleitament o materno até os 24 meses e os motivos Estudo da Coorte 1.377 mães rastreadas. Os principais motivos relatados para desmame foram leite insuficient e (57,3%), retorno ao Apesar da intenção de amamenta r, persistem barreiras estruturais e sociais que interferem no sucesso QUADRO 02- Informações referente aos 11 artigos selecionados para 02- Informações referente aos 11 artigos selecionados para pesquisa Pelotas, RS, 2014* para o desmame no primeiro ano de vida. trabalho/es cola (45,5%) e recusa inexplicáve l do bebê (40,1%). da amamenta ção, especialm ente as relacionad as ao trabalho materno. Dificuldad es relacionad as ao aleitament o materno: análisede um serviço especializa do em amamenta ção CARREIRO et al.,2018 Analisar a associação entre o tipo de aleitament o e as dificuldade s relacionad as a essa prática entre mulheres e crianças assistidas em um ambulatóri o especializa do em amamenta ção. Estudo Transversal Retrospect ivo, por meio de análise de prontuário s O aleitament o materno exclusivo foi praticado por 72,6% das mulheres atendidas, nos primeiros 30 dias após o parto. 3. RESULTADOS Houve associação significativ a entre esta prática e as dificuldade s O aleitament o materno exclusivo foi o mais prevalente nos primeiros 30 dias pós-parto e diversas variáveis maternas e neonatais Associação entre a depressão pós-parto e a prática do aleitament o materno exclusivo nos três primeiros meses de SILVA et al., 2016 Verificar a associação entre a depressão pós-parto e a ocorrência do aleitament o materno exclusivo. Estudo de coorte transversal A amamenta ção exclusiva foi observada em 50,8% das crianças e 11,8% das mulheres apresentar am sintomatol ogia indicativa de depressão pós-parto. A depressão pós-parto contribuiu para redução da prática do aleitament o materno exclusivo. Assim, esse transtorno deveria ser incluído nas orientaçõe s de apoio desde o pré-natal e nos primeiros meses pós- parto Amament ação e as intercorrên cias que contribue m para o desmame precoce. OLIVEIRA et al.,2015 Conhecer a vivência de mães em relação à amamenta ção e as intercorrên cias que Pesquisa do tipo descritivo- exploratóri o As principais alegações para interrupçã o do AME : Déficit de conhecime Reforçou a necessidad e de ajustes no modelo de atenção vigente, ultrapassa desmame precoce. cia/insegur ança; Banalizaçã o das angústias maternas; Intercorrên cias da mama puerperal; Interferênc ias familiares; Leite fraco/insufi ciente; Trabalho materno. ade de técnicas pré- definidas, incentivan do a criticidade perceptiva dos profissiona is de saúde na construção de novos saberes e condutas. Manutenç ão de aleitament o materno no retorno ao trabalho. SOUZA et al.,2019 Descrever se havia dificuldade s e estratégias realizadas para manutenç ão do aleitament o. Pesquisa Empírica, descritiva e exploratóri a do tipo pesquisa de campo. A introdução do leite pasteuriza do foi a causa do desmame precoce. Conclui-se que há inseguranç a para realização das ações de continuida de de aleitament o materno após o retorno ao trabalho. Sentiment os de mulheres com depressão pós-parto frente ao aleitament o materno OLIVEIRA et al.,2019 Descrever sentiment os de mulheres com depressão pós-parto frente ao aleitament o materno. Qualitativo, descritivo e exploratóri o. As dificuldade s da depressão pós-parto no aleitament o são: falta de condições psicológica s, Desencant amento e sentiment os como estresse, medo e tristeza. 3. RESULTADOS Constatou- se que mulheres com depressão pós-parto sofreram impacto negativo durante o aleitament o, e temforte relação ao desmame precoce Fortaleced ores e fragilizados da amamenta ção na ótica da nutriz e de sua família WAGNER et al.,2019 Descrever os fatores de fortalecim ento e enfraqueci mento da amamenta ção Qualitativa, estudos de caso com caráter descritivo Fatores de fortalecim ento: experiênci a anterior de amamenta ção e família história da amamenta ção etc; O processo de cuidar deve incluir a dimensão social e subjetiva, fortalecen do a rede de apoio do nutrizes, a fatores foram: expectativ as negativas; o mito do leite fraco; doença infantil; materno doença; experiênci as negativas práticas profissiona is mais satisfatória s que promovam a amamenta ção Condutas de gestores relacionad as ao apoio ao aleitament o materno nos locais de trabalho FERNAND ES et al.,2018 Identificar as condutas de gestores relacionad as ao apoio ao aleitament o materno realizadas em empresas públicas e privadas da região da Grande Florianópol Qualitativa, exploratóri o- descritiva. Evidenciou duas categorias, positivas: Importânci a da amamenta ção, disponibiliz ação de informaçõ es, acompanh amento e realização de atividades durante o processo Lacunas substanciai s no apoio à amamenta ção dos locais de trabalho foram identificad as através das condutas evidenciad as pelos gestores. Considera- se important is, Santa Catarina. de maternida de e etc. Prejudiciai s: se constatara m a indisponibi lidade de informaçõ es especializa das e flexibilidad e, visão negativa em relação à implantaçã o das salas de apoio à amamenta ção, desconhec imento das legislações e da situação da trabalhado ra. e a reflexão sobre quais condutas devem ser incentivad as e quais devem ser modificada s ou ampliadas, para que haja um aperfeiçoa mento do suporte à amamenta ção nos locais de trabalho. Autoeficáci VIEIRA et Avaliar a Estudo da As chances A amamenta ção e depressão pós-parto: estudo de coorte de sintomas de depressão no período pós-parto e a associação entre depressão pós-parto com a interrupçã o do aleitament o. interrupçã o do aleitament o materno exclusivo diminui em 48% com a melhora do nível baixo de autoeficáci a para médio e em 80% de médio para alto a para a amamenta ção revelou-se como fator de proteção para o aleitament o materno exclusivo, enquanto a depressão pós-parto configura- se como fator de risco. 3. RESULTADOS Desmame precoce na perspectiv a de puérperas: uma abordage m dialógica. PRADO, et al 2016 Identificar aspectos transforma dores e obstáculos para o desmame precoce com 12 mães que desmamar am precoceme nte. Metodolog ia Comunicat iva Crítica Os resultados mostram mães jovens, primíparas, casadas, ensino médio/téc nico completo e não trabalhava A amamenta ção revelou emoções boas e ruins.O diálogo igualitário mostrou que ainda há obstáculos enraizados m fora de casa na cultura, que precisam ser dialogados com a mãe para superar o desmame precoce. Fatores ass ociados à interrupçã o do aleitament o materno exclusivo em lactentes com até 30 dias MORAES et al.,2016 Identificar fatores associados à interrupçã o do aleitament o materno exclusivo (AME) em lactentes com até 30 dias de vida Estudo transversal, análise bivariada e multivaria da Prevalênci a de 79,5% de AME. Lactentes ≥ 21 dias, que receberam compleme nto lácteo no hospital, mães com dificuldade de amamenta ção pós- alta hospitalar e não- brancas apresentar am Os fatores associados à interrupçã o do AME direciona m os profissiona is de saúde a proporem ações de apoio à mãe e lactente em vista de suas dificuldade s, prevenind o a interrupçã FONTE: Autoria própria 4. DISCUSSÃO 4.1 ALEITAMENTO MATERNO E DESMAME PRECOCE: CONTEXTO E ASPECTOS CONCEITUAIS 4.1 ALEITAMENTO MATERNO E DESMAME PRECOCE: CONTEXTO E ASPECTOS CONCEITUAIS A importância do aleitamento materno para a idade da mãe-bebê é um assunto que vem sendo difundido trazendo benefícios em longo prazo, mas muitos são fatores que tem levado ao fim dessa prática. Pesquisas mostram que um grande número de mulheres afirma não possuir leite o suficiente causando a recusa da criança, retorno ao trabalho/escola foram os fatores mais corriqueiros para o desmame precoce. Vale ressaltar que a volta da mãe ao mercado de trabalho e a curta licença maternidade, faz com que esse fator esteja entre os principais motivos de desmame (AMARAL et al.,2019). O estudo feito por Carreiro et al. (2018) afirma que o desmame precoce está ligado a baixa produção de leite segundo a mãe associado ao choro do bebê e a consequente introdução de fórmulas lácteas e outros líquidos, a dificuldade da mãe quanto a mamada em si, levando ao aparecimento de lesões mamilares além das variáveis sociodemográficas como nível de escolaridade, já o fator de retorno da mãe ao trabalho/escola, não se destaca nessa pesquisa como influente no desmame precoce. Contrariando Carreiro et al.(2018), Fernandes et al.(2018) em sua pesquisa afirma que a entrada da mulher no mercado de trabalho tem influência nos baixos índices de aleitamento materno, o apoio oferecido pelo ambiente de trabalho associado ao apoio familiar podem ser capazes de manter o aleitamento materno por mais tempo, porém os suportes referentes as empresas são limitados, favorecendo o desmame precoce. O “papel da mulher” tem se expandido em comparação às décadas anteriores, além de mãe, trabalhadora e muitas vezes chefe de família, a mulher desde então enfrenta desafios diários, como por exemplo, amamentar seu filho mesmo estando grande parte do dia trabalhando, favorecendo então a introdução de fórmulas além do leite materno, diminuindo consideravelmente as chances do aleitamento exclusivo até os seis meses preconizados pela Organização Mundial de Saúde (TORRES et al.,2019). O “papel da mulher” tem se expandido em comparação às décadas anteriores, além de mãe, trabalhadora e muitas vezes chefe de família, a mulher desde então enfrenta desafios diários, como por exemplo, amamentar seu filho mesmo estando grande parte do dia trabalhando, favorecendo então a introdução de fórmulas além do leite materno, diminuindo consideravelmente as chances do aleitamento exclusivo até os seis meses preconizados pela Organização Mundial de Saúde (TORRES et al.,2019). 4.1 ALEITAMENTO MATERNO E DESMAME PRECOCE: CONTEXTO E ASPECTOS CONCEITUAIS Os problemas mamários se destacam como motivo para o desmame precoce principalmente em primíparas, o despreparo dessas mães por falta de informação dos profissionais acabam causando fissuras mamilares e mastites. Por outro lado o excesso de informação baseados em heranças culturais vinda de influência familiar em específico a avó materna é considerado um fator negativo, que acaba desestimulando o aleitamento materno (OLIVEIRA et al.,2015). A Depressão Pós Parto (DPP) é um transtorno de humor que afeta as mulheres nas semanas após o parto e pode durar até um ano, e esse transtorno pode estar ligado ao desmame precoce, que é explicado pela literatura como a perda da confiança materna em dar continuidade a prática de amamentar diminuindo então o tempo da amamentação. O resultado do estudo de Silva et al.(2016) mostra que pela Escala de Depressão Pós-Parto de Edimburgo, filhos de mães com DPP têm maior risco de interrupção da amamentação exclusiva. As mudanças psicológicas e biológicas que acontecem no corpo da mulher acabam trazendo mudanças emocionais por muitas vezes não saber lidar com a nova fase, a pesquisa feita na Escola Nacional de Saúde Pública da Fundação Oswaldo Cruz revela que uma em cada quatro mulheres apresentam sinais de depressão pós-parto (DPP). Segundo Oliveira et al.(2019) mulheres que possuíram DPP, apresentaram consequências negativas para a amamentação, e o sentimento revelado por essas mulheres era de tristeza, medo, estresse, desencantamento e ameaça. Vieira et al. (2018) afirma que há associação entre a prática de amamentar e saúde mental, a presença de depressão pós-parto pode causar o desmame, assim como a dificuldade na amamentação pode ser um fator de risco para a doença mental. O aparecimento de sintomas depressivos aumenta a chance de introdução de fórmulas lácteas, consequentemente causando o desmame precoce. A amamentação não depende apenas da mãe para sua continuidade, existe uma soma de fatores que influencia nessa prática, a condição de saúde da mãe/filho, trabalho materno e até a presença paterna. Segundo Wagner et al.(2019) o histórico familiar de amamentação onde as mulheres amamentavam seus filhos, influencia e incentiva diretamente na decisão da nutriz, assim como experiências anteriores da mãe. 4.1 ALEITAMENTO MATERNO E DESMAME PRECOCE: CONTEXTO E ASPECTOS CONCEITUAIS Moraes et al.(2016) afirma que complemento lácteo oferecido ainda hospital pode aumentar duas vezes mais as chances de interromper a amamentação exclusiva antes os 30 dias em comparação aos lactentes que não receberam o complemento, a forma como o leite é oferecido ao bebê também pode afetar negativamente caso seja utilizado a mamadeira, que deve ser substituída por um “copinho”, para não causar impacto no aleitamento materno. 4.2 BARREIRAS E ESTRATÉGIAS UTILIZADAS PELOS PROFISSIONAIS NA PROMOÇÃO DO ALEITAMENTO MATERNO Mesmo o aleitamento materno sendo um ato importante e a mulher ser a responsável pela continuação do mesmo, ainda sim as informações são passadas a elas pelos profissionais de forma deficiente sendo incapaz de prepará-la para essa nova fase, que muitas vezes são cercadas de dúvidas e inseguranças. O estudo realizado por Oliveira et al.(2015), mostra que segundo declaração de mulheres entrevistadas, há associação entre a falta de informações e a insegurança materna em manter o seu leite como alimento exclusivo para seus filhos. O enfermeiro segundo Prado; Fabbro; Ferreira. (2016) deve aprender a escutar de forma humanizada, observar as inseguranças apresentada pela nutriz, para então aplicar medidas individuais que protejam e promovam o aleitamento materno, ajudando a superar as dificuldades e inseguranças iniciais e assim construir estratégias que esclareçam e superem a inseguridade. materno, ajudando a superar as dificuldades e inseguranças iniciais e assim construir estratégias que esclareçam e superem a inseguridade. A assistência prestada durante o pré-natal de acordo com Silva et al.(2017) contribuem para decisão da mulher em amamentar ou manter por mais tempo, já que durante as consultas o profissional pode promover orientações, colaborando para a educação em saúde visando orientar e incentivar a prática e continuação da amamentação. O enfermeiro deve fazer com que tenha o aprimoramento de novas estratégias, técnicas e formular meios para que tenha uma aproximação entre paciente e profissional, fazendo a adaptação e esclarecendo dúvidas de forma simples para que tenha melhor compreensão. A elaboração de atividades educativas são importantes para a educação em saúde, promoção e proteção ao aleitamento materno ( TORRES et al.,2019). Segundo Wagner et al.(2019) o genograma sobre a permanência da amamentação das famílias pode ser utilizado pela Equipe da saúde da família como método para promoção do aleitamento materno. O histórico da família no aleitamento materno ou no desmame precoce influencia a nutriz, dessa forma caso identificado histórico negativo da família pelo genograma o profissional de saúde deve monitorar a então gestante no pré-natal assim diminuindo a influência da família nessa prática, ampliando e fortalecendo a rede de apoio, tendo o enfermeiro papel de destaque. 4.2 BARREIRAS E ESTRATÉGIAS UTILIZADAS PELOS PROFISSIONAIS NA PROMOÇÃO DO ALEITAMENTO MATERNO O estudo de Moraes et al.(2016) afirma que as questões referentes à amamentação devem ser abordadas desde o pré natal e que a assistência deve ser mantida mesmo após o parto, pois é onde surgem as dúvidas já que é nesse momento que se inicia de fato a ato de amamentar que vem acompanhado de dificuldades, insegurança e dúvidas. A abordagem multiprofissional, atendimento individualizado a mães com dificuldade e até mesmo visitas domiciliares, essas estratégias podem influenciar positivamente na continuidade do aleitamento materno. 5. CONSIDERAÇÕES FINAIS A pesquisa realizada se evidenciou a importância do aleitamento materno para saúde da mãe e bebê, os benefícios vão além da nutrição, possui capacidade imunológicas e psicológicas, porém existe uma soma de fatores que podem levar ao fim dessa prática, segundo pesquisas esses fatores pode estar relacionados a saúde mental da mãe, retorno ao mercado de trabalho, e principalmente assistência do profissional de saúde que influencia tanto ao sucesso quanto ao insucesso dessa prática. A introdução da mulher no mercado de trabalho se mostrou fator influente no desmame precoce, já que mesmo havendo leis trabalhistas, ainda sim esse é um fator que influencia negativamente na continuidade dessa prática. A saúde mental está relacionado ao desmame precoce, é um fator que causa medo e insegurança a nutriz, a assistência de Enfermagem tem um grande destaque quando se trata de aleitamento materno, já que o pré-natal tem papel fundamental em promover e proteger essa prática, o acolhimento e o acesso a educação em saúde são fatores determinantes para a manutenção e permanência. Medidas simples podem ser tomadas para evitar o desmame precoce, buscando informar de forma clara que são vários motivos que podem levar ao fim do aleitamento, e que muitas causas podem ser resolvidas por meio do acesso à informação, criação de estratégias, acolhimento e escuta humanizada. O esclarecimento de dúvidas, o pré-natal e assistência de qualidade, respeito a individualidade e anseios, buscando acima de todas as coisas influenciar de forma positiva para a continuidade de aleitamento materno. O presente trabalho visa mostrar que mesmo sendo uma prática importante, ainda existe falhas que devem ser corrigidas para que o aleitamento atinja índices mais elevados de adesão, que as queixas feitas pelas mulheres relacionadas a uma assistência deficiente sirvam de lição e incentivo para o Enfermeiro, como algo a ser melhorado para assim beneficiar a todos. N,L. Análise de Conteúdo.Lisboa: Presses Universitaires de France,1977 CARREIRO,J.A et al.Dificuldades relacionadas ao aleitamento materno: análise de um serviço especializado em amamentação. Acta Paul Enferm.v.31,n4.,p 430- 8.2018. FERNANDES, V.M.B et al. Condutas de gestores relacionadas ao apoio ao aleitamento materno nos locais de trabalho. Texto Contexto Enferm.v.27,n.3.2018. HIGASHI GC; SANTOS SS; SILVA RS; JANTSCHI LB; SODER RM; SILVA LAA. Práticas de enfermeiros e a influência sociocultural na adesão ao aleitamento materno. Rev baiana enferm. 2021. KALIL Irene Rocha; AGUIAR Adriana Cavalcanti de. Trabalho feminino, políticas familiares e discursos pró-aleitamento materno: avanços e desafios à equidade de gênero. Saúde debate. Rio de janeiro, v.40, p.208-223, jul./setembro. 2016. LIMA, A.P. C; NASCIMENTO, D.S; MARTINS, M.M.F. A pratica do aleitamento materno e os fatores que levam ao desmame precoce: uma revisão integrativa. J. Health Biol Sci. v.6,n.2, p.189-196. 2018. MARIANI, B.P.R; LOURENÇO,M.C; BARBA, P.C.S.D. Revisão Sistemática Integrativa da Literatura sobre modelos e práticas de intervenção precoce no Brasil. Rev Paul Pediatr ,v.35,n.4,p 456-463.2017. REFERÊNCIAS AMARAL, S.A et al. Intenção de amamentar, duração do aleitamento materno e motivos para o desmame: um estudo de coorte, Pelotas, RS, 2014.Epidemiol. Serv. Saúde. Brasília,v.29,n.1.2019. ARAÚJO, V.S. et al. Desmame precoce: aspectos da realidade e trabalhadoras informais.Enfermagem Referência,v.10,p.35-43,Jul.2013. BARDIN,L. Análise de Conteúdo.Lisboa: Presses Universitaires de France,1977. MINISTÉRIO DA SAÚDE. SAÚDE DA CRIANÇA: Aleitamento Materno e Alimentação Complementar. Brasília, 2015. Alimentação Complementar. Brasília, 2015. MOIMAZ, S.S.A. et al. Desmame Precoce: falta de conhecimento ou de acompanhamento?.Pesquisa Brasileira em Odontopediatria e Clínica Integrada, Paraíba, v.13, n.1, p.53-59, jan./mar.2013. MOIMAZ, S.S.A. et al. Desmame Precoce: falta de conhecimento ou de acompanhamento?.Pesquisa Brasileira em Odontopediatria e Clínica Integrada, Paraíba, v.13, n.1, p.53-59, jan./mar.2013. Enfermeira Enfermeiro Obstetra/Mestre em Saúde Coletiva Acadêmica de Medicina Acadêmica de Medicina Acadêmica de Medicina Acadêmica de Medicina Acadêmica de Medicina Acadêmica de Medicina Acadêmica de Medicina Acadêmica de Medicina Acadêmica de Medicina Acadêmico de Medicina Acadêmica de Medicina Acadêmica de Medicina Acadêmica de Odontologia ← Post anterior Post seguinte → 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Post seguinte → Post seguinte → MOIMAZ, S.S.A. et al. Desmame Precoce: falta de conhecimento ou de acompanhamento?.Pesquisa Brasileira em Odontopediatria e Clínica Integrada, Paraíba, v.13, n.1, p.53-59, jan./mar.2013. MOIMAZ, S.S.A. et al. Desmame Precoce: falta de conhecimento ou de acompanhamento?.Pesquisa Brasileira em Odontopediatria e Clínica Integrada, Paraíba, v.13, n.1, p.53-59, jan./mar.2013. MONTESCHIO, C.A.C; GAÍVA, M.A.M; MOREIRA, M.D.S. O enfermeiro frente ao desmame precoce na consulta de enfermagem à criança. Brás Enferm, v.68, n.5, p.587-93, set./out.2015. MORAES, B.A et al. Fatores associados à interrupção do aleitamento materno exclusivo em lactentes com até 30 dias. Revista Gaúcha de Enfermagem. v. 37.2016. OLIVEIRA, C.S et al.Amamentação e as intercorrências que contribuem para o desmame precoce.Revista Gaúcha de Enermagem.v.36,p 16-23.2015. OLIVEIRA, C.S et al.Amamentação e as intercorrências que contribuem para o desmame precoce.Revista Gaúcha de Enermagem.v.36,p 16-23.2015. OLIVEIRA, M.G et al. Sentimentos de mulheres com depressão pós-parto frente ao aleitamento materno. Enferm. Foco. v.10,n.3,.p 88-92.2019. OLIVEIRA, M.G et al. Sentimentos de mulheres com depressão pós-parto frente ao aleitamento materno. Enferm. Foco. v.10,n.3,.p 88-92.2019. PRADO, C.V.C; FABBRO, M.R.C; FERREIRA ,G.I. Desmame precoce na perspectiva de puérperas: uma bordagem dialógica. Texto Contexto Enferm.v.25,n.2.2016. SILVA, C.S et al. Associação entre a depressão pós-parto e a prática do aleitamento materno exclusivo nos três primeiros meses de vida. Jornal de Pediatria.Rio de janeiro. 2017; 93:356-64. SILVA, C.S et al. Associação entre a depressão pós-parto e a prática do aleitamento materno exclusivo nos três primeiros meses de vida. Jornal de Pediatria.Rio de janeiro. 2017; 93:356-64. SILVA, C.S et al. Associação entre a depressão pós-parto e a prática do aleitamento materno exclusivo nos três primeiros meses de vida. Jornal de Pediatria.Rio de janeiro. 2017; 93:356-64. SOUZA, M.M.L. et al . A Metodologia da Revisão Integrativa da Literatura em Enfermagem. Revista Investigação em Enfermagem ,p.17-26,Nov.2017. TORRES, F.C.A et al. Manutenção do aleitamento materno no retorno ao trabalho. Revista Nursing.v.22,n.255,p 3074-3077.2019. TORRES, F.C.A et al. Manutenção do aleitamento materno no retorno ao trabalho. Revista Nursing.v.22,n.255,p 3074-3077.2019. VIEIRA, E.S et al. Autoeficácia para amamentação e depressão pós-parto: estudo de coorte.Rev. Latino-Am. Enfermagem .26:e3035.2018. VIEIRA, F.S. et al. Educação em saúde para promoção do aleitamento materno: relato de um projeto de extensão.Rev.Enferm UFPI,v.5,n.2,p 80-83,abr./jun.2016. WAGNER, L.P.B et al. Fortalecedores e fragilizados da amamentação na ótica da nutriz e de sua família.Rev Esc Enferm USP.2020. RevistaFT A RevistaFT é uma Revista Científica Eletrônica Multidisciplinar Indexada de Alto Impacto e Qualis “B2” em 2023. Periodicidade mensal e de acesso livre. 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https://openalex.org/W3158751940	https://periodicos.feevale.br/seer/index.php/revistagestaoedesenvolvimento/article/download/2490/2861	Portuguese	null	RESPONSABILIDADE COMPARTILHADA DE RESÍDUOS SÓLIDOS: REFLEXÕES DA IMPLEMENTAÇÃO NO MUNICÍPIO DE TERESINA-PI	Gestão e Desenvolvimento	2,021	cc-by	9,379	RESUMO A implementação da Lei nº 12.305/10, que instituiu a Política Nacional de Resíduos Sólidos (PNRS), estabeleceu a regulamentação para o manejo de resíduos sólidos no país. Com a determinação das diretrizes para o gerenciamento compartilhado de resíduos, a PNRS esclareceu as atribuições e responsabilidades da sociedade, do setor público e do setor privado dentro do sistema de Logística Reversa brasileiro. O presente artigo busca analisar as medidas adotadas no município de Teresina-PI para atender as recomendações da Lei nº 12.305/10. Abordando o princípio da responsabilidade compartilhada, o objetivo geral desta pesquisa consiste em realizar o mapeamento e análise dos atores privados envolvidos na coleta, transporte e destinação de resíduos oriundos dos grandes geradores, de acordo com o capítulo III da supracitada lei. Considerando ainda a importância do setor público, a pesquisa pretende realizar uma análise da legislação municipal e das políticas públicas desenvolvidas para o gerenciamento de resíduos. Como principais resultados, observa-se a necessidade de intensificação na fiscalização e controle do manejo de resíduos no município. Nota-se que os setores envolvidos enfrentam dificuldades para o desenvolvimento de sistemas de logística reversa eficientes. Vale ressaltar que a produção científica pode auxiliar no planejamento e desenvolvimento de planos de ação para gestão de resíduos sólidos. Palavras-chave: Logística Reversa. Política Nacional de Resíduos Sólidos (PNRS). Responsabilidade Compartilhada. Geradores de Resíduos Sólidos. Recebido em: 5 de janeiro de 2021 Aprovado em: 19 de março de 2021 Sistema de Avaliação: Double Blind Review RGD \| v. 18 \| n. 2 \| p. 03-25 \| mai./ago. 2021 DOI: https://doi.org/10.25112/rgd.v18i2.2490 Luiza Gabrielle de Sousa Alves Acadêmica do curso de Bacharelado em Administração pela Universidade Estadual do Piauí (Teresina/Brasil). E-mail: luizagsalves013@gmail.com Ana Raissa Alcântara Noleto Acadêmica do curso de Bacharelado em Administração pela Universidade Estadual do Piauí (Teresina/Brasil). E-mail: contato.raissaadm@gmail.com Emanuel Alcântara da Silva Mestrando pelo Programa de Pós-Graduação em Administração de Empresas da Universidade de Fortaleza (Fortaleza/Brasil). Acadêmico do curso de Bacharelado em Administração pela Universidade Estadual do Piauí (Teresina/Brasil). E-mail: emanuelalcantara26@gmail.com Helano Diógenes Pinheiro Doutor pelo Programa de Pós-Graduação em Administração da Universidade Federal do Rio Grande do Norte (Natal/Brasil). Professor do curso de Bacharelado em Administração da Universidade Estadual do Piauí (Teresina/Brasil). E-mail: helanodp22@gmail.com Luiza Gabrielle de Sousa Alves Acadêmica do curso de Bacharelado em Administração pela Universidade Estadual do Piauí (Teresina/Brasil). E-mail: luizagsalves013@gmail.com Emanuel Alcântara da Silva Mestrando pelo Programa de Pós-Graduação em Administração de Empresas da Universidade de Fortaleza (Fortaleza/Brasil). Acadêmico do curso de Bacharelado em Administração pela Universidade Estadual do Piauí (Teresina/Brasil). E-mail: emanuelalcantara26@gmail.com Mestrando pelo Programa de Pós-Graduação em Administração de Empresas da Universidade de Fortaleza (Fortaleza/Brasil). Acadêmico do curso de Bacharelado em Administração pela Universidade Estadual do Piauí (Teresina/Brasil). E-mail: emanuelalcantara26@gmail.com Helano Diógenes Pinheiro Recebido em: 5 de janeiro de 2021 Aprovado em: 19 de março de 2021 Sistema de Avaliação: Double Blind Review RGD \| v. 18 \| n. 2 \| p. 03-25 \| mai./ago. 2021 DOI: https://doi.org/10.25112/rgd.v18i2.2490 Recebido em: 5 de janeiro de 2021 Aprovado em: 19 de março de 2021 Sistema de Avaliação: Double Blind Review RGD \| v. 18 \| n. 2 \| p. 03-25 \| mai./ago. 2021 DOI: https://doi.org/10.25112/rgd.v18i2.2490 1 INTRODUÇÃO A evolução dos sistemas produtivos, a intensificação do consumo e o crescimento populacional contribuíram para o aumento na geração de resíduos, ocasionando no comprometimento da qualidade de vida da sociedade e no agravamento dos problemas ambientais. As medidas sustentáveis adotadas acabam sendo insuficientes diante do elevado volume de resíduos descartados. Além disso, a ausência de políticas públicas efetivas favorece a destinação final inadequada. Considerando o contexto acima, pode-se dizer que a Logística Reversa surgiu diante da necessidade do desenvolvimento de sistemas de produção mais sustentáveis. Com a instauração dos processos de reaproveitamento e destinação de resíduos, a Logística Reversa estabeleceu que a gestão deve organizar estratégias e ações para produção de baixo impacto ambiental. No Brasil, visando a determinar as práticas e políticas adequadas para o manejo de resíduos sólidos, foi instituída a Lei nº 12.305/10 (regulamentada pelo Decreto nº 7.404, de 23 de dezembro de 2010), que criou a Política Nacional de Resíduos Sólidos (PNRS). A PNRS estabeleceu metas para erradicação dos chamados “lixões”, a classificação dos resíduos sólidos, determinou as diretrizes para responsabilidade compartilhada e instituiu os planos de gestão integrada (nos âmbitos nacional, estadual e municipal). O setor público tornou-se o principal agente regulamentador e fiscalizador das práticas do sistema de gestão de resíduos. A PNRS determinou que estados e municípios devem desenvolver planos de gestão de resíduos, os quais devem conter medidas para a disposição final ambientalmente adequada dos resíduos (BRASIL, 2010a). Com a instituição do princípio da responsabilidade compartilhada, a supracitada lei determinou que os grandes geradores realizem a destinação correta dos resíduos oriundos dos sistemas de produção. Visando a adequação às determinações legais, os grandes geradores de resíduos têm recorrido a outras organizações para realização do recolhimento dos resíduos sólidos produzidos. Diante desse cenário, surge o mercado dos atores privados, responsáveis pelo segundo nível da rede de logística reversa. Passados aproximadamente 10 (dez) anos da implementação da Lei nº 12.305/10, observa- se que o problema no manejo de resíduos sólidos ainda persiste. De acordo com dados da Associação Brasileira de Empresas de Limpeza Pública e Resíduos Especiais (ABRELPE, 2019), estima-se que no ano de 2018 foram geradas 79 milhões de toneladas de resíduos sólidos, representando um aumento de aproximadamente 1% em relação ao ano anterior. A ABRELPE (2019) destaca ainda que, do universo analisado, 6,3 milhões de toneladas de resíduos não foram coletadas junto aos locais de geração. e-ISSN: 2446-6875 p-ISSN: 1807-5436 e-ISSN: 2446-6875 p-ISSN: 1807-5436 ABSTRACT The implementation of Law nº. 12.305/10, which instituted Brazil’s National Solid Waste Policy (PNRS), established the regulation for the management of solid waste in the country. With the determination of the guidelines concerning shared waste management, PNRS clarified the roles and responsibilities of society, the public sector, and the private sector within the reverse logistics system in Brazil. This article seeks to analyze the measures adopted in the municipality of Teresina-PI in order to meet the recommendations of Law nº. 12.305/10. The general objective of this study is to carry out the mapping and analysis of the private actors involved in the collection, transportation, and disposal of waste from large generators, by addressing the principle of shared responsibility, and in accordance with Chapter III of the aforementioned law. Considering also the importance of the public sector, the research intends to carry out an analysis of the municipal legislation and public policies developed concerning waste management. As the main results have shown, there is a need to intensify the inspection and control of waste management in the municipality. It is noted that the sectors involved face difficulties in the development of efficient reverse logistics systems. It is worth mentioning that scientific production is able to assist in the planning and development of action plans for solid waste management. Keywords: Reverse Logistics. National Solid Waste Policy (PNRS). Shared Responsibility. Solid Waste Generators. Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 4 e-ISSN: 2446-6875 p-ISSN: 1807-5436 e-ISSN: 2446-6875 p-ISSN: 1807-5436 menor índice na cobertura de coleta de resíduos sólidos. Ademais, observou-se que na região, a maioria dos resíduos coletados são destinados a aterros controlados e lixões. De acordo com dados do Plano Municipal de Gestão Integrada de Resíduos Sólidos (PMGIRS) de Teresina (PMT, 2018), até o final do ano de 2032, estima-se que serão geradas 650 toneladas de resíduos por dia no município. O Plano Municipal (PMT, 2018) destaca ainda que a média de resíduos sólidos urbanos gerados por cada habitante corresponde a 1,53 Kg por dia. Tendo em vista este cenário, se pretende analisar os impactos dos mecanismos previstos na Lei nº 12.305/10. Este artigo objetiva analisar o gerenciamento de resíduos com base na responsabilidade do gerador ou responsabilidade compartilhada. Ademais, busca-se mapear os atores privados responsáveis pela coleta, transporte e destinação de resíduos oriundos dos grandes geradores, de acordo com o capítulo III da Lei nº 12.305/10. Para alcançar os objetivos estabelecidos, a pesquisa explora as medidas adotadas no município de Teresina-PI para atender as recomendações da supracitada lei e relacionar as situações que exigem a formulação de plano de gerenciamento de resíduos sólidos (conforme especificado no artigo 20 da lei 12.305/10) com a relação das empresas autorizadas. 1 INTRODUÇÃO Segundo o Panorama, a região Nordeste apresentou o Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 5 2 FUNDAMENTAÇÃO TEÓRICA Nesta seção serão discutidos tópicos referentes à implementação da Lei nº 12.305/10 e a relação entre as práticas de Logística Reversa e a responsabilidade compartilhada dos resíduos sólidos. e-ISSN: 2446-6875 p-ISSN: 1807-5436 outros ciclos produtivos, ou outra destinação final ambientalmente adequada (BRASIL, 2010a). De acordo com Macêdo e Rohlfs (2013, p. 8) “a implantação dos sistemas de logística reversa deve ser feita de forma independente dos serviços públicos de limpeza urbana e manejo de resíduos sólidos”. A instituição da PNRS visou a integrar o poder público, a sociedade civil e a iniciativa privada ao instituir as diretrizes relativas à “gestão integrada e ao gerenciamento de resíduos sólidos, incluindo os perigosos, às responsabilidades dos geradores e do poder público e aos instrumentos econômicos aplicáveis.” (BRASIL, 2010a). Destaca-se que o processo de retorno dos resíduos ao sistema de cadeia reversa envolve uma grande dispersão dos mesmos, o que torna mais econômico um sistema de coleta e reaproveitamento, ou destinação final, intermediado por empresas especializadas. Segundo Corrêa e Xavier (2013), a gestão de cadeias reversas favorece os envolvidos pelo fato da vantagem comercial em ambos os aspectos. No entanto, Mariath e Figueiró (2018, p. 138) apontam que “é notável a ampliação do debate sobre a logística reversa nas corporações, trazendo esta evidente preocupação e maior importância do tema nas organizações, contudo, ainda não é assunto prioritário.”. Nesse contexto, Mariath e Figueiró (2018) destacam que as falhas na fiscalização da Lei nº 12.305/10, bem como a falta de conhecimento quanto à estruturação do um sistema de Logística Reversa, contribuem para o baixo comprometimento das empresas na destinação correta dos resíduos. 2.1 LOGÍSTICA REVERSA E A IMPLEMENTAÇÃO DA LEI Nº 12.305/10 Tendo em vista a necessidade de desenvolvimento de processos voltados para destinação e reaproveitamento de resíduos, podendo ou não ocorrer a reincorporação dos mesmos ao sistema produtivo (ALVES, 2016, p. 113), surge a chamada Logística Reversa, caracterizada como “a parte da logística que gerencia os recursos e processos referentes aos fluxos reversos.” (CORRÊA; XAVIER, 2013, p. 5). No Brasil, em razão do crescimento no quantitativo dos resíduos gerados, foi implementada a Lei nº 12.305/10, que estabeleceu a Política Nacional de Resíduos Sólidos (PNRS). Segundo Corrêa e Xavier (2013, p. 80), no país, a referida Lei se caracteriza como “o principal instrumento regulamentador que define o conceito e a implementação da Logística Reversa (...).”. A Política Nacional de Resíduos Sólidos (PNRS) define a Logística Reversa como o Instrumento de desenvolvimento econômico e social caracterizado por um conjunto de ações, procedimentos e meios destinados a viabilizar a coleta e a restituição dos resíduos sólidos ao setor empresarial, para reaproveitamento, em seu ciclo ou em Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 6 2.2 RESPONSABILIDADE COMPARTILHADA DE RESÍDUOS SÓLIDOS Com a implementação da Lei nº 12.305/10, ficou atribuída às organizações a responsabilidade pelos resíduos que geram. No intuito de garantir a efetividade de suas ações, os geradores têm recorrido a outras organizações para o recolhimento dos resíduos sólidos produzidos. Alguns países, como o Brasil, optaram pela responsabilidade compartilhada, segundo a qual todos os agentes da cadeia são corresponsáveis pela gestão ambientalmente adequada dos resíduos. Isso tem possibilitado a consolidação de ações por acordos setoriais, dos quais participam as organizações produtoras, as empresas, recicladoras, a sociedade, academia e o governo. (CORRÊA; XAVIER, 2013, p. 188). Com base no contexto acima, bem como no art. 20 da Lei nº 12.305/10, entende-se que a prestação de serviços de coleta e destinação de resíduos sólidos enquadra-se na denominada “responsabilidade do gerador” ou “responsabilidade compartilhada” que estabelece às pessoas físicas ou jurídicas obrigação de elaborar seu plano de gerenciamento de resíduos sólidos. Ressalta-se também que a responsabilidade Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 7 e-ISSN: 2446-6875 p-ISSN: 1807-5436 e-ISSN: 2446-6875 p-ISSN: 1807-5436 e obrigatoriedade de planos de manejo, conforme o art. 27 da PNRS (BRASIL, 2010a), atribui aos atores a responsabilidade “pela implementação e operacionalização integral do plano de gerenciamento de resíduos sólidos aprovado pelo órgão competente”. Destaca-se que o princípio da responsabilidade compartilhada possibilita, diretamente ou por analogia, importantes avanços em outras matérias, como da responsabilização solidária dos causadores de quaisquer formas de poluição. Nesse contexto, ressalta- se que, a Lei nº. 12.305/10 ao estabelecer em seu artigo 27, caput e § 1º. que os agentes responsáveis pelo plano de gerenciamento de resíduos sólidos não terão sua responsabilidade excluída no caso de contratação de serviços de coleta, armazenamento, transporte ou destinação final de resíduos sólidos, ou disposição final de rejeitos em função do gerenciamento inadequado dos respectivos resíduos ou rejeitos, evidencia a aplicabilidade da solidariedade na responsabilidade civil por danos ambientais. (SINNOTT, 2012, p. 20). O setor público configura-se como o principal agente regulamentador e fiscalizador das práticas de gestão de resíduos. Segundo Mancini, Ferraz e Bizzo (2012, p. 348), “[...] a legislação brasileira aponta para o princípio do poluidor-pagador, responsabilizando cada gerador pelo seu resíduo.”. Segundo os parâmetros estabelecidos pela Política Nacional de Resíduos Sólidos (PNRS), cabe aos municípios articular a efetivação, organização e prestação direta ou indireta dos serviços envolvendo resíduos sólidos. Moreira et al. (2016, p. 1452) destacam que O que se pretende com os mecanismos previstos na PNRS, dentre os quais os acordos setoriais, destinados à promoção da responsabilidade ambiental pós-consumo é a concretização da orientação do princípio do poluidor-pagador, garantindo-se a justa e equitativa distribuição dos ônus e de bônus relativos à produção de determinados bens ao longo de sua cadeia de produção e consumo e, consequentemente, evitando a socialização dos respectivos custos ambientais. Em termos conceituais, o desenvolvimento da pesquisa aconteceu com base no princípio do poluidor- pagador, instituído pela Lei nº 12.305/10, no inciso II do art. 6º. Tendo avançado na área ambiental como a melhor forma de solucionar o problema da poluição, “o princípio do poluidor-pagador, como instituição basilar que objetiva a contenção do dano ambiental através de um caráter preventivo-repressivo, ao ser utilizado por várias nações, fortalece e influencia a legislação infraconstitucional brasileira.” (BORGES, MELLO; OLIVEIRA, 2010, p. 204). Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 e-ISSN: 2446-6875 p-ISSN: 1807-5436 Figura 1 - Responsabilidade compartilhada Fonte: Instituto Ethos de Empresas e Responsabilidade Social (p. 27, 2015). Figura 1 - Responsabilidade compartilhada Fonte: Instituto Ethos de Empresas e Responsabilidade Social (p. 27, 2015). O processo de gestão de resíduos sólidos envolve diversos aspectos e agentes para desenvolvimento das ações. Nesse sentido, Torres et al. (2016, p. 148) destaca que “a gestão de resíduos sólidos é compreendida como mecanismo viável e capaz de abranger as atividades referentes à tomada de decisões estratégicas e à organização do setor para esse fim, envolvendo instituições públicas, políticas, instrumentos e meios”. No princípio da responsabilidade compartilhada, ressalta-se que o gerador de resíduos domiciliares tem sua responsabilidade cessada na medida em que faz a disposição adequada dos resíduos para coleta. Ou seja, sua responsabilidade seria ampliada apenas nos casos que estivesse lidando com um dos resíduos especificados nos artigos 13 e 20 da PNRS. Nas situações de geração de resíduos advindo do consumo familiar, a obrigação é pela separação e acondicionamento adequado para realização da coleta domiciliar e a segregação no caso de resíduos recicláveis, conforme estabelece o artigo 35 da PNRS (BRASIL, 2010a). Ao analisar o sistema “Pay-As-You-Throw”, abreviado como PAYT, Oliveira (2018, p. 20) comenta sua semelhança com o princípio do poluidor-pagador e destaca que o Plano Nacional de Resíduos Sólidos, estabelecido pela PNRS, “apresenta Incentivos Econômicos como uma ferramenta aplicada à gestão de Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 9 9 Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 e-ISSN: 2446-6875 p-ISSN: 1807-5436 e-ISSN: 2446-6875 p-ISSN: 1807-5436 resíduos com a função de financiar os serviços de limpeza pública, orientar o comportamento da população e responsabilizar os impactos gerados pela quantidade de resíduos produzidos”. resíduos com a função de financiar os serviços de limpeza pública, orientar o comportamento da população e responsabilizar os impactos gerados pela quantidade de resíduos produzidos”. Nesse sentido, Oliveira (2018) destaca que a PNRS foi o principal meio para formalização dos pilares da gestão integrada de resíduos sólidos, proporcionando um ganho em escala por meio de uma gestão regionalizada. Segundo Oliveira (2018), a gestão regionalizada permite a redução de custos com limpeza urbana em razão da sinergia entre municípios no processo de compartilhamento do sistema de coleta, tratamento e destinação de resíduos sólidos. Para o estabelecimento do sistema de Logística Reversa previsto na PNRS, os diferentes stakeholders (setor público, setor privado e sociedade) devem interagir constantemente (ver Figura 1). Segundo a PNRS, o setor público ficou responsável por consolidar o incentivo do desenvolvimento social e econômico menos prejudicial ao meio ambiente. Com isso, os municípios obrigatoriamente necessitam contribuir com a efetivação, organização e prestação direta ou indireta dos serviços envolvendo resíduos sólidos. Neste contexto, o artigo analisou o cenário do Município de Teresina com base no Decreto nº 18.061, de 18 de outubro de 2018, que regulamenta a prestação de serviços de coleta e destinação final por meio do cadastramento de empresas transportadoras de resíduos sólidos (TERESINA, 2018a). O quadro abaixo possui a listagem e descrição da legislação municipal relacionada ao assunto em estudo. Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 10 10 e-ISSN: 2446-6875 p-ISSN: 1807-5436 Quadro 1 - Detalhamento da legislação municipal Quadro 1 - Detalhamento da legislação municipal Quadro 1 - Detalhamento da legislação municipal LEIS ASSUNTOS Lei nº 3.544/2006 Autoriza o Poder Executivo Municipal a criar o Programa de Reciclagem de Lixo em todos os órgãos da administração direta e indireta do município. Lei nº 3.646/2007 Institui o Código Sanitário do município de Teresina, e dá outras providências. Lei nº 3.923/2009 Dispõe sobre a implantação da coleta de lixo reciclável nos condomínios residenciais e comerciais; postos de gasolina e afins localizados no município de Teresina, e dá outras providências. Lei nº 4.224/2012 Dispõe sobre a criação do Disk Lixo no município, e dá outras providências. Lei nº 4.474/2013 Institui o “Programa Lixo Zero”, no âmbito do município de Teresina, e dá outras providências. Lei nº 4.684/2015 Institui diretrizes acerca da criação do programa de coleta seletiva de resíduos sólidos e inclusão social dos catadores de materiais recicláveis - Pró-catador, no município de Teresina, e dá outras providências. Lei nº 4.973/2016 Institui os objetivos e metas para o Plano Municipal de Saneamento Básico do município de Teresina e dá outras providências. Lei nº 4.974/2016 Institui o novo Código Tributário do município de Teresina e dá outras providências. Lei nº 5.229/2018 Institui o Programa de Sustentabilidade Ambiental na rede municipal de ensino, e dá outras providências. Decreto nº 17.733/2018 Institui e regulamenta o Plano Municipal de Gestão Integrada de Resíduos Sólidos – PMGIRS do Município de Teresina. Decreto nº 18.061/2018 Institui o cadastramento de empresas responsáveis pelo transporte de resíduos sólidos. Decreto nº 18.062/2018 Regulamenta o art. 120, §3º, da Lei Complementar nº 3.610/2007 (Código de Posturas), para disciplinar o cadastramento das áreas de destinação de resíduos sólidos, no âmbito do Município de Teresina, e dá outras providências. Fonte: Adaptado de COELHO (p. 60, 2019). e-ISSN: 2446-6875 p-ISSN: 1807-5436 e-ISSN: 2446-6875 p-ISSN: 1807-5436 abrangendo os resíduos sólidos domiciliares, os resíduos sólidos pós-consumo e aqueles provenientes da limpeza pública. Há também a geração determinada, que são os produzidos por gerador específico e identificável. Tal classificação permite ao poder público municipal taxar os geradores de resíduos sólidos identificáveis, de forma a manter a autossustentação do sistema. A análise do sistema teresinense abordou as chamadas empresas de segundo nível, ou seja, aquelas empresas autorizadas a atuar na coleta, transporte, segregação e destinação de resíduos em relações privadas, ou seja, ofertam seus serviços no âmbito municipal sem intermediação ou remuneração por parte do poder público. Tais serviços atendem à denominada “responsabilidade do gerador” ou “responsabilidade compartilhada” tratada pela Lei nº 12.305/10. O estudo busca nestas empresas uma descrição de como está se formando o mercado de serviços relacionados ao retorno de bens pós-consumo, material base para o processo de reaproveitamento dos resíduos produzidos no espaço urbano (LEITE, 2009; ROGERS; TIBBEN-LEMBKE, 1998). As empresas cadastradas junto ao poder público municipal para execução do serviço de coleta e transporte de resíduos sólidos, desde que não vinculadas por contrato e remuneradas pelo poder público, constituem o objeto do presente estudo. Dessa forma, procurou-se identificar como está sendo implementado o serviço de coleta dos resíduos junto aos geradores que possuem responsabilidade. Fonte: Adaptado de COELHO (p. 60, 2019). Conforme dispõe o art. 26 da Lei 12.305/10, o Plano Municipal de Gestão Integrada de Resíduos Sólidos (PMGIRS) baliza as ações do poder público para organização e prestação dos serviços limpeza urbana e manejo de resíduos sólidos (BRASIL, 2010a). De acordo com Coelho (2019, p. 63), o Plano Municipal de Gerenciamento Integrado de Resíduos Sólidos (PMGIRS) de Teresina (ver Quadro 1) (...) classifica os resíduos também quanto à identificação do gerador, em que há a geração difusa, que são os produzidos, individual ou coletivamente, por geradores dispersos e não identificáveis, por ação humana, animal ou por fenômenos naturais, Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 11 11 e-ISSN: 2446-6875 p-ISSN: 1807-5436 o objetivo de compreender as medidas adotadas pelo poder público para adequação da responsabilidade do gerador à PNRS. o objetivo de compreender as medidas adotadas pelo poder público para adequação da responsabilidade do gerador à PNRS. O estudo buscou captar o desenvolvimento do mercado privado de coleta de resíduos na cidade de Teresina-PI, identificando a implementação do serviço junto aos geradores que possuem responsabilidade. Com base nas informações coletadas no site da Secretaria Municipal de Desenvolvimento Urbano e Habitação (SEMDUH), órgão responsável pela gestão do sistema municipal de logística reversa (BRASIL, 2010a), pode-se constatar que as empresas do sistema de logística reversa teresinense atuam na coleta extradomiciliar, realizando o transporte, segregação e destinação de resíduos não-perigosos e perigosos, por meio de relações privadas com as entidades geradoras. Como fonte de informações, utilizou-se de dados documentais para uma análise da ação do poder público na implementação da ação em estudo e um retorno à pesquisa bibliográfica, desta vez manifesta na identificação de materiais publicados na internet noticiando a ação pública ou sua repercussão. Para tanto, foram selecionados os principais portais de notícias teresinenses em número de acessos de internautas, entre eles: o Portal Cidade Verde, Portal O Dia, 180 Graus, Meio Norte, G1/PI, GP1, que são os mais acessados de acordo com pesquisas em comunicação (CARVALHO; CARVALHO, 2018; VIANA; COELHO; RÊGO, 2011) e com o Ranking da Alexa da Amazon (EM FOCO, 2020; VOZ DO PIAUÍ, 2020). A utilização de informações da internet deve ser feita com cuidado, pois “[...] procurar e recuperar informações na internet é muito mais problemático do que procurar em um banco de dados bibliográfico. Não há campos padronizados, nenhuma hierarquia cuidadosamente definida, [...], nenhum critério de seleção e, em geral, nenhuma regra[...]” (COOPER; SCHINDLER, 2011, p. 129). Os dados utilizados na pesquisa são predominantemente de fontes secundárias e terciárias, caracterizadas por serem, respectivamente, interpretações de dados primários ou ainda uma referência a estudos que utilizaram dados secundários. De acordo com Michel (2009, p. 65), os dados secundários podem ser “coletados através de análise documental, ou seja, em documentos, relatórios, livros, revistas, jornais, sites, etc.”. Segundo Cooper e Schindler (2011, p. 104), “[...] enciclopédias, livros, manuais, artigos de revistas e jornais e a maioria das notícias são consideradas fontes secundárias de informação”. 3 METODOLOGIA A pesquisa buscou observar, registrar, analisar e relacionar a formação de um setor de serviços privados relacionados à coleta de resíduos na cidade de Teresina-PI, o que a caracteriza como uma pesquisa descritiva. De acordo com Cervo e Bervian (2002, p. 67), neste tipo de pesquisa “trata-se do estudo e da descrição das características, propriedades ou relações existentes na comunidade, grupo ou realidade pesquisada”. Durante a revisão bibliográfica, foram coletados autores com obras datadas dos anos de 2000 a 2019. As obras selecionadas referem-se ao objeto de estudo e abordam assuntos como a “responsabilidade compartilhada” e a “responsabilidade do gerador de resíduos”, o conceito e as aplicações do termo “poluidor-pagador”, da logística reversa, do direito ambiental, a Política Nacional de Resíduos Sólidos - PNRS (Lei 12.305/10) e o Plano de Gerenciamento de Resíduos Sólidos (PGRS). Durante o período de (três) meses, além da realização da revisão de literatura, ocorreu a revisão do material produzido em pesquisas científicas anteriores, a partir das diretrizes da Lei nº 12.305/10, com Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 12 12 12 e-ISSN: 2446-6875 p-ISSN: 1807-5436 e-ISSN: 2446-6875 p-ISSN: 1807-5436 Coletou-se as informações por meio da utilização de dados secundários, buscou-se informações referentes a coleta de resíduos sólidos, coleta seletiva ou simplesmente resíduos sólidos nos veículos noticiosos de teresina, bem como identificar empresas com “atividade de coleta de resíduos” a partir de lista disponibilizada pela Secretaria do Meio Ambiente (SEMAM) e pela relação de empresas cadastradas pela Secretaria Municipal de Desenvolvimento Urbano e Habitação (SEMDUH) e Coordenação Especial de Limpeza Urbana (CELIMP). Com isso, foi realizado o mapeamento de 5 (cinco) empresas que são envolvidas na coleta, transporte e destinação de resíduos oriundos de geradores privados, conforme o capítulo III da Lei nº 12.305/10. Devido às medidas de isolamento social, ocasionadas pelo vírus COVID-19, não se efetuou coletas de dados in loco. Chegou-se a elaborar um questionário on-line e a se identificar as empresas habilitadas a prestar o serviço em ofício da Secretaria do Meio Ambiente (SEMAM). Porém, o fechamento do varejo de contato influiria na coleta de informações consistentes sobre a aplicação da norma legal e, dessa forma, preferiu-se manter o artigo com base em informações públicas e privadas coletadas em meios noticiosos no período de 2019 a junho de 2020. Considerando as determinações do art.1º, §1º, §2º e §3º do Decreto Nº 18061, de 18 de outubro de 2018 (ver Quadro 1), verificou-se que as 5 (cinco) empresas analisadas possuem autorização para realizar o transporte de resíduos sólidos, têm sede no município e estão devidamente cadastradas na Secretaria Municipal de Desenvolvimento Urbano e Habitação (SEMDUH). Com base nos dados coletados nas relações citadas anteriormente, realizou-se a triagem das empresas de acordo com o tipo de atividade desenvolvida e o tipo de resíduos coletados. Ademais, para aprofundamento da pesquisa, foram selecionados dados dos sites institucionais das empresas. Considerados todos os dados recolhidos, foi realizada a comparação entre as empresas coletadas. 4 RESULTADOS E DISCUSSÃO Esta seção apresenta a exploração das medidas adotadas no município de Teresina-PI para atender as recomendações da Lei nº 12.305/10, bem como o mapeamento e análise dos atores privados envolvidos na coleta, transporte e destinação de resíduos oriundos dos grandes geradores. e-ISSN: 2446-6875 p-ISSN: 1807-5436 Quanto ao local de busca destas informações, utiliza-se cada vez mais a internet, pois os documentos on-line são cada vez mais comuns, tanto os documentos institucionais quanto informações transmitam por meios de comunicação diversos, porém estas informações são complicadas de coletar e interpretar por não estarem vinculadas e organizadas a uma base de dados. Em casos nos quais os processos sociais ainda estão se desenvolvendo, a busca na rede por informações ainda difusas pode fornecer informações sobre a ocorrência do processo, embora não se possa realizar análises ou correlações mais aprofundadas. Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 13 13 Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 4.1 AÇÕES DO PODER PÚBLICO MUNICIPAL A Política Nacional de Resíduos Sólidos (PNRS) proporcionou o avanço da Logística Reversa nos âmbitos público e privado, contribuindo para a transparência no gerenciamento de resíduos sólidos. Dentro desse contexto, a denominada responsabilidade do gerador ou responsabilidade compartilhada, Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 14 14 Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 e-ISSN: 2446-6875 p-ISSN: 1807-5436 estabelecidos pela PNRS, os municípios obrigatoriamente necessitam contribuir com a efetivação, organização e prestação direta ou indireta dos serviços envolvendo resíduos sólidos. No âmbito teresinense, a instituição e gestão do sistema municipal de logística reversa (BRASIL, 2010b) envolve a coleta de resíduos domiciliares, a varrição dos logradouros públicos, a coleta seletiva e o monitoramento dos aterros, fica sob responsabilidade da Secretaria Municipal de Desenvolvimento Urbano e Habitacional (SEMDUH), enquanto que a aprovação dos planos e a autorização de empresas para atuarem com atividade de potencial de impacto ambiental, incluindo o manejo de resíduos, requer um processo de licenciamento de responsabilidade da Secretaria Municipal de Meio Ambiente (SEMAM). Em cumprimento à Política Nacional de Resíduos Sólidos (PNRS), o município estabeleceu que os resíduos oriundos dos grandes geradores pertencem à coleta extradomiciliar, ou seja, de responsabilidade dos produtores. Por meio do Decreto Nº 18061, de 18 de outubro de 2018, a Secretaria Municipal de Desenvolvimento Urbano e Habitacional (SEMDUH) instituiu o cadastramento de empresas responsáveis pelo transporte de resíduos sólidos (coleta extradomiciliar), ou seja, os atores privados que atuam no segundo nível da rede de logística reversa. Considerando as determinações do art.1º, §1º, §2º e §3º do Decreto, as empresas devem possuir sede no município e estar devidamente cadastradas na SEMDUH. Vale destacar que, caso a Secretaria Municipal de Desenvolvimento Urbano e Habitacional (SEMDUH) realize a coleta de resíduos não enquadrados dentro da competência legal do município, por serem considerados extradomiciliares, conforme artigo 272 da Lei Complementar nº 4.974, de 26 de dezembro de 2016 (Código Tributário Municipal), acontecerá a cobrança da Taxa de Coleta, Transporte e Disposição Final de Resíduos Sólidos Extradomiciliares – TCRE (TERESINA, 2016). O Decreto Nº 18.062/2018, de 18 de outubro de 2018 (ver Quadro 1), disciplinou o cadastramento das áreas de destinação de resíduos sólidos, no âmbito do Município de Teresina (TERESINA, 2018b), em conformidade com as determinações do artigo 18, §4º da Lei nº 12.305/10. Atualmente, no município, a disposição final dos resíduos sólidos acontece em aterro controlado. Considerando as recomendações do Plano Municipal de Gerenciamento Integrado de Resíduos Sólidos (PMGIRS), instituído pelo Decreto nº 17.733/2018, pode-se observar que o poder público tem realizado ações voltadas para a Coleta Seletiva, com a instalação de Pontos de Entrega Voluntária (PEV’s) e Pontos de Recebimentos de Resíduos (PRR’s). Ressalta-se que cooperativas e ONGs têm contribuído para o recolhimento de resíduos sólidos. No entanto, segundo Coelho (2019, p. e-ISSN: 2446-6875 p-ISSN: 1807-5436 e-ISSN: 2446-6875 p-ISSN: 1807-5436 tratada no artigo 20 da Lei nº 12.305/10, estabelece que pessoas físicas ou jurídicas estão obrigadas a elaborar seu Plano de Gerenciamento de Resíduos Sólidos (PGRS). Ademais, a Lei atribuiu aos estados e municípios a função de regulamentar e fiscalizar as práticas de coleta e destinação final de resíduos. Considerando os critérios já presentes e consolidados no contexto brasileiro, a Política Nacional de Resíduos Sólidos (PNRS) instituiu várias classificações para os resíduos, sendo elas: quanto à destinação, quanto à origem, quanto à logística e quanto ao grau de periculosidade (BRASIL, 2010a). No artigo 27 da Lei nº 12.305/10, são estabelecidos os parâmetros quanto à responsabilidade e obrigatoriedade de planos de manejo para estabelecimentos que lidam com resíduos perigosos ou resultantes de processos produtivos. Por meio de solicitação à Secretaria Municipal de Desenvolvimento Urbano e Habitação (SEMDUH), obteve-se o Relatório Quantitativo do Programa de Educação Ambiental e a relação de resíduos removidos durante o ano de 2019. O referido possui informações a respeito da Coleta Seletiva e dos Pontos de Recebimento de Resíduos – PRR’s. O relatório não apresentava informações a respeito das empresas privadas, no entanto contribuiu para a visualização das ações governamentais para a instituição e gestão do sistema municipal de logística reversa. Contudo, com base nas informações presentes na relação de resíduos removidos durante o ano de 2019, pode-se visualizar a situação da coleta de resíduos feita no município. O gráfico a seguir foi desenvolvido conforme dados presentes na referida relação. Gráfico 1 - Resíduos removidos durante o ano de 2019 Fonte: Elaborado pelos autores com base nos dados obtidos da Secretaria Municipal de Desenvolvimento Urbano e Habitação (SEMDUH), 2019. C f it d t i t l d t úbli i t lid i ti Gráfico 1 - Resíduos removidos durante o ano de 2019 Fonte: Elaborado pelos autores com base nos dados obtidos da Secretaria Municipal de Desenvolvimento Urbano e Habitação (SEMDUH), 2019. Conforme citado anteriormente, o papel do setor público consiste em consolidar o incentivo do desenvolvimento social e econômico de baixo impacto ambiental. Tendo em vista os parâmetros Conforme citado anteriormente, o papel do setor público consiste em consolidar o incentivo do desenvolvimento social e econômico de baixo impacto ambiental. Tendo em vista os parâmetros Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 15 15 e-ISSN: 2446-6875 p-ISSN: 1807-5436 e-ISSN: 2446-6875 p-ISSN: 1807-5436 implementou a Lei nº 4.474/13 (ver Quadro 1), que instituiu o Programa “Lixo Zero”. Conforme disposto no art. 6º, parágrafo único, da Lei nº 4.477/13, os recursos provenientes das multas são destinados à educação e áreas de interesse social (TERESINA, 2013). Ressalta-se que, conforme dados extraídos do site da Prefeitura Municipal de Teresina, as pessoas jurídicas representam os maiores índices de autuações do Programa “Lixo Zero” (PMT, 2020). Vale destacar que, conforme dados extraídos do site da Prefeitura Municipal de Teresina (PMT, 2020), apenas em janeiro de 2020, houve um crescimento de 44,55% nas autuações do Programa “Lixo Zero”. Constatou-se que a maioria das infrações são cometidas por pessoas jurídicas, representando um percentual de 54% no universo coletado. e-ISSN: 2446-6875 p-ISSN: 1807-5436 93), “quanto à efetividade das ações estabelecidas no Plano, o município apresenta resultados inadequados.”, tendo em vista que o descarte de resíduos sólidos ainda acontece em aterro controlado. A aplicação de taxas ou multas pode funcionar como um instrumento de controle sobre a geração de resíduos. Neste sentido, visando reduzir o descarte irregular de resíduos, o poder legislativo municipal Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 16 16 4.2 REFLEXOS DA IMPLEMENTAÇÃO DA COLETA PRIVADA As determinações da PNRS, constantes na seção V, art. 20 ao 24 da Lei nº 12.305/10, estabelecem que os geradores de resíduos sólidos são responsáveis pela elaboração do Plano de Gerenciamento de Resíduos Sólidos (PGRS), documento regulamentador das medidas necessárias para o manejo dos resíduos produzidos. Contudo, todo o processo de manejo envolve uma grande dispersão de resíduos, fator que contribui para o desenvolvimento de um sistema de coleta e reaproveitamento, ou destinação final, intermediado por empresas especializadas, ou seja, as empresas que compõem o segundo nível de logística reversa. Conforme dados extraídos do Plano Municipal de Gestão Integrada de Resíduos Sólidos (PMGIRS) de Teresina (PMT, 2018), no município existe uma quantidade considerável de grandes geradores na área urbana. Conforme citado anteriormente, o Decreto Nº 18061, de 18 de outubro de 2018, instituiu o cadastramento das empresas responsáveis pelo transporte de resíduos sólidos (coleta extradomiciliar) oriundos dos grandes geradores, ou seja, atores privados que atuam no segundo nível. Com a passagem de aproximadamente 2 (dois) anos após a sanção do Decreto Municipal N° 18061, de 18 de outubro de 2018, o estudo não identificou impacto relevante no mercado das empresas do segundo nível, ou seja, as empresas autorizadas a atuar na coleta, transporte, segregação e destinação de resíduos em relações privadas, que ofertam seus serviços no âmbito municipal sem intermediação ou remuneração por parte do poder público. Considerando a relação de empresas com “atividade de coleta de resíduos” disponibilizada pela Secretaria do Meio Ambiente (SEMAM), bem como a relação de empresas cadastradas pela Secretaria Municipal de Desenvolvimento Urbano e Habitacional (SEMDUH) /Coordenação Especial de Limpeza Pública (CELIMP), foram verificados os cadastros de 30 (trinta) empresas que realizam os serviços de coleta de resíduos sólidos. Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 17 17 e-ISSN: 2446-6875 p-ISSN: 1807-5436 e-ISSN: 2446-6875 p-ISSN: 1807-5436 em vista a pandemia ocasionada pelo vírus COVID-19 e medidas de isolamento social adotadas, bem como a fragilidade do mercado limitado, pode-se dizer que as empresas coletadas sofreram impactos na captação de recursos. Constatou-se que, dentre a amostra coletada, 1 (uma) empresa teve suas atividades suspensas até o dia 31 de agosto de 2020, por determinação do Ministério Público. A medida foi adotada em virtude do processo de incineração de resíduos realizado pela empresa estar gerando poluição atmosférica, ocasionando em impactos ambientais e prejudicando a população local. A empresa encontra-se relacionada a inquérito civil público que apura possíveis irregularidades em seu funcionamento. Além dos geradores, os atores privados que atuam no segundo nível da rede de logística reversa enfrentam dificuldades para realizar adequadamente a destinação dos resíduos. Segundo dados extraídos do Plano Municipal de Gestão Integrada de Resíduos Sólidos (PMGIRS) de Teresina (PMT, 2018), no município existem 101 pontos de disposição irregular de resíduos. Ressalta-se que a regulamentação e cadastramento das empresas em análise acontece desde o ano de 2018. No entanto, apenas em março de 2019, a Prefeitura de Municipal de Teresina deixou de realizar o manejo de resíduos sólidos extradomiciliares. Segundo informações do Portal O Dia (2019), aproximadamente 200 estabelecimentos ainda recorriam ao serviço de coleta municipal. Entretanto, os referidos estabelecimentos foram devidamente comunicados e orientados a contratar empresas privadas para realização do manejo de seus resíduos. Considerando as informações citadas no Gráfico 1, sobre o quantitativo pequeno de empresas especializadas na coleta de resíduos, pode-se observar que o município ainda ocupa o espaço de maior agente no sistema de logística reversa. A fiscalização junto aos geradores de resíduos acontece de maneira retraída e pode ser um dos fatores que contribuem para os índices de disposição irregular elevados. Vale pontuar que, de acordo com o Plano Municipal de Gestão Integrada de Resíduos Sólidos (PMGIRS) de Teresina (PMT, 2018), o município também realiza o cadastramento dos geradores, podendo “validá- los ou não como grandes geradores passivos de coleta e taxação diferenciada.”. O cadastro possibilita a definição e informação a respeito da necessidade de desenvolvimento do Plano de Gerenciamento de Resíduos Sólidos (PGRS) por parte dos geradores. Tal fator pode estar contribuindo para a estagnação do desenvolvimento do mercado em análise, tendo em vista que os grandes geradores podem não estar recorrendo ao setor privado para destinação de seus resíduos. e-ISSN: 2446-6875 p-ISSN: 1807-5436 e-ISSN: 2446-6875 p-ISSN: 1807-5436 Com base nos dados coletados (apêndice A), observa-se que a maioria das empresas desenvolvem outras atividades além da coleta, transporte, segregação e destinação de resíduos. Aparentemente, durante o processo de cadastramento, não acontece uma triagem de empresas que trabalham exclusivamente com a destinação de resíduos. Portanto, para atingir os objetivos propostos, foram selecionadas empresas que desempenhavam apenas atividades relacionadas ao manejo de resíduos. Quadro 2 - Empresas cadastradas para coleta, transporte, segregação e destinação de resíduos Atores Privados CARACTERÍSTICAS DO MANEJO DE RESÍDUOS ATIVIDADES Coleta de resíduos perigosos Coleta de resíduos não- perigosos Transporte e disposição de resíduos perigosos Transporte e disposição de resíduos não- perigosos Possui atividades não relacionadas ao manejo de resíduos sólidos? Sim Não Empresa 01 x x x Empresa 02 x x x x x Empresa 03 x x x x x Empresa 04 x x x Empresa 05 x x x x x Fonte: Elaborado pelos autores (2021). Quadro 2 - Empresas cadastradas para coleta, transporte, segregação e destinação de resíduos Fonte: Elaborado pelos autores (2021). Fonte: Elaborado pelos autores (2021). Considerando as empresas identificadas (apêndice A), verificou-se 5 (cinco) empresas cujo objeto se enquadraria na coleta de resíduos empresariais (ver Quadro 2). Com base nos dados, observa-se um destaque na coleta e destinação de resíduos perigosos. Ressalta-se que, conforme disposto no art. 13 da Lei nº 12.305/10, a PNRS (BRASIL, 2010a) destaca que os resíduos perigosos, são aqueles que, em razão de suas características de inflamabilidade, corrosividade, toxicidade, patogenicidade, carcinogenicidade e mutagenicidade, apresentam significativo risco à saúde pública ou a qualidade ambiental, de acordo com a lei, regulamento ou norma técnica. Com relação a expansão territorial, apenas 2 (duas) empresas possuem filiais em outros municípios/ estados. Entre as empresas selecionadas, apenas 1 (uma) encontra-se localizada no polo industrial da cidade. Em consideração a importância do meio digital, foram verificados os sites institucionais das empresas. Todas alimentam os sites com informações básicas como listas de contatos, listas de endereços, listas com os principais clientes, dados sobre a história e sobre os tipos de serviços ofertados. Verificou-se que apenas 1 (uma) empresa não possui redes sociais. Ainda com base nos dados coletados nos sites das empresas, pode-se observar que as referidas atentem negócios e indústrias locais. Tendo Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 18 18 e-ISSN: 2446-6875 p-ISSN: 1807-5436 e-ISSN: 2446-6875 p-ISSN: 1807-5436 A produção científica a respeito dos assuntos como responsabilidade compartilhada, o conceito de poluidor-pagador, logística reversa, direito ambiental, a aplicabilidade da Lei 12.305/10, bem como a Política Nacional de Resíduos Sólidos (PNRS) é consideravelmente vasta. No entanto, são poucos que evidenciam a corresponsabilidade das empresas privadas contratadas pelas organizações geradoras. Ademais, não foram localizadas parcerias entre as empresas em análise e a comunidade acadêmica. e-ISSN: 2446-6875 p-ISSN: 1807-5436 Observa-se que a estruturação do mercado das empresas do segundo nível do sistema de logística reversa acontece de maneira lenta. Nota-se também a ausência de inovação tecnológica no mercado. Ademais, caso não haja o fortalecimento do princípio da responsabilidade compartilhada, os avanços gerados pela Lei nº 12.305/10 podem ser negligenciados. Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 19 19 e-ISSN: 2446-6875 p-ISSN: 1807-5436 e-ISSN: 2446-6875 p-ISSN: 1807-5436 Passados aproximadamente 10 (dez) anos da instauração da Lei nº 12.305/10, e aproximadamente 2 (dois) anos da sanção do Decreto Municipal N° 18061, de 18 de outubro de 2018, observa-se que os avanços são considerados pequenos diante das inúmeras falhas no controle e fiscalização da gestão de resíduos sólidos. Ademais, a baixa adesão por parte das empresas e da população contribuem para que a destinação correta dos resíduos ainda apresenta baixos quantitativos. Tendo em vista que o poder público enfrenta dificuldades quanto à realização e aplicação de novas políticas públicas, a implementação de parcerias público-privadas (PPP’s) poderia contribuir para o estabelecimento consistente da gestão compartilhada. Seria uma alternativa viável, possibilitando a diminuição de custos para o município e permitindo a inclusão de mais empresas no processo de manejo de resíduos. A realização do estudo destaca a importância da implementação de ações previstas em instrumentos legais, favorece a exploração do debate e contribui para análise da responsabilidade compartilhada dentro dos sistemas de logística reversa. Ressalta-se a importância de se analisar processos em andamento, captando as dificuldades de implementação que muitas vezes desaparecem quando se trata de relatos ex-post facto. A pesquisa possui limitações importantes, sendo a principal a ausência de dados relativos ao mercado. Por ser um processo ainda em formação, tanto as informações como as fontes ainda estão por serem mapeadas. Isto restringe o uso de instrumento de dados primários junto a atores que não o poder público. A busca por dados secundários na internet, sem uso de repositórios institucionais hierarquizados, compromete a representatividade e confiabilidade das informações, requerendo que a análise considere a existência, ou não, do fato, e não o conteúdo em si. Por fim, o momento em que a pesquisa estava em execução ocorreu no período de isolamento social provocado pela COVID-19, o que impossibilitou o acompanhamento da ação das empresas pioneiras. Como recomendações, tem-se tanto uma análise dos dados secundários referentes aos volumes coletados e tratados, que denotam o amadurecimento do sistema, quanto a realização de pesquisas qualitativas que acompanhem a formação do mercado privado, envolvendo empresas, cooperativas e movimentação social de conscientização e ação referente a redução e reaproveitamento dos resíduos urbanos. ABRELPE - Associação Brasileira de Empresas de Limpeza Pública e Resíduos Especiais. Panorama dos Resíduos Sólidos no Brasil - 2018/2019. São Paulo, 2019. Disponível em: <https://abrelp.org.br/pano rama/>. Acesso em: 15 ago. 2020. 5 CONSIDERAÇÕES FINAIS A implementação da Lei nº 12.305/10, que instituiu a Política Nacional de Resíduos Sólidos (PNRS), contribuiu para uma revolução nos termos ambientais do país. A Lei estabeleceu as diretrizes de classificação e destinação dos resíduos, introduziu o conceito de Logística Reversa nas organizações, atribuiu aos estados e municípios o poder de regulamentar e fiscalizar o seu cumprimento e estabeleceu a responsabilidade compartilhada sobre os resíduos. O princípio do poluidor-pagador, instituído no inciso II do art. 6º da referida, contribuiu para imputação da responsabilidade ao gerador com relação aos custos resultantes de seus sistemas de produção. No âmbito municipal, visando a adequar-se às medidas instauradas pela Lei nº 12.305/10, o Decreto Municipal N° 18061, de 18 de outubro de 2018, foi instituído para estabelecer o cadastramento e a fiscalização de empresas privadas para atuarem na coleta dos resíduos resultantes dos grandes geradores (coleta extradomiciliar). O presente estudo analisou o cenário municipal após a passagem de aproximadamente 2 (dois) anos da sanção do Decreto Municipal N° 18061, de 18 de outubro de 2018, bem como os impactos da Lei nº 12.305/10 e da Política Nacional de Resíduos Sólidos (PNRS) no sistema de logística reversa teresinense. Com base nos dados coletados, constata-se que o sistema de logística reversa teresinense referente a coleta de grandes geradores ainda é incipiente. Tendo em vista que o estudo buscou analisar apenas empresas especializadas na coleta, transporte, segregação e destinação de resíduos, nota-se que a grande maioria das empresas cadastradas nos órgãos públicos realizam outras atividades além da coleta de resíduos extradomiciliares. Observa-se que tanto os geradores, como o setor privado de coleta, enfrentam dificuldades para realizar a destinação correta de resíduos, persistindo a disposição irregular dos resíduos, desafiando a atuação dos órgãos públicos. Vale destacar que no município os resíduos coletados ainda são destinados a aterros sanitários. Ademais, ao analisar os prazos estabelecidos pela PNRS, pode-se constatar que as medidas adotadas pelo município são recentes, tendo sido estabelecidas após os períodos previstos em lei. Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 20 20 e-ISSN: 2446-6875 p-ISSN: 1807-5436 e-ISSN: 2446-6875 p-ISSN: 1807-5436 ALVES, Ricardo Ribeiro. Administração Verde: O caminho sem volta da sustentabilidade ambiental nas organizações. 1. ed. Rio de Janeiro: Elsevier, 2016. ALVES, Ricardo Ribeiro. Administração Verde: O caminho sem volta da sustentabilidade ambiental nas organizações. 1. ed. Rio de Janeiro: Elsevier, 2016. BORGES, Alexandre Walmott. MELLO, Giovanna Cunha. OLIVEIRA, Mário Angelo. Mecanismos Garantido res do Direito Fundamental ao Ambiente na Política Nacional de Resíduos Sólidos: análise dos princípios do Poluidor-Pagador e do Protetor-Recebedor. Veredas do Direito, v. 7, n. 13/14, p. 191-212, Belo Hori zonte, 2010. BRASIL. Lei nº 12.305, de 02 de agosto de 2010. Institui a Política Nacional de Resíduos Sólidos. Brasília: Presidência da República: Casa Civil, 2010a. BRASIL. Decreto nº 7.404, de 23 de dezembro de 2010. Regulamenta a Lei nº 12.305, de 2 de agosto de 2010, que institui a Política Nacional de Resíduos Sólidos; cria o Comitê Interministerial da Política Na cional de Resíduos Sólidos e o Comitê Orientador para a Implantação dos Sistemas de Logística Reversa; e dá outras providências. Brasília: Presidência da República, 2010b. CARVALHO, M. S. F. C.; CARVALHO, C. P. Discursos sobre a mulher no webjornalismo piauiense: análise crítica das notícias dos portais Cidade Verde e G1/PI. Revista Observatório, v. 4, n. 1, p. 779-802, 2018. CERVO, Amado L.; BERVIAN, Pedro A. Metodologia científica. 5. ed. São Paulo: Prentice Hall, 2002. COELHO, João Paulo Ferreira. Análise do Sistema de Gestão e Gerenciamento de Resíduos Sólidos Ur banos na Cidade de Teresina-PI. 2019. 119 f. Orientador: Prof. Dr. Denis Barros de Carvalho. Dissertação (Mestrado Profissional em Gestão Pública) - Universidade Federal do Piauí, Teresina, 2019. COOPER, Donald R.; SCHINDLER, Pamela S. Métodos de pesquisa em administração. 10a ed. Porto Ale gre: Bookman, 2011. CORRÊA, Henrique Luiz. XAVIER. Lúcia Helena. Sistemas de Logística Reversa: criando cadeias de supri mentos sustentáveis. São Paulo: Atlas, 2013. Gestão de resíduos sólidos são desafios para empresas em Teresina. Portal O Dia. Teresina, 09 de agos to de 2019. Disponível em: <https:www.portalodia.com/amp/noticias/politica/novas-regras-para-ges tao-de-residuos-impoem-desafio-a-empresas-na-capital-368110.html>. Acesso em 13 set. 2020. Gestão de resíduos sólidos são desafios para empresas em Teresina. Portal O Dia. Teresina, 09 de agos to de 2019. Disponível em: <https:www.portalodia.com/amp/noticias/politica/novas-regras-para-ges tao-de-residuos-impoem-desafio-a-empresas-na-capital-368110.html>. Acesso em 13 set. 2020. INSTITUTO ETHOS DE EMPRESAS E RESPONSABILIDADE SOCIAL. REFERÊNCIAS ABRELPE - Associação Brasileira de Empresas de Limpeza Pública e Resíduos Especiais. Panorama dos Resíduos Sólidos no Brasil - 2018/2019. São Paulo, 2019. Disponível em: <https://abrelp.org.br/pano rama/>. Acesso em: 15 ago. 2020. Gestão e Desenvolvimento \| Novo Hamburgo \| v. 18 \| n. 2 \| mai./ago. 2021 21 21 e-ISSN: 2446-6875 p-ISSN: 1807-5436 Princípios e Critérios para Imple mentação de Alianças Público-Privadas na Gestão de Resíduos Sólidos: como ações baseadas na colaboração entre os diversos atores sociais podem atender diretrizes da Política Nacional de Resíduos Sólidos (Lei 12.305/2010). São Paulo, 2015. 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https://openalex.org/W2293688677	https://inria.hal.science/hal-01789956/document	English	null	A version of LEACH Adapted to the Lognormal Shadowing Model	IFIP advances in information and communication technology	2,015	cc-by	4,461	To cite this version: Chifaa Tabet Hellel, Mohamed Lehsaini, Hervé Guyennet. A version of LEACH Adapted to the Lognormal Shadowing Model. 5th International Conference on Computer Science and Its Applications (CIIA), May 2015, Saida, Algeria. pp.465-475, 10.1007/978-3-319-19578-0_38. hal-01789956 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01789956 https://inria.hal.science/hal-01789956v1 Submitted on 11 May 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. 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Distributed under a Creative Commons Attribution 4.0 International License A Version of LEACH Adapted to the Lognormal Shadowing Model 1Tabet Hellel Chifaa, 1Lehsaini Mohamed, and 2Guyennet Hervé 1STIC Laboratory, University of Tlemcen, Algeria 2FEMTO-ST/DISC UFR ST, University of Franche-Comte, France tabetchifaa@gmail.com, m_lehsaini@mail.univ-tlemcen.dz, herve.guyennet@femto-st.fr A Version of LEACH Adapted to the Lognormal Shadowing Model 1Tabet Hellel Chifaa, 1Lehsaini Mohamed, and 2Guyennet Hervé 1STIC Laboratory, University of Tlemcen, Algeria 2FEMTO-ST/DISC UFR ST, University of Franche-Comte, France tabetchifaa@gmail.com, m_lehsaini@mail.univ-tlemcen.dz, herve.guyennet@femto-st.fr 1Tabet Hellel Chifaa, 1Lehsaini Mohamed, and 2Guyennet Hervé 1STIC Laboratory, University of Tlemcen, Algeria 2FEMTO-ST/DISC UFR ST, University of Franche-Comte, France tabetchifaa@gmail.com, m_lehsaini@mail.univ-tlemcen.dz, herve.guyennet@femto-st.fr Abstract. The most protocols designed for wireless sensor networks (WSNs) have been developed for an ideal environment represented by unit disc graph model (UDG) in which the data is considered as successfully received if the communicating nodes are within the transmission range of each other. Howev- er, these protocols do not take into account the fluctuations of radio signal that can happen in realistic environment. This paper aims to adapt LEACH protocol for realistic environment since LEACH is considered as the best cluster-based routing protocol in terms of energy consumption for WSNs. We have carried out an evaluation of LEACH based on two models; lognormal shadowing mod- el (LNS) in which the probability of reception without error is calculated ac- cording to the Euclidian distance separating the communicating nodes and probabilistic model in which the probability of reception is generated randomly. In both models, if the probability of successful reception is lower than a prede- fined threshold, a multi-hop communication is incorporated for forwarding data between cluster-heads (CHs) towards the base station instead of direct commu- nication as in original version of LEACH. The main aims of this contribution are minimizing energy consumption and guaranteeing reliable data delivery to the base station. The simulation results show that our proposed algorithm out- performs the original LEACH for both models in terms of energy consumption and ratio of successful received packets. Keywords: LEACH, Lognormal shadowing model, Multi-hop scheme, Proba- bilistic model, Unit Disc Graph model, WSNs. 1 Introduction 1 WSNs are composed of hundreds and thousands of small devices called "sensor nodes" distributed over a monitoring area for sensing data and sending it to a remote base station directly or via a multi-hop communication scheme depending on the ap- plication designed [1]. This novel technology has allowed the appearance of many applications such as; military, security, medical, environment monitoring, etc, due to the low cost of sensor nodes. Moreover, with this technology our way of life has been revolutionized since it allowed us to interact with the surrounded environment. Routing process is a fundamental operation in wireless sensor networks. It consists in establishing path to transmit a message from a source node to a remote base station according to the main routing schemes: hierarchical, location-based, data-centric and QoS-aware [2]. However, cluster-based routing in wireless sensor network is consid- ered as the perfect solutions for minimizing energy consumption [3,4]. In this scheme, the network is divided into clusters wherein each cluster contains a number of mem- bers which sense data from its environment and send it to its corresponding cluster- head (CH). The latter is responsible for gathering data received from its members. If the distance between the source node and the destination node will increase, the ener- gy consumption also increases, thereby a cluster-based routing scheme is recom- mended. Among the protocols proposed, LEACH (Low Energy Adaptive Clustering Hierarchy) [5] is considered as the best cluster-based protocol for saving energy. Nevertheless, the performance of these protocols may degrade in non-ideal environ- ments. In this paper, we used the lognormal shadowing model [6] and the probabilistic model to simulate a non-ideal environment, and we evaluated the performance of LEACH with these both models. Then, we proposed an improved version of LEACH to over- come the limitations of the original version. The proposed version involves a CH-to- CH routing scheme to guarantee reliable delivery. This routing scheme is used if the probability of reception of packets without error between cluster-heads and the base station is lower than a predefined threshold. Moreover, this scheme also permits to minimize energy consumption. The rest of paper is organized as follow; in section 2, we give an overview on LEACH protocol and discuss some works that improve LEACH related to our re- quirements. 1 Introduction Section 3 presents our improved version of LEACH to be adapted in realistic environment, and in section 4, we illustrate performance of LEACH and the proposed contribution in non-ideal environment. Finally, in section 5, we conclude our paper. 2 Related Work Since LEACH protocol is considered among the best cluster-based routing protocols in terms of energy efficiency, a lot of researches have been enhancing this protocol to reduce its limitations. In the following, we present briefly LEACH protocol and some variants of it. LEACH [5] is a cluster-based routing protocol that aims to minimize energy con- sumption and thereby increasing network lifetime. In LEACH, the network is divided into clusters and each cluster is headed by a cluster-head which is elected by itself by generating a random number between 0 and 1. If the number generated is lower than a predefined threshold, the concerned node becomes a CH for the current round. The threshold is computed by each sensor node according to the equation (1). ( ) { ( ) (1) (1) where p is the percentage of cluster-heads, r is the current round, G is the set of nodes that have not been selected as cluster-heads in the last (1/p) rounds. LEACH is performed in two phases: setup phase and steady phase. In setup phase, each CH broadcasts an advertisement message to construct its cluster, and each non- CH that receives this message joins the adequate cluster based on the RSSI (Received Signal Strength Indication) of the message received. Once the clusters are formed, a TDMA (Time Division Multiple Access) schedules are assigned to member nodes in each cluster. In steady phase, each member transmits its sensed data to its correspond- ing CH in its scheduled time-slot, and then the CH aggregates all data received from its members and sends it to the remote base station directly. To avoid interference between cluster-heads, each CH chooses a CDMA (Coding Division Multiple Access) code that is different from other clusters to communicate with the base station. In [7], the authors have proposed a multihop routing scheme with lower energy adaptive clustering hierarchy called MR-LEACH. In this scheme, the CHs are respon- sible to aggregate data sensed by their members and act as relay nodes for remote CHs from the base station. MR-LEACH increases network lifetime since it uses a multihop routing scheme. This protocol is performed in three phases: cluster for- mation at lowest level, cluster discovery at different levels and scheduling. 2 Related Work The proposed protocol is performed in three phases: cluster formation, data delivery and update of clusters. At the beginning of the first phase, each node generates a random number between 0 and 1 and compares it with a predefined threshold to which the energy factor is added as illustrated by the equation (2). ( ) { ( ) (2) where En is the residual energy and EAverage is the average residual energy of all nodes ( ) { ( ) (2) where En is the residual energy and EAverage is the average residual energy of all nodes. Each CH sets H to 0, where H is the number of hops from CH, and broadcasts a message containing (H=0, ID) in its vicinity. Each non-CH that receives this message joins the cluster to which it belongs the transmitter CH and sets its own CH to the CH of the message received and H to H+1, and PID with the ID of its parent. Then, this node also broadcasts a message containing (Hj,ID). If a node receives more than one message it compares its own H with H of the sending nodes and it updates its H with the lowest one among those received. For intra-cluster communication, each node sends its packet to its parent and this latter sends it to its parent until reach the CH, and in inter-cluster communication, CH transmits data in chain structure. The con- cerned CH chooses the closest CH from it as the next hop and this process is repeated until reach the base station. In update of clusters, at the end of a round the remaining energy of CH may not be sufficient for the next round so the CH must be replaced by the node with the greatest residual energy. g gy In [10], an enhanced version of LEACH is proposed whose aims are saving energy by using a CH-to-CH multihop scheme and creating a backup path between cluster- heads to achieve fault-tolerance in the presence of failures. At the beginning, the base station broadcasts a HELLO message and each CH that receives this message calcu- lates the RSSI. If RSSI is higher than a predefined threshold, this CH is closest to the base station and if not the CH is away from it and therefore it needs a relay node to reach the base station. 2 Related Work At the beginning of each round, cluster formation phase is lunched to construct a table in which each node maintains the information about its neighbors (node identifier, resid- ual energy and node status) by using a 'Hello' message. Then, CHs broadcast a HEAD-MSG message in its vicinity and each non-CH chooses its respective CH among those in its neighborhood based on the strength of RSSI. In cluster discovery, the base station broadcasts its identifier (ID). Each CH that receives this broadcasted message records the identifier of the base station and replies by a beacon signal with its ID. CHs that are closest to the base station are in level one i.e. they could reach the base station by single hop. Then, BS broadcasts again a control message, the CHs that are at level two reply to this message forwarded by CHs of level one and the BS would record cluster-head’s ID and its level. Similarly, this process is repeated until no new CH is discovered. After that, the BS will form a cluster of CHs. In scheduling phase; after cluster formation, a TDMA scheduling is used for communication be- tween CHs at different levels. In [8], the authors proposed MH-LEACH which uses a new scheme for multihop communication to minimize energy consumption. MH-LEACH is carried out in two phases to establish paths towards the base station. In the first phase, the cluster-heads are selected as in the original version of LEACH protocol, and then each CH broad- casts an announcement message within its vicinity. Each non-CH that receives this message chooses the closest one based on the RSSI of the message received. Moreo- ver, the base station performs the same process. In the second phase; each CH sends its initial route to reach the base station and the latter send back the route to the CH to confirm that there is a route between the considered CH and the base station. There- fore, a routing table is created by each CH that contains a list of available routes to the base station and the shortest one is used. In [9], the authors proposed an improved version of LEACH in which a multihop scheme is used and the election of the cluster-heads is done according to its residual energy. In this protocol, a multihop routing scheme for intra-cluster communication and a chain structure routing scheme for inter-cluster communication. 2 Related Work The relay node is one of the CHs that are closest to the base station and it is selected based on the RSSI of a message exchanged between CHs and a variable called Cred which is a random number comprised between 0 and 1. If each of these parameters is higher than a threshold, this CH is considered as perfect relay node. However, the failure of one CH on the multihop path can affect the entire path and then the information cannot reach the base station, in this case a backup path is incorporated to this multihop path to ensure fault-tolerance and reliable delivery. 3.1 Lognormal Shadowing Model The lognormal shadowing model [6] is considered as a realistic model. It takes into account the fluctuations of radio signal caused by several factors such as noise, the presence of obstacles, weather conditions, etc... to evaluate the link quality between communicating nodes. The link quality is used to determine the probability of suc- cessful reception between communicating nodes in order to know if the message is received or it is corrupted by the destination node. Since this probability implied sev- eral factors, it may be difficult to obtain an accurate evaluation for all these factors which are themselves prone to errors. Therefore, we assume that signal strength grad- ually decreases according to the distance; thereby the probability of reception without errors can be computed according to the distance separating two nodes. We used the fluctuation of the signal model described in [11] as presented by the equation (3). ( ) { ( ) ( ) ( ) ( ) { ( ) ( ) ( ) ( ) where  is the attenuation factor that depends on the environment and x is the dis- tance separating the two communicating nodes. Rc is the transmission range and if the distance between two nodes is equal to Rc, the probability of successful reception is 0.5. 3 Contribution Before presenting our contribution, we give a brief description of the lognormal mod- el and probabilistic model. Then, we evaluate the performance of LEACH protocol with the both models to point out its weaknesses over an ideal environment. 3.2 Probabilistic Model In this model, the probability of reception without errors is generated randomly be- tween each two communicating nodes. This model is used to illustrate the link quali- ty. Moreover, the probability of successful reception is independent of the distance separating the communicating nodes but it depends on the factors which exist in the environment such as the presence or the absence of obstacles. Fig. 1 shows that node A can communicate with the node B but it cannot communicate with the node C alt- hough the distance that separates it with the node C is lower than that of the node B. Fig. 1. Communication in probabilistic model A C B coverage area C B Fig. 1. Communication in probabilistic model 3.3 Proposed Scheme In WSNs, LEACH is considered one of the best protocols in terms of energy efficien- cy. Several proposed protocols compare its effectiveness to LEACH and a lot of im- proved versions of LEACH have been proposed to reduce its limitations but they cannot guarantee its functionalities in a realistic environment. Our proposed algorithm aims to minimize energy consumption and ensures reliable delivery to the base station in a realistic environment based on lognormal shadowing model and probabilistic model. We have proposed a multihop scheme instead of direct communication scheme be- tween clusterheads and the base station to overcome the limitations of LEACH, such as when a CH aggregates data received from its members it computes the probability of reception without error of its packet to the base station. If this probability is higher than a predefined threshold, the packet is received correctly via direct communication by the base station and if not it means that the communication link is unreliable and in this case a multihop communication scheme will be incorporated to ensure the suc- cessful reception of packets by the base station. The proposed routing schemes are performed according to the following algorithms: Algorithm 1: Routing scheme based on LNS model - CHS: Set of clusterheads - BS: Base station whose coordinates (xb, yb) - d: Euclidean distance between CH(x,y) and BS - d: Euclidean distance between CH(x,y) and BS - CHD: Set of clusterheads that can send data directly to the BS - CHR: Set of clusterheads that use relay nodes to reach the BS - CHR: Set of clusterheads that use relay nodes to reach the BS Begin For (CH  CHS) do - CH(x,y) calculates the Euclidean distance that sepa- rates it from the BS √( ) ( ) - CH computes the probability of reception without error ( ) ( ) if (Pr(d) > Threshold) then * + else * + end if end For - Let CH a clusterhead if (CH  CHD) then - CH sends directly aggregated data to BS else - CH sends directly aggregated data to BS else else - CH selects a (CHr  CHD) as relay node with minimum distance to BS - CH selects a (CHr  CHD) as relay node with minimum distance to BS Min =  For (CHi  CHD) do - Computes the distance between CH(x,y) and CHi(xi,yi) For (CHi  CHD) do - Computes the distance between CH(x,y) and CHi(xi,yi) √( ) ( ) if dd < Min then Min = dd CHr = CHi if dd < Min then Min = dd CHr = CHi CHr is selected as relay node by CH - CHr is selected as relay node by CH end if End Algorithm 2: Routing scheme based on probabilistic model Begin For (CH  CHS) do - CH generates a random number (rd_number) comprise be- tween 0 and 1 if (rd_number ≥ 0.5) then * + else * + end if end For if (rd_number ≥ 0.5) then - Let CH a clusterhead if (CH  CHD) then - CH sends directly aggregated data to BS else - CH selects a (CHr  CHD) as relay node such as CH and CHr have a maximum probability. pr = 0 For (CHi  CHD) do CH t d b ( d b ) b t - CH selects a (CHr  CHD) as relay node such as CH and CHr have a maximum probability. 0 For (CHi  CHD) do For (CHi  CHD) do - CH generates a random number (rd_number) between CH and CHi - CH generates a random number (rd_number) between CH and CHi if (rd_number > pr) then pr = rd_number CHr = CHi if (rd_number > pr) then pr = rd_number CHr = CHi if (rd_number > pr) then CHr is selected as relay node by CH CHr is selected as relay node by CH Algorithm 2: Routing scheme based on probabilistic model Algorithm 2: Routing scheme based on probabilistic model - CHS: Set of clusterheads - BS: Base station - CHD: Set of clusterheads that can send data directly to the BS - CHR: Set of clusterheads that use relay nodes to reach the BS - CHS: Set of clusterheads - BS: Base station - CHD: Set of clusterheads that can send data directly to the BS - CHR: Set of clusterheads that use relay nodes to reach the BS - CHS: Set of clusterheads - BS: Base station - CHD: Set of clusterheads that can send data directly to the BS - CHR: Set of clusterheads that use relay nodes to reach the BS Begin CHD =  CHR =  For (CH  CHS) do - CH generates a random number (rd_number) comprise be- tween 0 and 1 if (rd_number ≥ 0.5) then * + else * + end if end For 4 Simulation Results In improved LEACH the unreliability of links between a clusterhead and the base station can be treated by a multihop communication by against, in original LEACH the packet will be lost due to the unreliable links. Fig. 3. Energy consumption in LEACH and Improved-LEACH Fig.3 illustrates that energy consumption in improved LEACH based on LNS model or Probabilistic model is lower than in LEACH because in the improved version, the multihop transmission scheme minimizes energy consumption but the direct commu- nication in LEACH consumes more energy. 0 50 100 150 200 250 20 40 60 80 100 Energy consumption (joules) Number of nodes improved-LEACH (Probabilistic model) improved-LEACH (LNS model) LEACH Fig. 3. Energy consumption in LEACH and Improved-LEACH Fig.3 illustrates that energy consumption in improved LEACH based on LNS model or Probabilistic model is lower than in LEACH because in the improved version, the multihop transmission scheme minimizes energy consumption but the direct commu- nication in LEACH consumes more energy. Fig.3 illustrates that energy consumption in improved LEACH based on LNS model or Probabilistic model is lower than in LEACH because in the improved version, the multihop transmission scheme minimizes energy consumption but the direct commu- nication in LEACH consumes more energy. 4 Simulation Results Several simulations have been carried out to illustrate the performance of our contri- bution using TOSSIM simulator [12], and compared them with the original version of LEACH in terms of energy consumption and the ratio of successful received packets at the base station. For that, we used a network that contains respectively 20, 40, 60, 80 and 100 stationary nodes, which are randomly deployed on a 100m x 100m square area and the initial energy of each node is equal to 2 joules. The simulations were performed in 600 seconds, and we used a threshold p= 0.7 for probability of reception without error in LNS model. We used this threshold to avoid on the one hand the ideal model whose threshold is 0.5 and the other to avoid a highly disturbed environment. Moreover, for probabilistic model, we used a threshold of p=0.5 i.e. a clusterhead generates a random number comprise between 0 and 1 and if this number is higher than 0.5 we assume that this clusterhead can communicate directly with the base sta- tion. Table I summarize simulation parameters. Table 1. Simulation Parameters Parameter Value Deployment Area 100m x 100m Simulation Time 600 sec Number of nodes 20, 40, 60, 80, 100 Packet size 29 bytes Initial node energy 2 Joules Threshold for LNS model p = 0.7 Threshold for probabilistic model p = 0.5 Fig. 2. Evaluation of ratio of successful received packets at BS with p=0.7 20 40 60 80 100 20 40 60 80 100 Percentage of successful reception at BS (%) Number of nodes improved-LEACH (Probabilistic model) improved-LEACH (LNS model) LEACH Table 1. Simulation Parameters Parameter Value Deployment Area 100m x 100m Simulation Time 600 sec Number of nodes 20, 40, 60, 80, 100 Packet size 29 bytes Initial node energy 2 Joules Threshold for LNS model p = 0.7 Threshold for probabilistic model p = 0.5 20 40 60 80 20 40 60 80 100 Percentage of successful reception at BS (%) Number of nodes improved-LEACH (Probabilistic model) improved-LEACH (LNS model) LEACH Fig. 2. Evaluation of ratio of successful received packets at BS with p=0.7 Fig. 2 shows that the ratio of successful packets received at base station with a proba- bility of p=0.7 in improved LEACH is higher than in original LEACH and also the ratio is higher with probabilistic model compared with LEACH. 5 Conclusion In this paper, we have evaluated LEACH protocol in a realistic environment repre- sented by lognormal shadowing model and a probabilistic model. However, results obtained illustrated that the performance of LEACH degrades in this kind of envi- ronment. Therefore, we have proposed an improved version of LEACH to overcome its weaknesses with realistic environment. The proposed scheme aims to find an op- timal multihop path over links error which is modeled by LNS model and probabilis- tic model. The simulation results showed that our contribution outperforms the original version of LEACH in terms of energy consumption and ratio of successful packets received at the base station. Moreover, our contribution deals with fault tolerance in LEACH, ensures reliable delivery and minimizes energy consumption. 6 References 1. Chanak, P., Banerjee, I.: Energy efficient fault-tolerant multipath routing scheme for wire- less sensor networks. The Journal of China Universities of Posts and Telecommunications. 20(6), 42-61 (2013). 2. Akkaya, K., Younis, M. A.: Survey on routing protocols for wireless sensor networks. Ad Hoc Networks. 3(3), 325-349 (2005). 3. Tyagi, S., Kumar, N.: A systematic review on clustering and routing techniques based up- on LEACH protocol for wireless sensor networks. Journal of Network and Computer Ap- plications. 36(2), 623-645 (2013). 4. Vlajic, N. and Xia, D.: Wireless sensor networks: to cluster or not to cluster?. International Symposium on World of Wireless, Mobile and Multimedia Networks. pp260-268, 2006. 5. Heinzelman, W. R., Chandrakasan, A., Balakrishnan, H.: Energy-efficient communication protocol for wireless microsensor networks. In: Proceedings of the 33rd IEEE Annual Ha- waii International Conference on System Sciences, pp.1-10, Hawaii (2000). 6. Rappaport, T. S.: Wireless Communications Principles and Practice. Second Edition by Publisher: Prentice Hall Release (2001). 7. Farooq, M. O., Dogar, A. B., Shah, G. A.: MR-LEACH: Multi-hop Routing with Low En- ergy Adaptive Clustering Hierarchy. In: Proceeding of Fourth IEEE International Confer- ence on Sensor Technologies and Applications (IEEE), pp.262-268, Venice, Italy (2010). 8. Neto, J., Antoniel Rego, A., Andr-Cardoso, A., Jnior, J.: MH-LEACH: A Distributed Al- gorithm for Multi-Hop Communication in Wireless Sensor Networks. In Proceeding of The Thirteenth International Conference on Networks (ICN), pp.55-61, Nice, France (2014). 9. Yang, H., Xu, J., Wang, R., Qian, L.: Energy-Efficient Multi-hop Routing Algorithm Based on LEACH. In: R. Wang and F. Xiao (Eds): CWSN 2012, CCIS 334, vol. 334, pp.578-587. Springer-Verlag, Heidelberg (2013). 10. Tabet Hellel, C., Lehsaini, M., Guyennet, H.: An Enhanced Fault-tolerant Version of LEACH for Wireless Sensor Networks. International Journal of Advancements in Compu- ting Technology(IJACT). 6(6), 50-57 (2014). 11. Kurvilla, J., Nayak, A., Stojmenoviç, I.: Hop count optimal position based packet routing algorithms for ad hoc wireless networks with a realistic physical layer. 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https://openalex.org/W2056287310	https://hal.archives-ouvertes.fr/hal-01135040/document	English	null	Dynamic Quantitative Intravital Imaging of Glioblastoma Progression Reveals a Lack of Correlation between Tumor Growth and Blood Vessel Density	PloS one	2,013	cc-by	13,048	Dynamic quantitative intravital imaging of glioblastoma progression reveals a lack of correlation between tumor growth and blood vessel density C. Ricard, F. Stanchi, T. Rodriguez, Mc Amoureux, G. Rougon, F. Debarbieux Dynamic quantitative intravital imaging of glioblastoma progression reveals a lack of correlation between tumor growth and blood vessel density C. Ricard, F. Stanchi, T. Rodriguez, Mc Amoureux, G. Rougon, F. Debarbieux C. Ricard, F. Stanchi, T. Rodriguez, Mc Amoureux, G. Rougon, F. Debarbieux To cite this version: C. Ricard, F. Stanchi, T. Rodriguez, Mc Amoureux, G. Rougon, et al.. Dynamic quantitative intravital imaging of glioblastoma progression reveals a lack of correlation between tumor growth and blood vessel density. PLoS ONE, 2013, 8 (9), pp.e72655. 10.1371/journal.pone.0072655. hal-01135040 Distributed under a Creative Commons Attribution 4.0 International License Abstract The spatiotemporal and longitudinal monitoring of cellular processes occurring in tumors is critical for oncological research. We focused on glioblastoma multiforme (GBM), an untreatable highly vascularized brain tumor whose progression is thought to critically depend on the oxygen and metabolites supplied by blood vessels. We optimized protocols for orthotopic GBM grafting in mice that were able to recapitulate the biophysical constraints normally governing tumor progression and were suitable for intravital multiphoton microscopy. We repeatedly imaged tumor cells and blood vessels during GBM development. We established methods for quantitative correlative analyses of dynamic imaging data over wide fields in order to cover the entire tumor. We searched whether correlations existed between blood vessel density, tumor cell density and proliferation in control tumors. Extensive vascular remodeling and the formation of new vessels accompanied U87 tumor cell growth, but no strong correlation was found between local cell density and the extent of local blood vessel density irrespective of the tumor area or time points. The technique moreover proves useful for comparative analysis of mice subjected either to Bevacizumab anti-angiogenic treatment that targets VEGF or to AMD3100, an antagonist of CXCR4 receptor. Bevacizumab treatment massively reduced tumoral vessel densities but only transiently reduced U87 tumor growth rate. Again, there was no correlation between local blood vessel density and local cell density. Moreover, Bev applied only prior to tumor implantation inhibited tumor growth to the same extent as post-grafting treatment. AMD3100 achieved a potent inhibition of tumor growth without significant reduction in blood vessel density. These results indicate that in the brain, in this model, tumor growth can be sustained without an increase in blood vessel density and suggest that GBM growth is rather governed by stromal properties. Citation: Ricard C, Stanchi F, Rodriguez T, Amoureux M-C, Rougon G, et al. (2013) Dynamic Quantitative Intravital Imaging of Glioblastoma Progression Reveals a Lack of Correlation between Tumor Growth and Blood Vessel Density. PLoS ONE 8(9): e72655. doi:10.1371/journal.pone.0072655 Editor: Arrate Mun˜oz-Barrutia, University of Navarra, Spain Received May 13, 2013; Accepted July 12, 2013; Published September 12, 2013 Copyright: 2013 Ricard 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. HAL Id: hal-01135040 https://hal.science/hal-01135040v1 Submitted on 4 Oct 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Abstract Funding: This work was supported by grants from Association de Recherche sur le Cancer, Institut National du Cancer (INCa-DGOS-Inserm 6038) to GR, from Centre National de la Recherche Scientifique (CNRS), Agence Nationale de la Recherche (ANR JCJC PathoVisu3Dyn), Fe´de´ration de Recherche sur le Cerveau (FRC) to FD; and by fellowships from the Fe´de´ration de la Recherche Medicale (FRM) to FS, TR, CR and from Canceropole PACA to CR. 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: genevieve.rougon@univ-amu.fr (GR); franck.debarbieux@univ-amu.fr (FD) Dynamic Quantitative Intravital Imaging of Glioblastoma Progression Reveals a Lack of Correlation between Tumor Growth and Blood Vessel Density Cle´ment Ricard1,2, Fabio Stanchi1,3, Thieric Rodriguez1, Marie-Claude Amoureux1,2, Genevie`ve Rougon1,2, Franck Debarbieux1,2 Cle´ment Ricard1,2, Fabio Stanchi1,3, Thieric Rodriguez1, Marie-Claude Amoureux1,2, Genevie`ve Rougon1,2, Franck Debarbieux1,2 1 Developmental Biology Institute of Marseille-Luminy (IBDML), Aix Marseille University-CNRS 7288, Marseille, France, 2 European Center for Medical Imaging (CERIMED), Marseille, France, 3 VIB3 Vesalius Onderzoekscentrum, KU, Leuven, Belgium 1 Developmental Biology Institute of Marseille-Luminy (IBDML), Aix Marseille University-CNRS 7288, Marseille, France, 2 European Ce Marseille, France, 3 VIB3 Vesalius Onderzoekscentrum, KU, Leuven, Belgium September 2013 \| Volume 8 \| Issue 9 \| e72655 Dynamic Quantitative Imaging of Glioblastoma Cells medium was added to the fresh culture medium (1/4th volume). Spheroid with diameters of 150–200 mm were identified and sucked into the tip of a glass pipette with a few ml of culture medium. Pipette inner diameter was used to size spheroids. murine central nervous system has either involved the use of open- skull or thinned-skull preparations [2,15]. While the open-skull technique is versatile, it is intrinsically suboptimal for studying GBM because it is invasive and leads to tumor growth outside of the dura mater when grafted at imaging relevant depth. The thinned-skull approach is minimally invasive, but the repeated re- thinning of skull required for chronic imaging increases the risks of tissue injury and allows for a limited number of imaging sessions. A polished and reinforced thinned skull technique was recently implemented [16] but light diffusion and absorption at the interface with bone compromise spatial resolution and limit imaging depth. GL261 cells, a murine glioma cell line (National Cancer Institute, Charles River Labs), were transfected with a plasmid for DsRed expression (pDsRed2-N1, Clontech, Mountain View, USA). Clones that stably express DsRed were selected using Geneticin (0.5 mg/ml, Gibco). Cells derived from one clone were cultured as monolayers in RPMI1640+GlutaMAX-1 (Gibco 61870) supplemented with 10% Fetal Calf Serum (Thermo Scientific) and Geneticin (0.5 mg/ml, Gibco). Five passages following transfection, cells were massively expanded in the presence of Geneticin and aliquoted prior freezing. Cells were unfrozen when needed and kept at 37uC in a 5% CO2 atmosphere. We presented here a chronic window method for monitoring GBM growth and angiogenesis in the first millimeter below the dura and over an extended period of time. Importantly, whereas GBMs are heterogeneous with hypervascularized inner regions and peripheral regions from which the tumor cells invade the normal brain, our technique analyze most of the tumor volume at once while preserving a fully intracerebral growth. We established the methods for an appropriate quantification of such images that were repeatedly acquired from the same subjects to collect dynamic data. The weak correlation observed between tumor progression and blood vessel density was further evaluated under systemic pharmacological manipulations that further uncoupled the modulation of tumor cell densities from that of vascular density. Using two FDA approved molecules Bev and AMD3100 [5,17,18] which both inhibited tumor growth despite differential actions on vascularization, we provide evidence that GBM can develop without angiogenesis. Dynamic Quantitative Imaging of Glioblastoma Cells A possibility is that both anti-tumor drugs mainly act on the stroma to impair tumor progression. Animal model Mice were anaesthetized by intraperitoneal injection of a mixture of Xylazine/Ketamine (12 mg/kg and 120 mg/kg, respectively) and positioned on a stereotactic frame (Mouse adapter, Harvard Apparatus). A square craniotomy (diameter: 3–4 mm) was performed on the left parietal bone. An injection hole was performed in the dura using a 31G needle to prepare insertion of the glass pipette. The spheroid of U87 cells was then pressure injected in the cerebral cortex approximately 250 mm below the brain surface using micromanipulators. Following implantation, a moist half-Sephadex bead with diameter fitting with the dura opening was inserted in the injection wound and glued to the dura-mater and surrounding parenchyma using histo- compatible acrylic glue (Cyanolit). A round glass coverslip (diameter: 6 mm) was then glued on the surrounding bone that had been previously thinned to ensure close contact between glass and brain. Glass window was further fixed to the skull by dental cement (Figure 1C–D). Anti-inflammatory treatment was administered by means of subcutaneous injections of Dexameth- asone (0.2 mg/kg, Vetoquinol) and Carprofen (5 mg/kg, Rimadyl Pfizer) every 48 h for two weeks to help resuming post-surgical inflammation. Animals were first imaged when inflammation was resolved on day 16 post surgery as indicated by normal vascular pattern (Figure 1E–F). Animal care guidelines In accordance with the policy of the Developmental Biology Institute of Marseille-Luminy (IBDML) and the French legislation, experiments were done in compliance with the European Community Council Directive of November 24, 1986 (86/609/ EEC). The research on animals was approved by the Direction De´partementale des Services Ve´te´rinaires des Bouches-du-Rhoˆne (permit number 13.055.21) and has been approved and authorized by the National Committee for Ethic in Animal Experimentation (Section Nu14; project 87-04122012). Six to seven weeks old (male) NIH nude mice (Foxn1-null, n = 75, Charles River, France) and C57 Blak6 (n = 6) were housed in cages with food and water ad libitum in a 12 h light/dark cycle at 2261uC. Introduction The size of tumor burden, its molecular heterogeneity as well as the stage of development can be responsible for variability in disease outcome. Therefore, a better understanding of tumor physiology first requires studying analytically the influence of several parameters on simple models before studying more realistic ones. The development of dedicated imaging modalities and appro- priate animal models is needed to characterize cellular interactions in vivo [1] and to evaluate drug effects on specific cell populations [2]. We chose to focus on glioblastoma multiforme (GBM) an invariably fatal brain tumor accounting for approximately 40% of all primary malignant brain tumors. GBM are highly vascularized tumors, and preclinical data have suggested that GBM growth critically depends on the generation of tumor-associated blood vessels [3,4]. In particular, the vascular endothelial growth factor (VEGF) pathway has been a prime drug target underscored by the approval of bevacizumab (Bev, an anti-VEGF monoclonal antibody) for treatment of patients [5,6,7]. The effect of anti- VEGF on tumor cells that secrete high levels of VEGF [8] is however transient [9,10,11] and the majority of patients eventually relapse. The mode of action for its clinical benefits is still not fully understood and needs further investigations on preclinical models. Whereas MRI is currently used to follow GBM progression, the heterogeneity of patient populations, the sparseness of exams as well as the approximate relationship between tumor contrasts and actual tumor cell density ultimately lead to misinterpretations [12,13,14]. Consequently, there is considerable need to develop dedicated imaging modalities and appropriate animal models to investigate in vivo, the drug effects on specific cell populations with the aim to optimize the efficacy of treatments in humans. To evaluate interactions between tumor cells and tumor vessels at single cell resolution and over time in vivo we used intravital two- photon microscopy in an orthotopic xeno or allotransplanted mouse models. Traditionally, in vivo two-photon imaging of the 1 September 2013 \| Volume 8 \| Issue 9 \| e72655 PLOS ONE \| www.plosone.org Microscopy Mice were anaesthetized by an intraperitoneal injection of a mixture of Xylazine/Ketamine (10 mg/kg and 100 mg/kg, respectively) and injected intravenously with 100 ml of a RhodamineB conjugate dextran (70 kDa) solution (20 mg/ml in PBS, R9379, Sigma Aldrich) prior to each imaging session and positioned on a stereotactic frame. Height of each earbar and of the mouth piece was adjusted independently under microscope control to ensure planar repositioning of the animal brain surface based on visual landmarks. Anesthesia was supplemented hourly (40 mg/kg; 4 mg/kg) to prevent toe pinch reflex. Dynamic Quantitative Imaging of Glioblastoma Cells plastic (A) and forming spheroids on agarose substrate (B). (C) Drawings showing the position of the cranial window on the mouse parietal bone (pink circle) and tumor grafting: after craniotomy, spheroids of 150–200 mm diameter where injected focally ,250 mm below brain surface without removal of the dura mater (D) Dura sealing and glass window fixation: a Sephadex hemi-bead (blue) is glued (gray arrows) to the dura to clog the pipette trajectory (step1); the glass window is glued (gray arrow) to the surrounding bone in close contact with the dura (step2); the cement (dark gray) is used for long term seal to the skull (step3); the tumor is forced to develop inside the physically constrained brain parenchyma (output). (E) Progression of the acute inflammatory vascular response from the installation of the cranial window on post-surgery day 0 (PSD0) to PSD16. PSD16 corresponds to the day of first visualization (D0). A bicolor two-photon projection image is presented on the right. (F) Scheme summarizing the time lines of application of Bev, AMD3100 and vehicle with regard to the date of 2P observations. (G) Sagittal maximal intensity projection over the tumor (green) showing the spheroid 6 days post implantation and its evolution at 2 weeks intervals. Second harmonic signal from the dura mater is blue. Position of the Sephadex bead is outlined (star). The tumor cells remain below the dura mater into the brain parenchyma (left). When dura mater is not sealed extradural growth of the tumor is already detectable 6 days post implantation (arrows) and further developed over time. Extradural tumor progression is 3 times larger than that observed inside the brain and after 4 weeks the proliferation that occurs at the interface between the glass and the dura mater prevents further imaging of the tumor growing inside the brain (right). (H) Horizontal maximal intensity projection over the whole tumor volume of the same image stacks as for G (left). Single cell resolution is achieved (Inset: zoom of the white ROI). Scale bars: all scale bars: 300 mm, E: 1 mm & H inset 25 mm. doi:10 1371/journal pone 0072655 g001 doi:10.1371/journal.pone.0072655.g001 and the same profile of settings was used at all depths for all mice involved in quantitative fluorescence experiments. and the same profile of settings was used at all depths for all mice involved in quantitative fluorescence experiments. ImageJ software. Immunostaining Brain tissues fixed with paraformaldehyde 4% were cryosliced in 25 mm thick sections. Blood vessels were revealed using rabbit anti-Laminin antibody (Novus Biological, 1/1000) and proliferat- ing cells revealed with rat monoclonal anti-BrdU antibody (Abcam, 1/100). Donkey anti-rabbit and donkey anti-rat second- ary antibody flagged with Cy3 and Cy5 were respectively used at (1/100) and (1/200). Tiled images of the whole tumor sections were acquired on a Zeiss Axioimager fluorescence microscope with 106 (NA = 0.30) dry objective. Statistics All data are expressed as mean 6 SEM. Variation coefficient (VC) was used to estimate the stability and reproducibility of the measurements. Mann-Whitney one tail U-test was used to test for the reduction of cell density and blood vessel density between the treated and control groups. p,0.05 was used as a criterion for significance. The plot of red versus green fluorescence measure- ments was systematically fitted with a linear regression intercepting the vertical axis at y = 1. Correlation between the data was acknowledged by a p value,0.05 as calculated for a Pearson correlation. The quality of the correlation between cell density and blood vessel density was evaluated and interpreted using classical standards: strong correlation when determination coefficient (R2) of the regression was superior to 0.5; poor correlation for R2,0.25 Treatment A 10 mg/kg dose of Bevacizumab (Anti-VEGF, Roche) in PBS was injected i.v. every 2–3 days from the first day of visualization until the last. A 1.25 mg/kg dose of AMD3100 (CXCR4 antagonist, Sigma Aldrich) in PBS was subcutaneously injected twice daily from the first day of visualization until the last. PBS vehicle injected i.v. was used as a control. Animals groups for treatment were homogenized before starting the pharmacological protocol based on the size of the tumor on the first day of visualization. See Figure 1F for time table details. BrdU 50 mg/kg was administered in 200 ml PBS i.v. 7 days after initiation of Bev treatment and 2 days prior sacrifice. Images of immunostained sections were manually thresholded to get rid of the background and the densities of thresholded voxels were used as an index of vessel and proliferating cell densities, respectively. Dynamic Quantitative Imaging of Glioblastoma Cells A conserved volume of interest ranging from ,50 to ,250 mm below the brain surface was extracted for all time points. Green and red channels were then separated and analyzed separately using home-written macros. Each channel was sum projected over the whole volume and tumor epicenter automatically located from the 2D projection corresponding to the first day of visualization (D0). Concentric regions of interest (ROIs) conserved across all time points were defined on the projections for each mouse. Evolution of the mean fluorescence in each ROI was used as an index of local tumor cells density (green) and of local blood vessel density (red) (see Figure 3 for details). For reliable comparisons, green fluorescence values recorded for each session were normalized for each animal to the one measured at the tumor core (ROI1) on the first day. Similarly, the red fluorescence inside the tumor was normalized to the mean fluorescence in surrounding healthy brain (ROI4). In vitro experiments U87-GFP cells were seeded in a 96 wells culture plate (7000 cells per well) in (MEM+Earle’s+GlutaMAX-1, 10% Fetal Calf Serum, 1 mM pyruvate and 0.1 mm non essential amino-acids). Cells were divided in 5 sub-groups (n = 6 to 12 wells per sub- group) (day 0) and incubated with the different drugs in the same medium volume: VEGF (50 ng/ml), Bev (250 ng/ml), VEGF+Bev (50 ng/ml and 250 ng/ml, respectively), AMD3100 (10 mg/ml). The culture plate was positioned on an inverted Zeiss Observer Z1 with Colibri fluorescence system equipped with a motorized stage and an incubation chamber (37uC, 5% CO2). Visible and fluorescence images were acquired for each well every 30 min with a 106 (NA = 0.30) objective for 59 to 114 h using Zeiss Axiovision 4.8 software. Images were then processed using the ImageJ free software and the MTrackJ plugin. (Figure S1B). Other types of standard regressions were tested but resulted in no better R2 (Figure S2G). All statistical analyses were performed with Excel (Microsoft, USA) and GraphPad Prism4 (GraphPad software, USA). Cell culture and gene transfection g U87 MG cells (ATCC number HTB-14, P133) were transfected with a plasmid for GFP expression (pEGFP-C1, Clontech, Mountain View, USA). Cells that stably express GFP were selected using Geneticin (0.5 mg/ml, Gibco). Five passages following transfection, cells were massively expanded in the presence of Geneticin and aliquoted prior freezing. U87MG- GFP cells were cultured as monolayers in MEM+Earle’s+Gluta- MAX-1 (Gibco) supplemented with 10% Fetal Calf Serum (Thermo Scientific), 1 mM pyruvate (Gibco) and 0.1 mM non- essential amino-acids (Gibco). Cells were kept at 37uC in a 5% CO2 atmosphere. We used Zeiss two-photon microscopes (Axioskop 2FS LSM510 or LSM7MP) home-modified to allow animal positioning below the objective and coupled to a femtosecond pulsed infrared laser (Mai-Tai, Spectra Physics) tuned at 840 nm. 106 air objective (NA = 0.45) or 206 water immersion objective (NA = 1.0) were selected based on field of view/resolution considerations (Figure 2A–B). GFP (500–550 nm) and RhodamineB (575– 640 nm) signals were simultaneously collected on 2 non-descanned detectors. Images were typically acquired over a depth of 500 mm using 5 mm steps. Laser intensity was linearly increased with depth For each animal series, an aliquot was unfrozen and plated to reach confluence two days before their implantation in mice. Confluent cells were then replated on Petri dishes coated with 0.75% soft agarose to induce aggregation of the cells into spheroids due to physical interactions with the substrate. Conditioned September 2013 \| Volume 8 \| Issue 9 \| e72655 PLOS ONE \| www.plosone.org 2 Dynamic Quantitative Imaging of Glioblastoma Cells Figure 1. A truly intracerebral GBM model suitable for two photon imaging of tumor cell and blood vessels. U87-GFP cell cultures, o Figure 1. A truly intracerebral GBM model suitable for two photon imaging of tumor cell and blood vessels. U87-GFP cell cultures, on igure 1. A truly intracerebral GBM model suitable for two photon imaging of tumor cell and blood vesse Figure 1. A truly intracerebral GBM model suitable for two photon imaging of tumor cell and blood vessels. U87-GFP cell cultures, on September 2013 \| Volume 8 \| Issue 9 \| e72655 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 3 Data analysis We designed a GBM model compatible with two-photon in vivo imaging and mimicking physiological tumor development [19,20]. We prepared homogeneous groups of immunodeficient mice grafted 250 mm deep into the left parietal cortex with GFP-U87 (Figure 1A–B) or DsRed-GL261 (Figure S1) spheroids. Dura In vivo image stacks corresponding to individual yet overlapping field of views were stitched together (see Figure 2A–B) and semi- automatically registered based on stable vascular landmarks using the sets of plugins named ‘‘Align Stacks’’ and ‘‘Stitching’’ for PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e72655 4 Dynamic Quantitative Imaging of Glioblastoma Cells Figure 2. Imaging blood vessel network and interactions with tumor cells. (A) Mean projection image of a tumor and associated vascularization acquired through a 206Water Immersion objective (N.A. = 1.0). One field of view corresponds to the dotted line box; 49 such fields were acquired to cover the full tumor extent. (B) For quantitative pharmacological experiments, a 106dry objective (N.A. = 0.45) was used to increase data throughput by a factor 4. An overlap of 50 mm between fields of view ensured qualitative image stitching performed with ImageJ 3D Stitching macro, ‘‘pcm3D’’. (C) Upper part, 3D projection of the vascular network over a depth of 150 mm of the tumor (right of the plain line) and of surrounding healthy brain (left of the plain line). Lower part: zooms of the outlined ROIs. Thin and straight normal brain vessels (left); tortuous and swollen tumor vessels (right). (D) Example of bicolor images showing at a 10 day interval the average vascular density (red) and tumor cell density (green) in a 25 mm thick horizontal section of the tumor located 200 mm below the brain surface. Note that the lower vascular density observed after 10 days is not associated with a reduction of tumor cell density. (E,F) Horizontal mean projection images over 100 mm subdural layers 15–19 days post implantation showing perivascular invasion (arrow heads). (G) Sagittal 3D rendering projection showing superficial tumor cells (arrow heads) migrating toward deeper brain regions along a large vertical vessel. (H) Mean projection of the volume of interest scanned for vascular changes occurring at a 1 day interval. All the changes (white dots) were found inside or within 150 mm off the tumor margin but none in the healthy brain region. Insets show examples of microvascular changes (arrows) at several locations. Data analysis injection of Rhodamine Dextran in healthy brain of n = 19 to 25 mice over repeated imaging sessions a healthy brain, vascularization is stable and average fluorescence in the raw images varied within 19% over 3 successive visua include all the methodological biases such as inter-animal variability, variations of Rhodamine dextran injected volumes repositioning, changes of tissue optical properties, laser and detector variability. Stability over time allows for quantitative a E) Analysis of 3D images stacks. (C) 3D rendering image (left) and zoom (right) of a bicolor 3D stack acquired over a dept view covering the whole extend of the tumor in the horizontal plane (2.4 mm2). Volumes of interest acquired every we registered based on several vascular landmarks (example: white arrow) and size adjusted by adding NaN (Not a Number) vo corresponding to the superficial tumor volume between ,50 mm and ,250 mm below the brain surface are projected by s are separated to obtain a set of vascular projections (D) and its corresponding set of brain tumor projections. (E) Coord center of mass in the green projection are then used to locate tumor epicenter on D0 (orange arrow, E). Tumor epicente Figure 3. Quantitative fluorescence measurements. (A) Average green fluorescence recorded from U87-GFP in culture wells was highly correlated (R2.0.95) with the number of cells. Green fluorescence was then used as an index of tumor cell density. (B) Raw fluorescence counts recorded after i.v. injection of Rhodamine Dextran in healthy brain of n = 19 to 25 mice over repeated imaging sessions and corresponding SEM. In healthy brain, vascularization is stable and average fluorescence in the raw images varied within 19% over 3 successive visualizations. These variations include all the methodological biases such as inter-animal variability, variations of Rhodamine dextran injected volumes, tilt angle during mouse repositioning, changes of tissue optical properties, laser and detector variability. Stability over time allows for quantitative analysis of our images. (C– E) Analysis of 3D images stacks. (C) 3D rendering image (left) and zoom (right) of a bicolor 3D stack acquired over a depth of 400 mm and a field of view covering the whole extend of the tumor in the horizontal plane (2.4 mm2). Volumes of interest acquired every week are semi-automatically registered based on several vascular landmarks (example: white arrow) and size adjusted by adding NaN (Not a Number) voxels. Data analysis Scale bars, A&B: 1 mm; C&D: 300 mm, F&G: 50 mm,; H:300 mm, inset 25 mm. doi:10.1371/journal.pone.0072655.g002 Dynamic Quantitative Imaging of Glioblastoma Cells Figure 2. Imaging blood vessel network and interactions with tumor cells. (A) Mean projection image of a tumor and associated vascularization acquired through a 206Water Immersion objective (N.A. = 1.0). One field of view corresponds to the dotted line box; 49 such fields were acquired to cover the full tumor extent. (B) For quantitative pharmacological experiments, a 106dry objective (N.A. = 0.45) was used to increase data throughput by a factor 4. An overlap of 50 mm between fields of view ensured qualitative image stitching performed with ImageJ 3D Stitching macro, ‘‘pcm3D’’. (C) Upper part, 3D projection of the vascular network over a depth of 150 mm of the tumor (right of the plain line) and of surrounding healthy brain (left of the plain line). Lower part: zooms of the outlined ROIs. Thin and straight normal brain vessels (left); tortuous and swollen tumor vessels (right). (D) Example of bicolor images showing at a 10 day interval the average vascular density (red) and tumor cell density (green) in a 25 mm thick horizontal section of the tumor located 200 mm below the brain surface. Note that the lower vascular density observed after 10 days is not associated with a reduction of tumor cell density. (E,F) Horizontal mean projection images over 100 mm subdural layers 15–19 days post implantation showing perivascular invasion (arrow heads). (G) Sagittal 3D rendering projection showing superficial tumor cells (arrow heads) migrating toward deeper brain regions along a large vertical vessel. (H) Mean projection of the volume of interest scanned for vascular changes occurring at a 1 day interval. All the changes (white dots) were found inside or within 150 mm off the tumor margin but none in the healthy brain region. Insets show examples of microvascular changes (arrows) at several locations. Scale bars, A&B: 1 mm; C&D: 300 mm, F&G: 50 mm,; H:300 mm, inset 25 mm. doi:10.1371/journal.pone.0072655.g002 September 2013 \| Volume 8 \| Issue 9 \| e72655 PLOS ONE \| www.plosone.org 5 Dynamic Quantitative Imaging of Glioblastoma Cells Figure 3. Quantitative fluorescence measurements. (A) Average green fluorescence recorded from U87-GFP in correlated (R2.0.95) with the number of cells. Green fluorescence was then used as an index of tumor cell density. (B) recorded after i.v. Imaging tumor growth, angiogenesis dynamics and heterogeneity Rhodamine B dextran (70 kDa) was injected intravenously prior to every imaging session to visualize vasculature (Figure 2). Several fields of view (up to n = 49) were acquired to cover the full tumor extent (Figure 2A, Video S1). An overlap of 50 mm between fields of view ensured qualitative image stitching performed with ImageJ 3D Stitching macro, ‘‘pcm3D’’ (Figure 2B). Tumor growth was accompanied by extensive vascular remodeling and the formation of new vessels (Figure 2C– H) that were dense, tortuous and swollen compared to healthy brain vessels (Figure 2C) as described for GBM patient tissue specimens [17]. Increases of cell density observed over time were not necessarily associated with increases in vascular density (Figure 2D) despite tumor cells migrating in close vicinity of blood vessels. Interactions between tumor cells and local blood vessels were sometimes obvious (Figure 2E–G, Video S2). The ability to follow individual blood vessels by high resolution imaging of the same field of view at 24 h intervals revealed several spots of marked and rapid vascular changes (Figure 2H). These plastic spots were exclusively located inside the tumor or within a ,150 mm margin (Figure 2H). Weak correlation between tumor proliferation and local blood vessel density 2 We indeed found no strong correlation (R2 = 0.0464, p,0.0001) between local cell density and blood vessel density when considering the group of mice as a whole (Figure 4B). Analyzing data from individual subjects confirmed the overall independence of tumor growth with regard to blood vessel density since correlation was extremely weak in 5 out of 7 animals (21.099,R2,0.298, p.0.05 n = 4 out of 5), and good only for the 2 others (R2 = 0.573 and 0.6718 respectively, p,0.0001 Figure S2A). We further investigated whether the strength of correlation was dependent on the level of tumor cell density (Figure S2B), on the stage of tumor evolution (Figure S2C), as well as on the location with respect to tumor center (Figure S2D). Linear correlation was poor in all cases, while no simple mathematical model performed better in describing the weak correlation between the two parameters (Figure S2G). We also took into consideration the hypothesis that an increase in cell density might be either concomitant, precede or succeed to an increase in blood vessel density. We did not found correlations between changes in local blood vessel density either resultant ((R2 = 20.143, p,0.0001) preceding (R2 = 20.057, p,0.05) or subsequent (R2 = 20.342, p = 0.57) to changes in cell densities (Figure 4C, Figure S2E, S2F). Dynamic Quantitative Imaging of Glioblastoma Cells followed as an index of mean blood vessel density and an index of tumor cell density in the 4 ROIs. doi:10.1371/journal.pone.0072655.g003 tumor (D0, D7, D14, D21). Despite implantation of calibrated spheroids of cells from the same culture passage, in the same cortical brain area of mouse littermates, tumor burden differed between animals on the first day of visualization (D0, VC = 58%, n = 19). For reliable comparisons, green fluorescence values recorded for each session were normalized for each animal to the one measured at the tumor core (ROI1) on the first day. Similarly, the red fluorescence inside the tumor was normalized to the mean fluorescence in surrounding healthy brain (ROI4). Relative changes of fluorescence values for equivalent ROIs were then averaged across animals. Exponential-like increase of tumor cell densities was observed in all regions with more or less delay at onset (Figure 4A & 5D). Although the corresponding blood vessel densities increased asymptotically by 64618% on average within 3 weeks, the dynamics of vascular changes were highly heteroge- neous between ROIs and animals and appeared unrelated to the local changes in tumor cell densities (Figure 4B, 5C–D, S2, S3). mater was essentially intact everywhere but at the site of injection where it was sealed at the end of the tumor implantation process. Sealing consisted in gluing a Sephadex hemibead to the meninges and surrounding tissue in order to block the pipette trajectory (Figure 1C–D). The repulsive Sephadex surface thus prevented tumor cells escape from deep brain layers toward the glass surface, a process that is otherwise responsible for unphysiological tumor cell proliferation in the absence of biophysical constraints (Figure 1G). Over time a tumor mass formed and expended in the 3 directions under the intact dura matter with cells infiltrating the peripheral regions as a common histological hallmark of malignant gliomas (Figure 1G–H). Imaging protocol started two weeks after grafting (Figure 1F) once animals had recovered from the transient surgical vascular inflammation (Figure 1E). Imaging tumor growth, angiogenesis dynamics and heterogeneity Data analysis The set of 40 images corresponding to the superficial tumor volume between ,50 mm and ,250 mm below the brain surface are projected by summation. Then channels are separated to obtain a set of vascular projections (D) and its corresponding set of brain tumor projections. (E) Coordinates of the fluorescence center of mass in the green projection are then used to locate tumor epicenter on D0 (orange arrow, E). Tumor epicenter is then repositioned on every projection (orange circle in D & E). Concentric circles with 330 mm radius increments are then virtually superimposed on the projections. The area between two successive circles defines a conserved region of interest (ROI) across time points. Evolution of mean fluorescence has thus been September 2013 \| Volume 8 \| Issue 9 \| e72655 September 2013 \| Volume 8 \| Issue 9 \| e72655 PLOS ONE \| www.plosone.org 6 Dynamic Quantitative Imaging of Glioblastoma Cells Quantitative analysis of proliferation and local vessel density y To search whether correlations could be found between vascular plasticity, vascular density, tumor cell density and proliferation we devise a technique to quantify our dynamic imaging data. To this end, we first established on GFP-U87 cell cultures that green fluorescence intensity was linearly correlated with cell numbers (R2.0.95, Figure 3A). Second, we confirmed that stable brain vascularization gave constant red fluorescence values over repeated imaging sessions (VC (Variation Coeffi- cient) = 12% over 3 consecutive imaging sessions n = 19). Inter- animal variability was small (VC = 19%, n = 19, Figure 3B) including all sources of variations such as hardware stability, windows optical properties and animal repositioning or success of i.v. injection. We then used the mean fluorescence values on the green and red channels in concentric regions of interest (ROIs, Figure 3D–E) as indexes of local cell density and local blood vessel density, respectively. A circular field of 2.5 mm diameter was thus routinely analyzed over a depth of at least 200 microns. For each mouse, fluorescence was quantified for every individual ROI from the central (ROI1) to the peripheral (ROI4) relative to the tumor epicenter at 4 different times of development of the To finally search whether a correlation could be found between blood vessel density and proliferation we injected tumor bearing mice with BrdU two days prior sacrifice and performed immunolabeling experiments (Figure 4D). Since tumor hetero- geneity can often bias data obtained from subject biopsies, we performed multiscale analysis of the bicolor fluorescence signals in the whole tumor area for sagital cryosections taken from the central part of the tumor. Similarly to in vivo results, vascular density was 1.7 times larger inside the tumor compared to surrounding healthy brain regions (5767 a.u. compared to 3465 a.u. n = 4), and a poor correlation (R2 = 0.344, p,0.05) was found between BrdU incorporation and the extent of local vasculariza- tion (Figure 4E) further supporting independence of tumor growth and vascularization. Independence of tumor progression with regard to blood vessel density does not appear to be a specific feature of U87 cells since it was also observed for the murine GL261 cell line grafted in C57Bl6 mice (Figure S1). September 2013 \| Volume 8 \| Issue 9 \| e72655 7 PLOS ONE \| www.plosone.org Dynamic Quantitative Imaging of Glioblastoma Cells Figure 4. Quantitative analyses and lack of correlation between blood vessel density and tumor cell density. Quantitative analysis of proliferation and local vessel density (A) Green (tumo and red channel (vessel) images of the same plane showing the whole tumor section and its surrounding healthy brain obtained on D7 (day D14 respectively (depth, 40 mm below the brain surface). Small clusters of tumor cells were detectable (thin arrow in green channel, D14). vascular landmarks (thick arrows in red channel) have been used to register the corresponding 3D image stacks. Initial tumor epicent systematically relocated to define 4 conserved circular regions of interests (ROI) (increment, 330 mm) whose mean fluorescence was monitore index of tumor cell density or blood supply. Evolutions of the cell density (green) and blood vessel density (red) in the 4 ROIs of two differen (dots versus triangles). Note that local cell densities and local blood vessel densities are uncorrelated. (B–C) Correlative analysis of the data co on mice (n = 7) over 4 sessions in 4 ROIs each time (B) Local blood vessel density is poorly correlated with local cell density (R2 = 0.0464). (C) Th changes in cell density ((Dfluo) over time (t2-t1 = Dt)) are also uncorrelated (R2,0) with the initial (t1) (black), resultant (t2) (grey) or subsequen vessel density (t3) (dashed line). (D) Immunostaining of vessels (anti-laminin, red) and BrdU positive cells (green) in a section of the whole White square boxes are enlarged on the right. (E) A poor correlation (R2,0.35) was found between vessel density and cell proliferation qua from thresholded images of immunostained brain slices (n = 4) taken at D9 from 2 mice. Scale bars, 500 mm. doi:10.1371/journal.pone.0072655.g004 Figure 4. Quantitative analyses and lack of correlation between blood vessel density and tumor cell density. (A) Green (tumor cells) and red channel (vessel) images of the same plane showing the whole tumor section and its surrounding healthy brain obtained on D7 (day 7) and D14 respectively (depth, 40 mm below the brain surface). Small clusters of tumor cells were detectable (thin arrow in green channel, D14). Stable vascular landmarks (thick arrows in red channel) have been used to register the corresponding 3D image stacks. Initial tumor epicenter was systematically relocated to define 4 conserved circular regions of interests (ROI) (increment, 330 mm) whose mean fluorescence was monitored as an index of tumor cell density or blood supply. Evolutions of the cell density (green) and blood vessel density (red) in the 4 ROIs of two different mice (dots versus triangles). Quantitative analysis of proliferation and local vessel density Note that local cell densities and local blood vessel densities are uncorrelated. (B–C) Correlative analysis of the data collected on mice (n = 7) over 4 sessions in 4 ROIs each time (B) Local blood vessel density is poorly correlated with local cell density (R2 = 0.0464). (C) The local changes in cell density ((Dfluo) over time (t2-t1 = Dt)) are also uncorrelated (R2,0) with the initial (t1) (black), resultant (t2) (grey) or subsequent blood vessel density (t3) (dashed line). (D) Immunostaining of vessels (anti-laminin, red) and BrdU positive cells (green) in a section of the whole tumor. White square boxes are enlarged on the right. (E) A poor correlation (R2,0.35) was found between vessel density and cell proliferation quantified from thresholded images of immunostained brain slices (n = 4) taken at D9 from 2 mice. Scale bars, 500 mm. doi:10.1371/journal.pone.0072655.g004 September 2013 \| Volume 8 \| Issue 9 \| e72655 PLOS ONE \| www.plosone.org 8 Dynamic Quantitative Imaging of Glioblastoma Cells Figure 5. Dynamics of Bev treatment effects on tumor development and blood vessel density. (A–B) Example of 3D projections (over 200 mm) of high-resolution images of the microvascularization observed at the margin (yellow line) between tumor and healthy brain in an untreated mouse (A) and a mouse treated with Bev for 1 week (B). Tumor (star) exhibits a high density of tortuous vessels in the untreated compared to less dense and straighter vessels in the Bev-treated mouse. (C–D) Evolutions of the mean blood vessel density (C) and the mean tumor cell density (D) in corresponding brain volumes of control (n = 7) and Bev-treated (n = 7) mice (see methods). Tumor induced changes in blood vessel density are sustainably blocked by Bev (C) and mean tumor cell density is significantly reduced at day 21 (D) (, p,0.05). (E) % reduction of blood vessel density relative to mean control values as a function of corresponding % reduction of tumor cell density. The absence of correlation (R2,0) indicates that effect of Bev on tumor development is not mediated by its effect on blood vessel density. (F) Evolution of the increase rate of tumor cell densities in control and in mice treated with Bev for 3 weeks. Increase rate is expressed as the average of the ratios of fluorescence intensities recorded at one week intervals in the same areas. Quantitative analysis of proliferation and local vessel density Increase rate is significantly reduced during the first 2 weeks but recovers the control increase rate on the third week despite a sustained blockade of vascular changes. (G–I) Immunostaining of vessels (anti-laminin, red) and BrdU positive cells (green) on D9 in untreated (G) or Bev-treated (H) tumors. Quantifications of BrdU(+) cells and blood vessels (I) in sections covering the whole tumor indicate that proliferation is not correlated with the amount of blood vessel density. Scale bars, 200 mm. Error bars represent SEM; (, p,0.05). doi:10.1371/journal.pone.0072655.g005 Figure 5. Dynamics of Bev treatment effects on tumor development and blood vessel density. (A–B) Example of 3D projections (over 200 mm) of high-resolution images of the microvascularization observed at the margin (yellow line) between tumor and healthy brain in an untreated mouse (A) and a mouse treated with Bev for 1 week (B). Tumor (star) exhibits a high density of tortuous vessels in the untreated compared to less dense and straighter vessels in the Bev-treated mouse. (C–D) Evolutions of the mean blood vessel density (C) and the mean tumor cell density (D) in corresponding brain volumes of control (n = 7) and Bev-treated (n = 7) mice (see methods). Tumor induced changes in blood vessel density are sustainably blocked by Bev (C) and mean tumor cell density is significantly reduced at day 21 (D) (, p,0.05). (E) % reduction of blood vessel density relative to mean control values as a function of corresponding % reduction of tumor cell density. The absence of correlation (R2,0) indicates that effect of Bev on tumor development is not mediated by its effect on blood vessel density. (F) Evolution of the increase rate of tumor cell densities in control and in mice treated with Bev for 3 weeks. Increase rate is expressed as the average of the ratios of fluorescence intensities recorded at one week intervals in the same areas. Increase rate is significantly reduced during the first 2 weeks but recovers the control increase rate on the third week despite a sustained blockade of vascular changes. (G–I) Immunostaining of vessels (anti-laminin, red) and BrdU positive cells (green) on D9 in untreated (G) or Bev-treated (H) tumors. Quantifications of BrdU(+) cells and blood vessels (I) in sections covering the whole tumor indicate that proliferation is not correlated with the amount of blood vessel density. Scale bars, 200 mm. Error bars represent SEM; (, p,0.05). Role of tumor micro-environment on GBM progression Role of tumor micro-environment on GBM progression The absence of correlation between cell densities and the levels of blood vessel density therefore suggests that the effect of Bev is either direct on tumor cells or indirect by inducing a non- permissive stromal environment. As VEGF is expressed both by brain cells [21] and by U87 cells [2,22], first we monitored in real time the effect of exogenous VEGF and Bev on cultured U87 cell densities (Figure S4A–B& S4D) In culture, Bev in fact increased rather than decreased cell density by up to 12% in the first 48 h and still 7% in the following days. We thus concluded that a direct effect on tumor cells could not account for the 25% reduction in tumor growth observed in vivo in the same time line (Figure S4B). Cell tracking moreover showed that neither Bev, nor VEGF significantly affected U87 velocities, or migration on a given substrate (Figure S4C). Efforts were made to improve the reproducibility of the imaging protocols and to take full advantage of quantitative fluorescence measurements. Whereas in most studies, tumor progression is estimated from the size of the long and short axis of the tumor [1,2,29], we have monitored the evolution of cell densities in ROIs that were conserved over the whole experiment. Besides providing an accurate evaluation of tumor progression, the methodology allowed the characterization of tumor heterogeneities as we systematically imaged a major part of the tumor volume. Whereas cells of the same passage were orthotopically implanted, we evidenced that the Bev tumor inhibitory effect was variable between animals and tumor areas, an effect unlikely due to heterogenous molecular profiles of the cells. Our U87 GBM model revealed several lines of evidence indicating that increases in tumor cell densities were in fact not fueled by blood vessels as initially thought [4,30]. First, in the absence of Bev treatment we found cases where tumor vascular- ization disappeared within a few days without affecting tumor cell densities. Second, although in some instances blood vessel density and tumor cell density showed some degree of interdependence as indicated by the low p value for Pearson correlation for two different GBM cell lines, we never observed a strong correlation between local cell densities and local blood vessel density as indicated by the weakness of their determination coefficients. Reduction in blood vessel density by Bev is without direct impact on tumor growth rate blood vessel density (Figures 7C & S5). Growth rate reduction was sustained and always 50% more pronounced than under Bev (Figure 7B & 7D). Unlike Bev, AMD3100 was equally efficient at the core and at the periphery of the tumor (Figure S3). For comparison with in vivo data we used linear regressions to extrapolate a possible cumulative effect of the drug during one week, but the modest direct effect observed in culture (Figure S4E) was unable to account for the 46% reduction observed in vivo after one week. This was in agreement with a major stromal action of AMD3100. To confirm the minor influence of blood vessel density on tumor progression in this model we tested whether angiogenesis could be blocked without modifying tumor growth rate. Mice (n = 13) were continuously treated with Bev for 3 weeks (10 mg/kg, i.v. every two days). One week treatment was sufficient to block vascular plasticity in tumor areas (362 vascular changes/mm3/24 h versus 71619 in control tumors) and to massively reduce tumoral microvessel densities (Figure 5A–B). As a result, the average blood vessel density in tumor areas remained similar to the one observed at D0 for the whole duration of the treatment (Figure 5C). Under high dose of Bev [2], average tumor cell density was significantly reduced after 3 weeks (.50%, Figure 5D). As for untreated tumors, quantitative and correlative analysis of images revealed a complete lack of correlation between local blood vessel density and local cell density as well as between intersession changes in tumor cell density and the corresponding changes in blood vessel density (R2 = 20.0267, p = 0.28) (Figure 5E). By the end of the second week moreover, tumor cells escaped Bev treatment and recovered control growth rates despite a sustained blockade of angiogenesis (Figure 5C–F). BrdU immunostaining confirmed that tumor cell proliferation was similar in control and Bev treated animals despite a significant effect of Bev on tumor vascular density (Figure 5G–I). Interestingly, as illustrated (Figure 6A–B) the Bev tumor inhibitory effect was variable between animals and within given tumor areas cell densities were not related to the observed local blood vessel densities (Figure 6C–D). We finally found no indication of hypoxic necrotic area under Bev treatment, as no fluorescence holes were observed in tumor cells patterns regardless of tumor sensitivity to Bev (Figure 6A–B). Discussion We have developed a dedicated orthotopic GBM model and an imaging methodology to study the progression of genetically labeled tumor cells with respect to the evolution of local micro- vascularization. The presented model was validated using xeno- grafts of U87, a widely used human GBM cell line and GL261, a murine GBM cell line. The approach can be extended to virtually any cell line derived from patient biopsies previously transfected to stably express a fluorescent reporter. The advantage of our model resides in the enforcement of 3D isotropic tumor growth orthotopically implanted in the superficial layers of the brain. Compared to previously described models in which the dura mater is removed [2], we here avoid unwanted deposit of sub- and supra-dural cells, whose proliferation is overwhelming in absence of 3D matricial constraints. As such, our model resembled patients’ GBM, for whom extradural growth is rare [26]. This also likely explains the slower progression we observed compared to previously published models using the same cell line [2]. Moreover, injecting cells as spheroids helped to confine the GBM tumor within a determined brain area, since micro-regional heterogeneities inside the brain likely influence proliferation and invasion patterns [27,28]. Quantitative analysis of proliferation and local vessel density doi:10.1371/journal.pone.0072655.g005 September 2013 \| Volume 8 \| Issue 9 \| e72655 PLOS ONE \| www.plosone.org 9 Dynamic Quantitative Imaging of Glioblastoma Cells Dynamic Quantitative Imaging of Glioblastoma Cells Dynamic Quantitative Imaging of Glioblastoma Cells Dynamic Quantitative Imaging of Glioblastoma Cells Reduction in blood vessel density by Bev is without direct impact on tumor growth rate September 2013 \| Volume 8 \| Issue 9 \| e72655 Role of tumor micro-environment on GBM progression Max intensity projection of the tumor cell densities (green outline) at D14 in a 30 mm section of the tumors presented in A and B, side by side with the corresponding projection images of the vascular network (red outline). Tumor margins are highlighted with dotted yellow lines. Highest tumor cell densities are preferentially located in the central area (between vertical lines in C) with no bias toward the most vascularized sub-regions 1 or 3 compared to 2; High tumor cell densities are found in less vascularized subregions of the tumor (simple arrow versus double arrows in D). Scale bars, 400 mm. doi 10 1371/journal pone 0072655 g006 Dynamic Quantitative Imaging of Glioblastoma Cells Figure 6. Heterogeneity of the Bev effect on tumor cell proliferation despite successfu Progression of the tumor under continuous Bev treatment. Mean projection images over a dept densities under Bev (10 mg/kg) treatment in 2 mice whose tumor burdens on D0 were similar (s shown). Treatment was initiated after the first visualization on D0. Tumor cells were more invasive are the zoom of the region of interest outlined with dashed line. After 3 weeks the tumor cell dens D) Independence of tumor progression from local blood vessel density. Max intensity projection of 30 mm section of the tumors presented in A and B, side by side with the corresponding project Tumor margins are highlighted with dotted yellow lines. Highest tumor cell densities are preferen lines in C) with no bias toward the most vascularized sub-regions 1 or 3 compared to 2; High t subregions of the tumor (simple arrow versus double arrows in D). Scale bars, 400 mm. doi:10.1371/journal.pone.0072655.g006 y Figure 6. Heterogeneity of the Bev effect on tumor cell proliferation despite successful blockade of blood vessel formation. (A–B) Progression of the tumor under continuous Bev treatment. Mean projection images over a depth of 200 mm showing the evolution of tumor cell densities under Bev (10 mg/kg) treatment in 2 mice whose tumor burdens on D0 were similar (same mean fluorescence intensities within 6%, not shown). Treatment was initiated after the first visualization on D0. Tumor cells were more invasive and spread in B compared to A (see insets). Insets are the zoom of the region of interest outlined with dashed line. After 3 weeks the tumor cell density was more than 2 times larger in B than in A. Role of tumor micro-environment on GBM progression Therefore an increase or reduction of blood vessel density cannot be predictive of a proportional change in cell density. Pharma- cologically targeting blood vessel density might thus be a sub- efficient strategy to fight tumor. Supporting this view, tumor inhibition under Bev was not correlated with the amplitude of blood vessel density reduction. The maximal effect on tumor cells Chemical signals from the environment play an important role in refractoriness or acquired resistance to anti-angiogenic therapy [23,24]. We investigated whether Bev-induced depletion of stromal VEGF affected tumor progression. When GFP-U87 spheroids were grafted in mice pretreated with high doses of Bev for one week prior to tumor implantation, tumor growth was inhibited by 58610% (n = 5 Bev, n = 8 Control) already at its earliest stage of development when angiogenesis is still low (Figure 7A). As VEGF producing cells often release Stromal Derived Factor 1 alpha that modulates microenvironment and acts on tumor growth [18,25] we used an antagonist of its CXCR4 receptor, AMD3100 (1.25 mg/kg, twice daily), as an alternative to Bev to reduce tumor growth. As observed with Bev, tumor sensitivity to AMD3100 was variable (Figure 8), but potent inhibition was observed on average (Figure 7B) without significant reduction in September 2013 \| Volume 8 \| Issue 9 \| e72655 September 2013 \| Volume 8 \| Issue 9 \| e72655 PLOS ONE \| www.plosone.org 10 Dynamic Quantitative Imaging of Glioblastoma Cells Figure 6. Heterogeneity of the Bev effect on tumor cell proliferation despite successful blockade of blood vessel formation. (A–B) Progression of the tumor under continuous Bev treatment. Mean projection images over a depth of 200 mm showing the evolution of tumor cell densities under Bev (10 mg/kg) treatment in 2 mice whose tumor burdens on D0 were similar (same mean fluorescence intensities within 6%, not shown). Treatment was initiated after the first visualization on D0. Tumor cells were more invasive and spread in B compared to A (see insets). Insets are the zoom of the region of interest outlined with dashed line. After 3 weeks the tumor cell density was more than 2 times larger in B than in A. (C– D) Independence of tumor progression from local blood vessel density. September 2013 \| Volume 8 \| Issue 9 \| e72655 Dynamic Quantitative Imaging of Glioblastoma Cells volume was observed on D14 until it reached 58% on D28 similar to what observed upon continuous Bev treatment. (B–C) Tumor development and blood vessel density under AMD3100 treatment to prevent signaling by Stromal Derived Factor 1 alpha. Evolutions of mean tumor cell density (B) and mean blood vessel density (C) in control (n = 7) or AMD3100-treated (n = 6) mice (, p,0.05). The average evolution under Bev is reported in gray for comparison. Blood vessel density was not significantly affected by AMD3100 treatment and remained significantly larger than the one observed under Bev on D21. (D) Evolution of the average tumor increase rate in control conditions and under AMD3100 treatment. Ratios between fluorescence intensities recorded in the same areas at one week interval have been averaged and evolution of this average was followed over the 3 weeks of treatment. Bev data are reported in gray for comparison. Reduction of tumor size is almost twice larger under AMD3100 than under Bev. doi:10.1371/journal.pone.0072655.g007 Figure 7. Contribution of the micro-environment on tumor development. (A) Evolution of the tumor volume in mice pretreated with i.v. injections every 2 days of PBS (n = 8) or Bev (n = 5) for 1 week before tumor implantation. Tumor volume was normalized on the first day of visualization (day 0) and interpolated linearly between imaging sessions to take into account day of visualization mismatches between the two groups of mice. Interpolated data are indicated with squares linked together with dashed lines. A significant reduction of tumor growth on blood vessel density. This finding might be explained by the fact that GBM tumor cells grow inside the already densely vascularized healthy brain (Video S1) where 20% of the most oxygenated blood of the body is drained and distributed in real time according to the needs. Moreover, tumor cells have been shown to depend more on nutrients supply than on oxygen for their energy production [33]. GBM growth would therefore not require increased access to oxygen, but access to nutrients whose supply is already optimally organized in the brain to support energy expensive neuronal activity. A dense network of astrocytes [34] and a rich cerebrospinal fluid [35,36] can provide nutrients in addition to blood. Angiogenesis independent tumor growth has already been reported [37,38] and in that case average blood supply inside the tumor was even lower than in healthy brain. Dynamic Quantitative Imaging of Glioblastoma Cells Altogether our results obtained on a U87 tumor model support the challenging view that GBM growth is not directly related to blood vessel density. Yet the reason for such tumoral vascular remodeling remains to be elucidated. A possibility would be that tumor angiogenesis as well as tumor growth are promoted by inflammation [6,39,40,41]. Inflammatory cells indeed shape the tumor micro-environment [42,43] eventually promoting tumor growth [24,40,44,45]. Our methodology should serve future studies to test whether the reported immuno-modulatory effect of anti-VEGF therapies [24,40,46,47] or anti-SDF1 alpha therapies [45,48] could account for the anti-angiogenic and anti- tumoral effects reported here. Role of tumor micro-environment on GBM progression (C– D) Independence of tumor progression from local blood vessel density. Max intensity projection of the tumor cell densities (green outline) at D14 in a 30 mm section of the tumors presented in A and B, side by side with the corresponding projection images of the vascular network (red outline). Tumor margins are highlighted with dotted yellow lines. Highest tumor cell densities are preferentially located in the central area (between vertical lines in C) with no bias toward the most vascularized sub-regions 1 or 3 compared to 2; High tumor cell densities are found in less vascularized subregions of the tumor (simple arrow versus double arrows in D). Scale bars, 400 mm. doi:10.1371/journal.pone.0072655.g006 tumor cannot be detected due limitations of the imaging field of view; additional analysis based on tissue sectioning and vimentin immunochemical tumor cell detection should further document this issue. Finally under AMD3100, massive angiogenesis was maintained despite a strong reduction of tumor cell densities. was observed in the first week of treatment at a time when angiogenesis was weak in the control group. Progression rate then recovered to control values within two weeks despite the sustained blockade of angiogenesis. This observation dismissed the hypoth- esis that tumor escape might result from activation of VEGF independent angiogenic pathways [9,17,31]. Although anti-angio- genic treatments are often reported to trigger tumor cell invasion [32], we did not find any evidence for increased invasiveness in our U87 model. However, cells that would have migrated far from the Our observations indicated that angiogenesis was not required to improve tumor metabolism in our experiments. No specific difference between central, intermediary or peripheral regions of the tumor was observed regarding the dependence of tumor September 2013 \| Volume 8 \| Issue 9 \| e72655 September 2013 \| Volume 8 \| Issue 9 \| e72655 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 11 Supporting Information Treatment was initiated a weeks, the tumor cell density was more than 2.5 times larger in B than in A. (C–D) Max intensity projection o at D14 in a 30 mm section of the tumors presented in A, side by side with the corresponding projection ima Tumor margins are highlighted with yellow dotted lines. (C) Note the presence of tumor blood vessels com completely devoid of blood vessels shown in Figure 6C. (D) Dense tumor vascularization is even more o vascular densities (double arrows) are not correlated with the highest tumor densities (single arrow). Scale doi:10.1371/journal.pone.0072655.g008 Dynamic Qu Dynamic Quantitative Imaging of Glioblastoma Cells Figure 8. Sensitivity to AMD3100 is heterogeneous and independent of blood vessel density. (A–B) Mean projection images over a depth of 200 mm showing the evolution of tumor cell densities under AMD3100 (1.25 mg/kg twice daily) treatment in two mice whose tumor burden on D0 were similar (same mean fluorescence intensities within 16%, not shown). Treatment was initiated after the first visualization on D0. After 3 weeks, the tumor cell density was more than 2.5 times larger in B than in A. (C–D) Max intensity projection of the tumor cell densities (green outline) at D14 in a 30 mm section of the tumors presented in A, side by side with the corresponding projection images of the vascular network (red outline). Tumor margins are highlighted with yellow dotted lines. (C) Note the presence of tumor blood vessels compared to a similar size Bev treated tumor completely devoid of blood vessels shown in Figure 6C. (D) Dense tumor vascularization is even more obvious in the larger tumor. The highest vascular densities (double arrows) are not correlated with the highest tumor densities (single arrow). Scale bars, 400 mm. doi:10.1371/journal.pone.0072655.g008 Figure 8. Sensitivity to AMD3100 is heterogeneous and independent of blood vessel density. (A–B) Mean projection images over a depth of 200 mm showing the evolution of tumor cell densities under AMD3100 (1.25 mg/kg twice daily) treatment in two mice whose tumor burden on D0 were similar (same mean fluorescence intensities within 16%, not shown). Treatment was initiated after the first visualization on D0. After 3 weeks, the tumor cell density was more than 2.5 times larger in B than in A. Supporting Information Figure S1 Lack of correlation between blood vessel density and tumor cell density in a syngenic orthotopic GBM model. (A) Grafted GL261 DsRed tumor (red) visualized 16 days post-implantation. The vasculature is enlightened by the intravenous injection of Cascade-blue dextran 70 kDa (blue). Side images present a zoomed out version of each channel. (B) Absence of correlation between local tumor cell density and local intra- tumoral vascular density (R2,0. 1, n = 6 mice). Figure S2 Weak correlation between tumor growth and blood vessel density in U87 GBM. (A) Correlative analysis of the cell density and blood vessel density collected on a weekly basis for 3 weeks in all the different ROIs for each individual mouse (n = 7) according to the protocol described in Figure 3. Correlation is predominantly poor or inexistent (R2,0.3, 5 out 7 mice) which explained the average R2 = 0.046 observed in Figure 4B. (B) Correlation is poor irrespective of the range of tumor cell densities considered. Data presented in Figure 4B were binned using cell density ([0,1] dark gray; [1,2] light gray; [2,5] black); linear regression was determined in each bin. (C) Figure 7. Contribution of the micro-environment on tumor development. (A) Evolution of the tumor volume in mice pretreated with i.v. injections every 2 days of PBS (n = 8) or Bev (n = 5) for 1 week before tumor implantation. Tumor volume was normalized on the first day of visualization (day 0) and interpolated linearly between imaging sessions to take into account day of visualization mismatches between the two groups of mice. Interpolated data are indicated with squares linked together with dashed lines. A significant reduction of tumor September 2013 \| Volume 8 \| Issue 9 \| e72655 PLOS ONE \| www.plosone.org 12 Correlation is poor at every time point of tumor development. For each week, the dataset consisted of the measurements obtained on two consecutive sessions for all ROIs and all mice. (D) Correlation between cell density and blood vessel density as a function of most central area of the tu periphery (ROI4, white). Th from center to periphery. (E cell density and the resulta Figure 8. Sensitivity to AMD3100 is heterogeneous and independent of blood vessel density depth of 200 mm showing the evolution of tumor cell densities under AMD3100 (1.25 mg/kg twice daily) tre on D0 were similar (same mean fluorescence intensities within 16%, not shown). References 1. Winkler F, Kienast Y, Fuhrmann M, Von Baumgarten L, Burgold S, et al. (2009) Imaging glioma cell invasion in vivo reveals mechanisms of dissemination and peritumoral angiogenesis. Glia 57: 1306–1315. 12. Fink J, Born D, Chamberlain MC (2011) Pseudoprogression: relevance with respect to treatment of high-grade gliomas. Curr Treat Options Oncol 12: 240– 252. p g g 2. von Baumgarten L, Brucker D, Tirniceru A, Kienast Y, Grau S, et al. (2011) Bevacizumab has differential and dose-dependent effects on glioma blood vessels and tumor cells. Clin Cancer Res 17: 6192–6205. 13. Gerstner ER, Chen PJ, Wen PY, Jain RK, Batchelor TT, et al. (2010) Infiltrative patterns of glioblastoma spread detected via diffusion MRI after treatment with cediranib. Neuro Oncol 12: 466–472. 3. Folkman J (2002) Role of angiogenesis in tumor growth and metastasis. Semin Oncol 29: 15–18. 14. Verhoeff JJ, van Tellingen O, Claes A, Stalpers LJ, van Linde ME, et al. (2009) Concerns about anti-angiogenic treatment in patients with glioblastoma multiforme. BMC Cancer 9: 444. 4. Jain RK, di Tomaso E, Duda DG, Loeffler JS, Sorensen AG, et al. (2007) Angiogenesis in brain tumours. Nat Rev Neurosci 8: 610–622. 15. Zhang L, Lapierre A, Roy B, Lim M, Zhu J, et al. (2012) Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window. J Vis Exp: 4201–4205. 5. Chamberlain MC (2011) Bevacizumab for the treatment of recurrent glioblastoma. Clin Med Insights Oncol 5: 117–129. 6. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144: 646–674. 16. Zhang L, Lapierre A, Roy B, Lim M, Zhu J, et al. (2012) Imaging glioma initiation in vivo through a polished and reinforced thin-skull cranial window. J Vis Exp. 7. Plate KH, Breier G, Weich HA, Risau W (1992) Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 359: 845–848. 17. di Tomaso E, Snuderl M, Kamoun WS, Duda DG, Auluck PK, et al. (2011) Glioblastoma recurrence after cediranib therapy in patients: lack of ‘‘rebound’’ revascularization as mode of escape. Cancer Res 71: 19–28. 8. Bao S, Wu Q, Sathornsumetee S, Hao Y, Li Z, et al. (2006) Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor. Cancer Res 66: 7843–7848. 18. Rubin JB, Kung AL, Klein RS, Chan JA, Sun Y, et al. Acknowledgments We thank S. Bensemmane, J. Bonnardel, M. Hocine, P. Weber, and the staff of the animal and PicSIL imaging facilities of the IBDML for skillfull technical assistance; Pr D. Figarella-Branger and Dr K.K. Fenrich for helpful critical feedback on the study and manuscript. We thank S. Bensemmane, J. Bonnardel, M. Hocine, P. Weber, and the staff of the animal and PicSIL imaging facilities of the IBDML for skillfull technical assistance; Pr D. Figarella-Branger and Dr K.K. Fenrich for helpful critical feedback on the study and manuscript. Supporting Information The modest increase in density observed in vitro cannot account for the 25% decrease in cell density observed in vivo after 1 week Bev treatment (gray arrow). (C) Instantaneous velocity (mm/min) of U87 cells measured every 30 min for 59 h under various conditions (control, VEGF or Bev). Note the absence of effect on cell migratory behavior (D) Average ratio of cell densities observed under treatment versus control conditions calculated over a 4.5 day experiment. Treatments were VEGF, Bev, a combination of Bev and VEGF (: P,0,05; Figure S4 U87 cells behavior under VEGF, Bev and AMD3100 treatments. (A) Comparative evolution of tumor cell densities in control and Bev (250 ng/ml) treated cultures (n = 2 independent experiments, 120 wells) (B) Ratios of mean cell densities in Bev versus control conditions (presented in A) are plotted over time. Green line represents the linear extrapolation of cell densities ratio until day 7. The modest increase in density observed in vitro cannot account for the 25% decrease in cell density observed in vivo after 1 week Bev treatment (gray arrow). (C) Instantaneous velocity (mm/min) of U87 cells measured every 30 min for 59 h under various conditions (control, VEGF or Bev). Note the absence of effect on cell migratory behavior (D) Average ratio of cell densities observed under treatment versus control conditions calculated over a 4.5 day experiment. Treatments were VEGF, Bev, a combination of Bev and VEGF (*: P,0,05; Author Contributions Conceived and designed the experiments: FD GR FS. Performed the experiments: FD CR FS. Analyzed the data: TR CR FD MCA. Contributed reagents/materials/analysis tools: FD FS TR. Wrote the paper: FD GR MCA. Supporting Information (C–D) Max intensity projection of the tumor cell densities (green outline) at D14 in a 30 mm section of the tumors presented in A, side by side with the corresponding projection images of the vascular network (red outline). Tumor margins are highlighted with yellow dotted lines. (C) Note the presence of tumor blood vessels compared to a similar size Bev treated tumor completely devoid of blood vessels shown in Figure 6C. (D) Dense tumor vascularization is even more obvious in the larger tumor. The highest vascular densities (double arrows) are not correlated with the highest tumor densities (single arrow). Scale bars, 400 mm. doi:10.1371/journal.pone.0072655.g008 Correlation is poor at every time point of tumor development. For each week, the dataset consisted of the measurements obtained on two consecutive sessions for all ROIs and all mice. (D) Correlation between cell density and blood vessel density as a function of distance to the center of the tumor. Correlation is not better in the most central area of the tumor (ROI1, black) compared to its periphery (ROI4, white). The gray value of data points is lighter from center to periphery. (E) Poor correlation between the local cell density and the resultant local blood vessel density (gray), between the local cell density and the local blood vessel density September 2013 \| Volume 8 \| Issue 9 \| e72655 PLOS ONE \| www.plosone.org 13 Dynamic Quantitative Imaging of Glioblastoma Cells observed in the preceding session (black) as well as between the local cell density and the local blood vessel density observed in the subsequent session (white). Although weak, the correlation is better with the preceding local blood vessel density than with the resultant blood vessel density; it is worse with the subsequent than with the resultant blood vessel density. This suggests that blood vessel density does not increase to sustain the metabolic needs of tumor cells but that highly supplied areas trigger densification of tumor cells. (F) The amplitude of local changes of cell density measured between two consecutive time points are always positive and uncorrelated with the previous blood vessel density, uncorrelated with the resultant blood vessel density and uncorre- lated with the subsequent blood vessel density. (G) Data presented in Figure 4B were not better fitted by other simple mathematical functions. (TIFF) one-tailed Student t-test). Supporting Information (E) Evolution of the ratio between tumor cell densities observed in AMD3100 (10 mg/ml) treated cultures relative to control cultures. Linear extrapolation (green line) of the AMD3100 induced reduction in cell density indicate that the direct effect of AMD3100 on tumor cells (,20%) cannot account for the 46% reduction of tumor cell density observed in vivo. (TIFF) Figure S5 Effect of AMD3100 and Bev on healthy and tumor blood vessel densities. (A,C,E) Two weeks continuous treatment with either drug does not affect blood vessel density in healthy brain regions (AMD3100, A; Control = CTR, C; BEV, E). Max intensity projections of typical 30 mm thick sections of brain, before (D0) and 14 days (D14) after continuous treatment. (B, D, F) Similar projections as in (A, C, E) for tumor areas imaged 14 days after starting treatments. Tumor vascularization is strongly inhibited by Bev (F) and only weakly affected by AMD3100 (B) when compared to control tumor (D). Scale bar, 200 mm. (TIFF) Figure S3 Regional efficacy of AMD3100 and Bev treatments on tumor cell density and tumor blood vessel density. Data presented in Fig. 5 & 7 for the whole tumor are here presented for every individual ROI from the central (ROI1) to the peripheral (ROI4) relative to the tumor epicenter. The largest differences between the two treatments on cell densities are observed in the most central ROIs whereas only Bev affects blood vessel density. Error bars represent S.E.M. and stars indicate significant differences relative to control values (p,0.05). (TIFF) Video S1 3D volume rendering of a U87-GBM tumor. Tumor cells are highlighted by GFP (green) and blood vessels by i.v. injection of Rhodamine Dextran 70 kD (red). Scale bar 400 mm. Video S2 Perivascular infiltration of U87-GBM cells. Z- stack of horizontal images at 5 mm interval over 100 mm showing perivascular infiltration of the brain at the periphery of the primary tumor. Scale bar 100 mm. (AVI) Figure S4 U87 cells behavior under VEGF, Bev and AMD3100 treatments. (A) Comparative evolution of tumor cell densities in control and Bev (250 ng/ml) treated cultures (n = 2 independent experiments, 120 wells) (B) Ratios of mean cell densities in Bev versus control conditions (presented in A) are plotted over time. Green line represents the linear extrapolation of cell densities ratio until day 7. Dynamic Quantitative Imaging of Glioblastoma Cells 20. 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https://openalex.org/W3005044948	https://journal2.unusa.ac.id/index.php/JHS/article/download/1288/1005	Indonesian	null	Akses media dan perilaku seksual pranikah beresiko infeksi menular seksual (IMS)pada siswa SMA di Kabupaten Malang	Jurnal Ilmiah Kesehatan	2,020	cc-by-sa	3,846	Rifzul Maulina Politeknik Kesehatan RS dr Soepraoen Kesdam V/Brawijaya, rifzulmaulina3@gmail.com Rifzul Maulina Politeknik Kesehatan RS dr Soepraoen Kesdam V/Brawijaya, rifzulmaulina3@gmail.co Zainal Alim Politeknik Kesehatan RS dr Soepraoen Kesdam V/Brawijaya, zainaliem@gmail.com Zainal Alim Politeknik Kesehatan RS dr Soepraoen Kesdam V/Brawijaya, zainaliem@gmail.com Zainal Alim Politeknik Kesehatan RS dr Soepraoen Kesdam V/Brawijaya, zainaliem@gmail.com Abstrak Usia remaja adalah masa dimana terdapat perubahan yang signifikan secara pemikiran, psikologis dan bentuk tubuh.Perubahan yang signifikan ini menyebabkan usia remaja memiliki sifat yang sama yaitu memiliki jiwa ingin tahu yang besar, menyenangi tantangan serta petualangan dan mengarah berani memikul dampak atas perlakuannya tanpa didahului oleh pemikiran yang dewasa. Tujuan penelitian ini adalah untuk mengetahui faktor-faktor lingkungan apa saja yang berhubungan dengan perilaku seksual pranikah beresiko IMS. Penelitian ini merupakan penelitian penjelasan (explanatory research) dengan desain penelitian yang digunakan adalah cross sectional. Sampel dipilih secara random sampling, yaitu siswa sekolah menengah yang meliputi 2 Sekolah Menengah Atas di Kabupaten Malang yang dekat dengan eks- lokalisasi sebanyak 318 responden. Variabel penelitian yang diteliti adalah karakteristik responden (umur dan jenis kelamin), sikap orang tua, sikap teman sebaya dan akses media. Instrumen yang digunakan adalah kuesione yang telah teruji realibilitas dan validitas. Analisa data yang digunakan yaitu analisa univariat, bivariat (chi-square), dan multivariat (regresi logistik) Hasil Penelitian menunjukkan variabel yang berhubungan dengan perilaku seksual pranikah remaja berisiko IMS pada responden laki-laki adalah umur (p=0,047), sikap orang tua (p=0,014) dan akses media (p=0,03). Variabel yang paling berpengaruh terhadap perilaku seksual beresiko IMS pada responden laki-laki adalah akses media dengan OR 2,378 yang artinya responden laki-laki dengan tingkat akses media tinggi memiliki peluang 2,3 kali untuk melakukan perilaku seksual beresiko IMS. Bagi orang tua hendaknya selalu memberikan bimbingan yang positif terhadap anak-anaknya agar berperilaku sesuai dengan agamanya. Kata Kunci: akses media, perilaku seksual pranikah Submit : 22 November 2019, Accept : 8 January 2020, DOI https://doi.org/10.33086/jhs.v13i01.1288 Abstract Adolescence is a period where there are significant changes in thought, psychology and body shape. This significant change causes adolescence to have the same nature of having a big soul of curiosity, likes challenges and adventures and leads to dare to bear the impact of its treatment without precedence by mature thinking. The purpose of this study are what environmental factors that correlated premarital sexual behavior at risk for STIs. This research is an explanatory research with a cross sectional study design. The sample was chosen by random sampling, namely high school students which included 2 high schools in Malang Regency close to ex-localization of 318 respondents. Determination of 2 high school research sites due to a small sample. The research variables studied were the characteristics of respondents (age and sex), attitudes of parents, attitudes of peers and media access. The instrument used was a questionnaire. Analysis of the data used is univariate, bivariate (chi square), and multivariate (logistic regression) analyzesThe results showed variables related to premarital sexual behavior of adolescents at risk for STIs in male respondents were age (p = 0.047), parental attitudes (p = 0.014) and media access (p = 0.03). The variable that most influences sexual behavior at risk for STIs in male respondents is media access with OR 2,378, which means male respondents with high media access Submit : 22 November 2019, Accept : 8 January 2020, DOI https://doi.org/10.33086/jhs.v13i01.1288 40 Jurnal Ilmiah Kesehatan (Journal of Health Sciences), Vol. 13, No. 1, Februari 2020, Hal. 39-48 40 levels have 2.3 times the opportunity to engage in sexual behavior at risk of STIs. Whereas in female respondents there are no related variables. Suggestions for schools to provide intensive information to students about reproductive health in order to improve teenage understanding. Keywords: media access, premarital sexual be Keywords: media access, premarital sexual be yang biasanya orang dewasa lakukan hal ini didorong oleh keinginan remaja untuk merasakan kegiatan seksual. Minimya pemberian informasi ini membuat remaja mencari tahu dan mencoba sendiri (Tukan, 2010). PENDAHULUAN Usia remaja adalah masa dimana terdapat perubahan yang signifikan secara pemikiran, psikologis dan bentuk tubuh. Hal ini menyebabkan usia remaja memiliki sifat yang sama yaitu memiliki jiwa ingin tahu yang besar, menyenangi tantangan serta petualangan dan mengarah berani memikul dampak atas perlakuannya tanpa didahului oleh pemikiran yang dewasa. Perubahan ini juga diikuti dengan terdapatnya sarana yang ada disekitar remaja untuk memenuhi kebutuhan dan perubahn sifat tersebut. Kondisi seperti ini sering kali menyebabkan pertentangan batin pada diri remaja. Jika keputusan yang dipilih dalam menghadapi pertentangan tidak benar, mereka akan terjerumus ke dalam perilaku beresiko, masalah kesehatan psikosoial dan fisik seumur hidupnya (Ahmadi, 2009). Penyampaian informasi masalah seksual merupakan hal yang sangat penting dikarenakan remaja berada dalam potensi seksual yang aktif, disebabkan berhubungan dengan rangsangan seksual yang disebabkan oleh perubahan hormon endokrin serta remaja sering tidak mempunyai informasi yang cukup mengenai aktivitas seksual mereka sendiri (Tukan, 2010). Hal ini akan membuat berbahaya untuk perkembangan jiwa remaja jika remaja tidak mempunyai informasi serta pengetahuan yang benar. Berdasarkan realita yang ada menunjukkan jika hampir sebagian besar remaja sangat tidak memahami dampak negatif dari perilaku seksual yang remaja lakukan, berdasarkan usia remaja dikategorikan tidak matang apabila melakukan hubungan seksual terlebih lagi jika remaja harus menanggung dampak negatif dari hubungan seksual yang dilakukan remaja (Sarwono, 2014). Sebagian besar remaja mempunyai rasa ingin tahu yang besar dan tinggi (high curiousity). Karena disebabkan memiliki rasa ingin tahu yang besar dan tinggi, remaja condong ingin mencoba dan menjelajahi sesuatu hal yang belum pernah dilakukan atau dirasakan. Remaja pada umumnya ingin mencoba melakukan apa Nomor p-ISSN: 1978-6743, Nomor e-ISSN: 2477-3948 Rifzul Maulina, Zainal Alim Akses Media dan Perilaku Seksual Pranikah Beresiko Infeksi Menular Seksual (IMS) Pada Siswa SMA di Kabupaten Malang Rifzul Maulina, Zainal Alim Akses Media dan Perilaku Seksual Pranikah Beresiko Infeksi Menular Seksual (IMS) Pada Siswa SMA di Kabupaten Malang 41 Berdasarkan data salah satu Tim Survei dari Sebaya dan FK Unair pada tahun 2005 di kota Surabaya dari 126 responden yang berusia 19-23 tahun mendapat hasil bahwa 13,5% responden mengaku pernah melakukan hubungan seks pranikah. Saat tidak ada pasangan untuk melakukan hubungan seks, beberapa di antaranya melakukan dengan PSK. Survei menyebutkan bahwa 45,7% responden yang ditemui dilokalisasi mengaku pertama kali ketika berusia 16-20 tahun (Asfriyati, 2010). diasumsikan ada tiga faktor yang mempengaruhi adanya kekhawatiran terjadinya risiko seksual pada remaja. HASIL PENELITIAN pertimbangan informasi tersebut benar atau tidak. (Suryoputro, 2006). Tabel 1. Distribusi Frekuensi Perilaku Seksual berdasarkan Umur PENDAHULUAN Pertama, suatu kecenderungan remaja mengalami kematangan seksual lebih awal karena pergaulan sosial yang sangat permisif dan usia pernikahan semakin tertunda karena melanjutkan pendidikan. Selanjutnya yang kedua sebagian besar remaja tidak mengetahui cara mencari informasi yang tepat baik di rumah ataupun di sekolah tentang kesehatan reproduksi. Sangat terbatasnya kemungkinan untuk peluang diskusi tentang kesehatan reproduksi, dan bahkan sebagian besar guru dan orang tua menganggap hal tabu untuk membicarakan tentang seksualitas. Ketiga, perkembangan teknologi informasi dan komunikasi serta arus deras globalisasi mempunyai dampak positif dan negatif. Hasil penelitian yang dilakukan di 4 kota besar yaitu yaitu Jakarta, Bandung, Surabaya dan Medan pada bulan September 2004 yang dilakukan oleh Synovate Research tentang perilaku seksual remaja menunjukkan bahwa 65% remaja mendapatkan informasi tentang seks dari teman, 35% dari film porno dan hanya 5% dari orang tua. Bahkan 44% pernah mempunyai pengalaman seks usia 16-18 tahun. Efek sampingdari perilaku seks bebas, setiap tahun diperkirakan ada 2,3 juta kasus aborsi, yang 20% diantaranya dilakukan remaja, berdasarkan penelitian Perkumpulan Keluarga Berencana Indonesia (PKBI), dan ada 73% yang melakukan aborsi tidak aman. (Kemenkes, 2013). Dampak negatif dari perkembangan teknologi informasi dan komunikasi diantaranya timbulnya budaya permisif (kebebasan tanpa batas) mengakses media yang bersifat eksploitasi seksual ataupun penyajian kekerasan. Juga berubahnya culture ignore (budaya acuh) sebagai contoh remaja seharusnya mendapatkan informasi yang tepat karena kesibukan orang tua maka upaya pendampingan kurang diperhatikan sehingga informasi yang diperoleh diterima begitu saja tanpa Beberapa penelitian yang dilakukan mengenai resiko perilaku seksual remaja Nomor p-ISSN: 1978-6743, Nomor e-ISSN: 2477-3948 42 Jurnal Ilmiah Kesehatan (Journal of Health Sciences), Vol. 13, No. 1, Februari 2020, Hal. 39-48 42 HASIL PENELITIAN Tabel 1. Distribusi Frekuensi Perilaku Seksual berdasarkan Umur Umur Perilaku Seksual Laki-laki Perempuan Beresiko Tidak Beresiko Bersiko Tidak Beresiko f % f % f % f % Remaja Awal 27 41,5 38 58,5 21 22,1 74 77,9 Remaja Akhir 20 24,7 61 75,3 13 16,9 64 83,1 Jumlah 47 32,2 99 67,8 34 19,8 138 80,2 p=0,047 Ho ditolak p= 0,508 Ho diterima METODE Penelitian ini merupakan penelitian penjelasan (explanatory research) dengan desain penelitian yang digunakan adalah cross sectional, penelitian ini hanya dilakukan pada saat bersamaan saja dan satu kali saja. Data dikumpulkan melalui metode kuantitatif. Populasi dalam penelitian ini adalah siswa SMA di Kabupaten Malang sejumlah 1.828 siswa. Sampel dipilih secara simple random sampling dengan membuat gulungan kertas dan memberi label X sebanyak 318 responden. Variabel bebas dalam penelitian ini adalah Faktor lingkungan meliputi sikap teman sebaya, sikap orang tua, dan akses media sedangkan Variabel terikat dalam penelitian ini adalah perilaku seksual pranikah beresiko IMS. Berdasarkan tabel 1 menunjukkan bahwa responden laki-laki dengan umur remaja awal (<17 tahun) memiliki perilaku seksual pranikah beresiko (41,5%) lebih besar jika dibandingkan dengan remaja akhir (≥17 tahun) (24,7%). Hasil uji chi- square menunjukkan bahwa ada hubungan yang signifikan antara umur dengan perilaku seks pranikah beresiko IMS pada responden laki-laki. Demikian juga pada responden perempuan dengan umur remaja awal (<17 tahun) memiliki perilaku seksual pranikah beresiko (22,1%) lebih besar jika dibandingkan dengan remaja akhir (≥17 tahun) (16,9%). Instrumen dalam penelitian ini yang digunakan adalah kuesioner yang berisikan pertanyaan tertutup dan sudah dilakukan uji validitas dan reliabilitas. Hasil penelitian dianalisis secara univariat, bivariat, dan multivariat. Analisis bivariat menggunakan chi-square dan analisis multivariat menggunakan regresi logistik untuk mengetahui faktor yang paling berpengaruh terhadap perilaku seksual pranikah beresiko IMS. Tabel 2. Distribusi Frekuensi Perilaku Seksual berdasarkan Jenis Kelamin Tabel 2. Distribusi Frekuensi Perilaku Seksual berdasarkan Jenis Kelamin Jenis Kelamin Perilaku Seksual Total Beresiko Tidak Beresiko f % f % f % Laki-laki 47 32,2 99 67,8 146 100 Perempuan 43 25 129 75 172 100 Uji chi suare p= 0,196 Ho diterima Berdasarkan tabel 2 menunjukkan bahwa laki-laki berperilaku seksual Nomor p-ISSN: 1978-6743, Nomor e-ISSN: 2477-3948 Rifzul Maulina, Zainal Alim Akses Media dan Perilaku Seksual Pranikah Beresiko Infeksi Menular Seksual (IMS) Pada Siswa SMA di Kabupaten Malang Rifzul Maulina, Zainal Alim Akses Media dan Perilaku Seksual Pranikah Beresiko Infeksi Menular Seksual (IMS) Pada Siswa SMA di Kabupaten Malang Rifzul Maulina, Zainal Alim 43 43 Rifzul Maulina, Zainal Alim Akses Media dan Perilaku Seksual Pranikah Beresiko Infeksi Menular Seksual (IMS) Pada Siswa SMA di Kabupaten Malang pranikah beresiko terhadap IMS sebesar 32,2%, sedangkan pada responden perempuan berperilaku seksual pranikah beresiko terhadap IMS sebesar 25%. METODE Hasil uji chi-square menunjukkan bahwa nilai p value > 0,05 jadi Ho di terima, sehingga dapat disimpulkan bahwa tidak ada hubungan antara jenis kelamin dengan perilaku seksual pranikah beresiko terhadap IMS pada remaja SMA di Kabupaten Malang. laki terdapat hubungan antara sikap orang tua dengan perilaku seks pranikah beresiko IMS, sedangkan pada responden perempuan tidak ada hubungan antara sikap orang tua dengan perilaku seks pranikah beresiko IMS. Tabel 4. Distribusi Frekuensi Perilaku Seksual berdasarkan Sikap Teman Sebaya Sikap Teman Sebaya Perilaku Seksual Laki-laki Perempuan Beresiko Tidak Beresiko Bersiko Tidak Beresiko f % f % f % F % Tidak Permisif 29 40,3 43 59,7 22 20,8 84 79,2 Permisif 18 24,3 56 75,7 12 18,2 54 81,8 Jumlah 47 32,2 99 67,8 34 19,8 138 80,2 p=0,059 Ho diterima p= 0,830 Ho diterima Tabel 4. Distribusi Frekuensi Perilaku Seksual berdasarkan Sikap Teman Sebaya Sikap Teman Sebaya Perilaku Seksual Laki-laki Perempuan Beresiko Tidak Beresiko Bersiko Tidak Beresiko f % f % f % F % Tidak Permisif 29 40,3 43 59,7 22 20,8 84 79,2 Permisif 18 24,3 56 75,7 12 18,2 54 81,8 Jumlah 47 32,2 99 67,8 34 19,8 138 80,2 p=0,059 Ho diterima p= 0,830 Ho diterima Faktor Lingkungan meliputi sikap orang tua, sikap teman sebaya dan akses media. Tabel 4 menunjukkan bahwa responden laki-laki (40,3%) dan responden perempuan (20,8%) memiliki teman sebaya yang sikapnya tidak permisif. Mereka yang memiliki teman sebaya dengan sikap tidak permisif memiliki proporsi melakukan perilaku seksual beresiko IMS. Hasil uji chi-square menunjukkan bahwa pada responden laki-laki maupun perempuan tidak terdapat hubungan yang signifikan antara sikap teman sebaya dengan perilaku seks pranikah beresiko IMS Tabel 3. Distribusi Frekuensi Perilaku Seksual berdasarkan Sikap Orang Tua Sikap Orang tua Perilaku Seksual Laki-laki Perempuan Beresiko Tidak Beresiko Bersiko Tidak Beresiko f % f % f % f % Tidak Permisif 30 42,9 40 57,1 13 16 68 84 Permisif 17 22,4 59 77,6 21 23,1 70 76,9 Jumlah 47 32,2 99 67,8 34 19,8 138 80,2 Uji chi square p=0,014 Ho diterima p= 0,335 Ho diterima Tabel 3. Distribusi Frekuensi Perilaku Seksual berdasarkan Sikap Orang Tua Tabel 3 menunjukkan bahwa responden laki-laki (42,9%) memiliki orang tua yang sikapnya tidak permisif, sedangkan pada responden perempuan (23,1%) memiliki orang tua yang sikapnya tidak permisif. Mereka yang memiliki orang tua dengan sikap permisif memiliki proporsi melakukan perilaku seksual beresiko IMS. Hasil uji chi-square menunjukkan bahwa pada responden laki- Tabel 5. METODE Distribusi Frekuensi Perilaku Seksual berdasarkan Akses Media Akses media Perilaku Seksual Laki-laki Perempuan Beresiko Tidak Beresiko Bersiko Tidak Beresiko f % f % f % f % Rendah 24 41,4 34 58,6 12 18,2 54 81,8 Tinggi 23 26,1 65 73,9 22 20,8 84 79,2 Jumlah 47 32,2 99 67,8 34 19,8 138 80,2 p=0,030 Ho diterima p= 0,830 Ho diterima Nomor p-ISSN: 1978-6743, Nomor e-ISSN: 2477-3948 44 Jurnal Ilmiah Kesehatan (Journal of Health Sciences), Vol. 13, No. 1, Februari 2020, Hal. 39-48 44 Jurnal Ilmiah Kesehatan (Journal of Health Sciences), Vol. 13, No. 1, Februari 2020, Hal. 39-48 44 Jurnal Ilmiah Kesehatan (Journal of Health Sciences), Vol. 13, No. 1, Februari 2020, Hal. 39-48 Tabel 5 menunjukkan bahwa responden laki-laki (41,4%) memiliki akses media rendah, sedangkan pada responden perempuan (20,8%) memiliki akses media tinggi. Hasil uji chi-square menunjukkan bahwa pada responden laki-laki ada hubungan antara akses media dengan perilaku seks pranikah beresiko IM. usia 17 tahun merupakan masa remaja akhir dan selanjutnya akan masa dewasa. Jadi, responden dalam penelitian ini adalah remaja yang berada pada masa remaja awal dan remaja akhir, yang duduk di bangku SMA. Pada masa ini remaja masih mencari identitas diri. Pada saat usia remaja seharusnya remaja sudah memahami tentang seksualitas adalah hal yang merupakan upaya untuk membentuk identitas yang bagus dan tidak labil, karena dengan identitas yang bagus dan tidak labil sebagai makhluk seksual, setiap orang juga paham tentang sikap, keyakinan, batasan-batasan, nilai-nilai yang sudah dimiliki. (Mutia, 2008) Dari hasil penelitian variabel yang memiliki hubungan yang signifikan terhadap variabel dependen pada remaja laki-laki adalah umur (p=0,047), sikap orang tua (p=0,014) dan akses media (p=0,03). Sedangkan pada responden perempuan adalah tidak ada variabel yang berhubungan. Variabel yang paling berpengaruh terhadap perilaku seksual beresiko IMS pada responden laki-laki adalah akses media dengan OR 2,378 yang artinya responden laki-laki dengan tingkat akses media tinggi memiliki peluang 2,3 kali untuk melakukan perilaku seksual beresiko IMS. Hasil uji chi-square menunjukkan bahwa pada responden laki-laki (p value=0,047) ada hubungan yang signifikan, sedangkan pada responden perempuan (p value=0,508) tidak ada hubungan yang signifikan antara umur dengan perilaku seks pranikah beresiko IMS. Sikap Teman Sebaya Hasil penelitian ini menunjukkan bahwa responden yang melakukan intercourse multipartner, bahwa responden laki-laki (40,3%) dan responden perempuan (20,8%) memiliki teman sebaya yang sikapnya tidak permisif. Mereka yang memiliki teman sebaya dengan sikap tidak permisif memiliki proporsi melakukan perilaku seksual beresiko IMS. Hasil uji chi square menunjukkan bahwa pada responden laki-laki (p value=0,059) maupun responden perempuan (p value=0,830), tidak ada hubungan antara sikap teman sebaya dengan perilaku seks pranikah beresiko IMS. Rifzul Maulina, Zainal Alim Akses Media dan Perilaku Seksual Pranikah Beresiko Infeksi Menular Seksual (IMS) Pada Siswa SMA di Kabupaten Malang Rifzul Maulina, Zainal Alim Akses Media dan Perilaku Seksual Pranikah Beresiko Infeksi Menular Seksual (IMS) Pada Siswa SMA di Kabupaten Malang 45 dorongan seksual yang muncul, mereka dikhawatirkan akan melakukan perilaku seksual pranikah, mulai dari masturbasi hingga intercourse. (Notoatmojo, 2003) yang ada, hal ini merupakan hasil dari penelitian PKBI (Djoerban, 2009) Umur Hal ini disebabkan bahwa responden pada penelitian ini mempunyai rentang umur yang sangat bervariasi sehingga mempunyai cara pandang yang berbeda dalam menyikapi dan berperilaku terhadap seksualitas. Selain itu, dengan usia menstruasi yang dini saat ini menyebabkan umur menjadi lebih cepat dewasa, sehingga apabila tidak mampu mengendalikan Sampel dalam penelitian ini adalah siswa yang sedang duduk di bangku SMA kelas 1, 2, dan 3 yang berusia 15 – 18 tahun. Berdasarkan umur, responden yang termasuk dalam kategori umur remaja awal (<17 Tahun) sebanyak 50,3% sedangkan umur remaja akhir (≥17 Tahun) sebanyak 49,7%. Menurut Kemenkes (2011) bahwa Nomor p-ISSN: 1978-6743, Nomor e-ISSN: 2477-3948 Rifzul Maulina, Zainal Alim Akses Media dan Perilaku Seksual Pranikah Beresiko Infeksi Menular Seksual (IMS) Pada Siswa SMA di Kabupaten Malang Rifzul Maulina, Zainal Alim Akses Media dan Perilaku Seksual Pranikah Beresiko Infeksi Menular Seksual (IMS) Pada Siswa SMA di Kabupaten Malang Sikap Orang Tua Hasil penelitian menunjukkan bahwa responden yang melakukan intercouse multipartner bahwa responden laki-laki (42,9%) memiliki orang tua dengan sikap tidak permisif, sedangkan pada responden perempuan (23,1%) memiliki orang tua yang sikapnya permisif. Mereka yang memiliki orang tua dengan sikap permisif memiliki proporsi melakukan perilaku seksual beresiko IMS. Hal ini dikarenakan responden laki-laki dan responden perempuan memiliki efikasi diri rendah. Hasil uji chi-square menyatakan bahwa pada sampel laki-laki (p value=0,014) menunjukkan bahwa ada hubungan antara sikap orang tua dengan perilaku seksual pranikah beresiko IMS, sedangkan pada responden perempuan (p value=0,335) tidak ada hubungan yang signifikan antara sikap orang tua dengan perilaku seks pranikah beresiko IMS. Pada masa perkembangan, proses remaja adalah masa dimana remaja mulai belajar sosialisasi dan belajar mandiri keluar dari keluarga. Beberapa sifat remaja yaitu dengan membentuk kelompok sosial yang terdiri dari teman-teman terdekatnya, sehingga memiliki rasa yang dapat diterima oleh golongannya dan hal ini merupakan suatu hal yang sangat penting pada saat usia remaja (Nugrahawati, 2016). Keluarga yang sehat mampu memberikan fungsi secara maksimal dan optimal serta membantu remaja agar bisa mengalihkan dorongan seksual secara produktif dengan cara sesuai dengan nilai yang berlaku serta selaras dengan norma Sehingga remaja akan mengupayakan agar bisa diterima oleh kelompok sosialnya dengan berbagai cara diantaranya mengikuti perilaku, nilai dan sikap kelompoknya Meskipun remaja sudah Nomor p-ISSN: 1978-6743, Nomor e-ISSN: 2477-3948 46 Jurnal Ilmiah Kesehatan (Journal of Health Sciences), Vol. 13, No. 1, Februari 2020, Hal. 39-48 46 Jurnal Ilmiah Kesehatan (Journal of Health Sciences), Vol. 13, No. 1, Februari 2020, Hal. 39-48 46 responden dengan akses media tinggi juga memiliki proporsi melakukan perilaku seksual beresiko IMS. dalam tahap perkembangan kognitif yang memadai sehingga dapat menentukan perbuatannya sendiri, namun perilaku remaja untuk menentukan jati dirinya banyak dipengaruhi oleh lingkungan salah satunya adalah teman sebayanya (Asfriyati, 2010). Hal yang bisa mempengaruhi terhadap keinginan remaja untuk berperilaku seksual diantaranya adalah buku dan meja berdasarkan Leaner dan Spainer. Pada era saat ini yaitu dengan adanya teknologi yang sangat canggih menyebabkan informasi terkait seks mudah ditemui sehingga bisa menyebabkan dapat memberikan rangsangan secara visual (Mamahit, 2000). Sehingga remaja dalam masa ini ingin mengetahui dan ingin mencoba menirukan apa yang didengarkan dan apa yang dilihat dari media massa yang pernah diakses (Suryoputro, 2006). Komunitas teman sebaya remaja dapat diakui mempengaruhi keputusan serta pertimbangan seorang remaja tentang sikap dan perilakunya. Komunitas teman sebaya adalah sumber rujukan penting bagi remaja dalam hal sikap dan persepsi tentang gaya hidup remaja. Sikap Orang Tua Untuk remaja, teman sebaya merupakan sumber informasi misalnya tentang kehidupan sehari- hari.(Mutia, 2008) Dengan demikian, remaja yang sedang dalam periode ini ingin mengetahui akan mencoba menirukan apa yang dilihat dan apa yang didengarnya dari media massa. Hal ini disebabkan karena remaja merasa aneh dan tidak nyaman sehingga tabu untuk membicarakan tentang seksualitas dan kesehatan reproduksinya sehingga remaja berinisiatif sendiri untuk mencari informasi salah satunya yaitu lewat media massa dimana informasi tersebut belum sepenuhnya benar (Bandura, 1997). Akses Media Akses media adalah berbagai media informasi tentang kesehatan reproduksi, seksual dan pornografi yang pernah dibaca, didengar ataupun dilihat oleh responden. Hasil penelitian menunjukkan bahwa responden yang melakukan intercourse multipartner bahwa responden laki-laki (41,4%) memiliki akses media rendah, sedangkan pada perempuan (20,8%) memiliki akses media tinggi. Hasil uji chi square menunjukkan bahwa pada responden laki-laki (p value=0,030) dan perempuan (p value=0,830), menunjukkan untuk laki-laki ada hubungan antara akses media dengan perilaku seks pranikah beresiko IMS. Sehingga ada kecenderungan Remaja seringkali merasa tidak nyaman atau tabu untuk membicarakan masalah seksualitas dan kesehatan reproduksinya. Akan tetapi karena faktor keingintahuannya mereka akan berusaha Nomor p-ISSN: 1978-6743, Nomor e-ISSN: 2477-3948 Rifzul Maulina, Zainal Alim Akses Media dan Perilaku Seksual Pranikah Beresiko Infeksi Menular Seksual (IMS) Pada Siswa SMA di Kabupaten Malang 47 Rifzul Maulina, Zainal Alim Akses Media dan Perilaku Seksual Pranikah Beresiko Infeksi Menular Seksual (IMS) Pada Siswa SMA di Kabupaten Malang Faktor yang Mempengaruhinya .Laporan Penelitian Dosen Muda Universitas Airlangga. Surabaya. 2010. untuk mendapatkan informasi ini. Seringkali remaja merasa bahwa orang tuanya menolak membicarakan masalah seks kemudian mereka akan mencari alternatif sumber informasi lain seperti teman atau media massa (Kemendiknas, 2019). Faktor yang Mempengaruhinya .Laporan Penelitian Dosen Muda Universitas Airlangga. Surabaya. 2010. Dinas Kesehatan Kota Malang. Analisis Situasi IMS, HIV dan AIDS Tahun 2013. (2013). Dinas Kesehatan Kota Malang. Malang. Direktorat Jenderal Pengendalian Penyakit dan Penyehatan Lingkungan, Kementrian Kesehatan Republik Indonesia.. (2013) Laporan Perkembangan HIV-AIDS Triwulan II 2013. Kementrian Kesehatan Republik Indonesia. Jakarta. SIMPULAN Responden laki-laki dengan umur remaja awal (<17 tahun) memiliki perilaku seksual pranikah beresiko lebih besar jika dibandingkan dengan remaja akhir (≥17 tahun). Responden laki-laki berperilaku seksual pranikah beresiko terhadap IMS sebesar 32,2%, sedangkan pada responden perempuan berperilaku seksual pranikah beresiko terhadap IMS sebesar 25%. Responden laki-laki (42,9%) memiliki orang tua yang sikapnya tidak permisif, sedangkan pada responden perempuan (23,1%) memiliki orang tua yang sikapnya tidak permisif. Responden laki-laki (40,3%) dan responden perempuan (20,8%) memiliki teman sebaya yang sikapnya tidak permisif. Responden laki-laki (41,4%) memiliki akses media rendah, sedangkan pada responden perempuan (20,8%) memiliki akses media tinggi. Djoerban, Z. (2009). Membidik AIDS, Ikhtiar Memahami HIV dan ODHA. Galang Press Yogyakarta. Yogyakarta. Kementrian Kesehatan (Kemenkes) Republik Indonesia. (2011). Surveilans Terpadu Biologis Perilaku (STBP) 2011. Kementrian Kesehatan Republik Indonesia. Jakarta. Komisi Penanggulangan AIDS Provinsi Jawa Timur (KPAP Jatim). (2013). Kondisi HIV & AIDS di Jawa Timur s/d Juni 2013. Komisi Penanggulangan AIDS Provinsi Jawa Timur. Surabaya. Mamahit, E. (2000). Validasi Pemeriksaan Infeksi Menular Seksual secara Pendekatan Sindrom pada Kelompok Berperilaku Resiko Tinggi. Buletin Penelitian Kesehatan. Jakarta. Mutia, Y. (2008). Perilaku Seksual Beresiko Terkait HIV-AIDS pada Buruh Bangunan di Proyek P Perusahaan Konstruksi K tahun 2008. Universitas Diponegoro . Semarang. Nomor p-ISSN: 1978-6743, Nomor e-ISSN: 2477-3948 DAFTAR PUSTAKA Notoatmojo, S. (2003). Pendidikan dan Perilaku Kesehatan. Rineka Cipta . Jakarta. Ahmadi, H. (2009). Psikologi Sosial. Rineka Cipta. Jakarta. 2009. Asfriyati, D. (2010). Prilaku Seksual Remaja Surabaya serta Faktor- Nomor p-ISSN: 1978-6743, Nomor e-ISSN: 2477-3948 48 Jurnal Ilmiah Kesehatan (Journal of Health Sciences), Vol. 13, No. 1, Februari 2020, Hal. 39-48 Notoatmojo, S. (2013) Metodologi Penelitian Kesehatan. Rineka Cipta . Jakarta. Notoatmojo, S. (2013) Metodologi Penelitian Kesehatan. Rineka Cipta . Jakarta. Nugrahawati, E. (2016). Profil Teman Sebaya, Religiusitas, dan Perilaku Seksual Pranikah pada Mahasiswa. Universitas Islam Bandung. Bandung. Sarwono, S. (2014) Psikologi Remaja. Raja Grafindo Persada . Jakarta. Suryoputro, A;. Faktor-Faktor yang Mempengaruhi Perilaku Seksual Remaja di Jawa Tengah. Implikasinya terhadap Kebijakan dan Layanan Kesehatan Reproduksi. Makara, Kesehatan Vol. 10, No. 1, Juni, 2006. 29-40. Tukan, J. (2010). Etika Seksual dan Perkawinan. Intermedia. Jakarta. Nomor p-ISSN: 1978-6743, Nomor e-ISSN: 2477-3948

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